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The tissue-preferential distributed calcium sensors, SOS3 and SCaBP8, play important roles in SOS pathway to cope with saline conditions. Both SOS3 and SCaBP8 interact with and activate SOS2. However the regulatory mechanism for SOS2 activation and membrane recruitment by SCaBP8 differs from SOS3. SCaBP8 is phosphorylated by SOS2 at plasma membrane (PM) under salt stress. This phosphorylation anchors the SCaBP8-SOS2 complex on plasma membrane and activates PM Na+/H+ anti-porter, such as SOS1. Here, we describe that SOS2 has high binding affinity and catalytic efficiency to SCaBP8, suggesting that phosphorylation of SCaBP8 by SOS2 perhaps occurs rapidly in salt condition. SCaBP8 is also phosphorylated by PKS5 (SOS2-like Protein Kinase5) which negatively regulates PM H+-ATPase activity and functions in plant alkaline tolerance, providing a clue to roles of SCaBP8 in both salt and alkaline tolerance. SOS2 interacts with SOS3 and SCaBP8 with its FISL motif at C-terminus. However, luciferase activity complement assay indicates that SOS2 N-terminal is also essential for interacting with these proteins in plant.Key words: calcium signal, kinase activity, luciferase complementDue to their sessile nature, plants have developed elaborate strategies to deal with a number of environmental challenges. One overwhelming constraint is high salinity in the soil, which inhibits plant growth and decreases the agricultural productivity. Efflux and/or sequestering of sodium ion to apoplastic space/vacuolar are well-known cellular mechanisms that plants protect them from saline stress.1 Recently identified SOS (salt overly sensitive) pathway plays critical roles in maintaining ion homeostasis in response to high salinity.2 Two calcium sensors, SOS3 and SCaBP8 (SOS3-like calcium binding protein8), perceive cytosolic calcium signature triggered by salt, interact with and activate a Thr/Ser protein kinase, SOS2 and recruit it to the plasma membrane. Then, the formed SOS3-SOS2 or SCaBP8-SOS2 complex activates a PM Na+/H+ anti-porter, SOS1.24 Moreover, SOS2 also regulates vascular Na+/H+ antiporter activity.5 Previously, we reported that SCaBP8 and SOS3 function distinctly in activation of SOS2.3 For instance, N-terminal myristoylation of SOS3 plays an important role in salt tolerance.6 However, there is no consensus myristoylated motif in SCaBP8. Instead, an N-terminal hydrophobic domain is sufficient to facilitate the association of SCaBP8 to plasma membrane.3 In addition, SCaBP8 is phosphorylated by SOS2 under salt stress and this phosphorylation stabilizes the interaction of SOS2 and SCaBP8.4 In this report, we describe that SCaBP8 possibly is rapidly phosphorylated by SOS2 under salt stress and also phosphorylated by another stress responsible protein kinase, implying additional roles of SCaBP8 in stress responses.  相似文献   

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The SLC38 family of solute transporters mediates the coupled transport of amino acids and Na+ into or out of cells. The structural basis for this coupled transport process is not known. Here, a profile-based sequence analysis approach was used, predicting a distant relationship with the SLC5/6 transporter families. Homology models using the LeuTAa and Mhp1 transporters of known structure as templates were established, predicting the location of a conserved Na+ binding site in the center of membrane helices 1 and 8. This homology model was tested experimentally in the SLC38 member SNAT2 by analyzing the effect of a mutation to Thr-384, which is predicted to be part of this Na+ binding site. The results show that the T384A mutation not only inhibits the anion leak current, which requires Na+ binding to SNAT2, but also dramatically lowers the Na+ affinity of the transporter. This result is consistent with a previous analysis of the N82A mutant transporter, which has a similar effect on anion leak current and Na+ binding and which is also expected to form part of the Na+ binding site. In contrast, random mutations to other sites in the transporter had little or no effect on Na+ affinity. Our results are consistent with a cation binding site formed by transmembrane helices 1 and 8 that is conserved among the SLC38 transporters as well as among many other bacterial and plant transporter families of unknown structure, which are homologous to SLC38.The sodium-coupled neutral amino acid transporter, SNAT2,2 belongs to the SLC38 gene family of solute carrier proteins (1). Together with SNAT1 and -4 (2), it is believed to mediate Na+-dependent amino acid transport activity that was classically assigned to System A transporters (38). In addition to SNAT1 and -2, the SLC38 family has four other known members, two of which predominantly mediate glutamine transport (SNAT3 and -5, System N (911)). SNAT2 is widely expressed in mammalian tissue (1, 7), but it may play a particularly critical role in the brain (12), where it may help shuttle glutamine from astrocytes to neurons via the glutamate-glutamine cycle (1). This process is essential for recycling the neurotransmitter glutamate (13). However, the exact contribution of SNAT2 to the glutamate-glutamine cycle is still controversially discussed (14).Despite this physiological importance, surprisingly little is known about the functional properties and the structural basis of amino acid transport by the SLC38 proteins. Although hydropathy analysis predicts 11 transmembrane helices (TMs), with an intracellular N terminus and an extracellular C terminus (1), it is not clear whether the transporters belong to a large superfamily of transporters, of which members have been characterized structurally through x-ray crystallography. At present, sequence homology has only been established with transporters of the mammalian SLC32 and SLC36 families as well as with the more distantly related plant auxin carriers and the bacterial amino acid-polyamine-organocation (APC) family (15, 16). High resolution crystal structures are not available for any of the transporters from these families, although low resolution projection structures were recently reported for the APC family members AdiC (17) and SteT (18). However, these structures do not allow the assignment of transmembrane helices. Thus, it remains unknown whether the SLC38 fold is similar to established transport protein folds, although homology to the major facilitator superfamily seems unlikely.We have recently identified a conserved amino acid residue in SNAT2, Asn-82, which is involved in controlling the Na+ affinity of the transporter (19). Interestingly, Asn-82 is localized in the predicted TM1 of SNAT2. This first transmembrane helix was recently found to contribute ligands to a Na+ binding site in several bacterial transporters, which are related to the SLC5 (sodium glucose symporter) and SLC6 (sodium- and chloride-dependent neurotransmitter transporter) family members (2022), which also comprises bacterial members (23, 24). Although sequence similarity with SLC5 and -6 is not detectable, SLC38 may be a member of a possibly very large superfamily with the same general fold, which also contains many amino acid transport proteins.Here, we used a homology modeling approach based on profile-based sequence alignment (25, 26). A search against sequences deposited in the Protein Data Bank (PDB (27)) revealed that the transporters with the highest likelihood to share an analogous fold are a leucine transporter from Aquifex aeolicus, LeuTAa, and a homologous hydantoin transporter from Microbacterium liquefaciens, Mhp1. We established a homology model based on these structures, which predicts Asn-82 to be part of a Na+ binding site. Furthermore, another conserved hydrophilic amino acid residue in TM8, Thr-384, was predicted to be near this cation binding site. When Thr-384 was mutated to alanine, a dramatic loss of the affinity of SNAT2 for Na+ was observed, whereas mutations to other sites that were spatially removed from the predicted Na+ binding site had little or no effect on Na+ affinity. We hypothesize that the SLC38 family is a member of a large superfamily of cation/organic substrate transporters which includes the mammalian SLC5 and -6 proteins and which has a conserved cation binding site formed by TMs 1 and 8.  相似文献   

5.
The glutamine transporter SLC38A3 (SNAT3) plays an important role in the release of glutamine from brain astrocytes and the uptake of glutamine into hepatocytes. It is related to the vesicular GABA (γ-aminobutyric acid) transporter and the SLC36 family of proton-amino acid cotransporters. The transporter carries out electroneutral Na+-glutamine cotransport-H+ antiport. In addition, substrate-induced uncoupled cation currents are observed. Mutation of asparagine 76 to glutamine or histidine in predicted transmembrane helix 1 abolished all substrate-induced currents. Mutation of asparagine 76 to aspartate rendered the transporter Na+-independent and resulted in a gain of a large substrate-induced chloride conductance in the absence of Na+. Thus, a single residue is critical for coupled and uncoupled ion flows in the glutamine transporter SNAT3. Homology modeling of SNAT3 along the structure of the related benzyl-hydantoin permease from Microbacterium liquefaciens reveals that Asn-76 is likely to be located in the center of the membrane close to the translocation pore and forms part of the predicted Na+ -binding site.The amino acid and auxin permease superfamily comprises a wide variety of transport proteins. In mammals, three distinct solute carrier families (SLC) belong to this superfamily, namely SLC32, SLC36, and SLC38 (1). Despite belonging to the same superfamily, the three solute carrier families have different transport mechanisms. The SLC32 family has only one member, the vesicular inhibitory amino acid transporter, which supposedly carries out a H+-GABA (γ-aminobutyric acid) antiport (2). The SLC36 family comprises four members, two of which have been characterized in more detail. These are the proton amino acid cotransporters 1 and 2 (PAT1 and 2) that carry out glycine and proline uptake in kidney and intestine and are mutated in iminoglycinuria (3, 4). The SLC38 family is comprised of 11 members, 5 of which have been characterized in more detail (5). Two different transport mechanisms are found within this family, namely the Na+-amino acid cotransporters SNAT1, SNAT2, and SNAT4 and the Na+-amino acid cotransporters-H+-antiporters SNAT3 and SNAT5. Transporters of the superfamily play a key role in inhibitory and excitatory neurotransmission, metabolite absorption, and liver metabolism. Despite their important roles in mammalian physiology, relatively little is known about the structure and function of these transporters.The activity of ion-coupled membrane transporters is frequently associated with currents which de- or hyperpolarize the cell membrane. These currents may be due to electrogenic transport stoichiometry and/or to a non-stoichiometric ion conductance (6). Transport-associated ion conductances have been identified in a number of transporters but have been particularly well studied in several Na+-coupled neurotransmitter transporters (711). Transport-associated conductances have also been observed in electroneutral transporters that do not carry out net charge movement (8, 1215). The glutamine transporter SNAT3, for instance, has a transport mechanism in which glutamine uptake is coupled to the cotransport of 1Na+ and the antiport of 1H+ and, hence, is unaffected by changes of the membrane potential (13, 16). Despite the electroneutral transport mechanism, substrate uptake is accompanied by inward currents, which are carried by cations below pH 7 and by protons at alkaline pH. In addition, a substrate-independent cation conductance and a Na+/H+ exchange activity has been observed (17). Non-stoichiometric currents can be mediated by the same ions that are involved in the coupled transport process, such as in the case of SNAT3, but may also be carried by different ions. Stoichiometric glutamate transport, for instance, involves Na+, H+, and K+ ions, whereas the glutamate transport-associated conductance is carried by chloride (18).A crucial question concerning transporter-associated ion conductances is whether the conducting pore coincides with the translocation pathway of the substrate and whether both use the same critical residues. In the case of the glutamate transporters, evidence has been presented suggesting that different residues are critical for the anion conductance than for substrate transport (19, 20) but that they all line the same pathway (21). Here we show that asparagine 76 of SNAT3 is critical for substrate-induced ion conductance and affects binding of the cosubstrate Na+. In addition we show that this residue is likely to be localized in the translocation pore in the center of the membrane.  相似文献   

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Alkalosis impairs the natriuretic response to diuretics, but the underlying mechanisms are unclear. The soluble adenylyl cyclase (sAC) is a chemosensor that mediates bicarbonate-dependent elevation of cAMP in intracellular microdomains. We hypothesized that sAC may be an important regulator of Na+ transport in the kidney. Confocal images of rat kidney revealed specific immunolocalization of sAC in collecting duct cells, and immunoblots confirmed sAC expression in mouse cortical collecting duct (mpkCCDc14) cells. These cells exhibit aldosterone-stimulated transepithelial Na+ currents that depend on both the apical epithelial Na+ channel (ENaC) and basolateral Na+,K+-ATPase. RNA interference-mediated 60-70% knockdown of sAC expression comparably inhibited basal transepithelial short circuit currents (Isc) in mpkCCDc14 cells. Moreover, the sAC inhibitors KH7 and 2-hydroxyestradiol reduced Isc in these cells by 50-60% within 30 min. 8-Bromoadenosine-3′,5′-cyclic-monophosphate substantially rescued the KH7 inhibition of transepithelial Na+ current. Aldosterone doubled ENaC-dependent Isc over 4 h, an effect that was abolished in the presence of KH7. The sAC contribution to Isc was unaffected with apical membrane nystatin-mediated permeabilization, whereas the sAC-dependent Na+ current was fully inhibited by basolateral ouabain treatment, suggesting that the Na+,K+-ATPase, rather than ENaC, is the relevant transporter target of sAC. Indeed, neither overexpression of sAC nor treatment with KH7 modulated ENaC currents in Xenopus oocytes. ATPase and biotinylation assays in mpkCCDc14 cells demonstrated that sAC inhibition decreases catalytic activity rather than surface expression of the Na+,K+-ATPase. In summary, these results suggest that sAC regulates both basal and agonist-stimulated Na+ reabsorption in the kidney collecting duct, acting to enhance Na+,K+-ATPase activity.Maintenance of intracellular pH depends in part on the extracellular to intracellular Na+ gradient, and elevation of intracellular [Na+] can lead to acidification of the cytoplasm. It has been shown that acidification of the cytoplasm of cells from frog skin and toad bladder by increased partial pressure of CO2 reduces Na+ transport and permeability (1, 2). Conversely, the rise in plasma bicarbonate caused by metabolic alkalosis with chronic diuretic use has been shown to increase net renal Na+ reabsorption independently of volume status, electrolyte depletion, and/or increased aldosterone secretion (3, 4). However, the underlying mechanisms involved in these phenomena remain unclear.The soluble adenylyl cyclase (sAC)2 is a chemosensor that mediates the elevation of cAMP in intracellular microdomains (5-7). Unlike transmembrane adenylyl cyclases (tmACs), sAC is insensitive to regulation by forskolin or heterotrimeric G proteins (8) and is directly activated by elevations of intracellular calcium (9, 10) and/or bicarbonate ions (11). Thus, sAC mediates localized intracellular increases in cAMP in response to variations in bicarbonate levels or its closely related parameters, partial pressure of CO2 and pH. Mammalian sAC is more similar to bicarbonate-regulated cyanobacterial adenylyl cyclases than to other mammalian nucleotidyl cyclases, which may indicate that there is a unifying mechanism for the regulation of cAMP signaling by bicarbonate across biological systems. Although sAC appears to be encoded by a single gene, there is significant isoform diversity for this ubiquitously expressed enzyme (11, 12) generated by alternative splicing (reviewed in Ref. 13). sAC has been shown to regulate the subcellular localization and/or activity of membrane transport proteins such as the vacuolar H+-ATPase (V-ATPase) and cystic fibrosis transmembrane conductance regulator in epithelial cells (14, 15). Functional activity of sAC has been reported in the kidney (16), and sAC has been localized to epithelial cells in the distal nephron (14, 17).Given that natriuresis is decreased during metabolic alkalosis, when bicarbonate is elevated, and Na+ reabsorption is impaired by high partial pressure of CO2, we hypothesized that bicarbonate-regulated sAC may play a key role in the regulation of transepithelial Na+ transport in the distal nephron. Reabsorption of Na+ in the kidney and other epithelial tissues is mediated by the parallel operation of apical ENaC and basolateral Na+,K+-ATPase, and both transport proteins can be stimulated by cAMP via the cAMP-dependent protein kinase (PKA) (18, 53). The aims of this study were to investigate the role of sAC in the regulation of transepithelial Na+ transport in the kidney through the use of specific sAC inhibitors and electrophysiological measurements. We found that sAC inhibition blocks transepithelial Na+ reabsorption in polarized mpkCCDc14 cells under both basal and hormone-stimulated conditions. Selective membrane permeabilization studies revealed that although ENaC activity appears to be unaffected by sAC inhibition, flux through the Na+,K+-ATPase is sensitive to sAC modulation. Inhibiting sAC decreases ATPase activity without affecting plasma membrane expression of the pump; thus, tonic sAC activity appears to be required for Na+ reabsorption in kidney collecting duct.  相似文献   

8.
Human concentrative nucleoside transporter 3 (hCNT3) utilizes electrochemical gradients of both Na+ and H+ to accumulate pyrimidine and purine nucleosides within cells. We have employed radioisotope flux and electrophysiological techniques in combination with site-directed mutagenesis and heterologous expression in Xenopus oocytes to identify two conserved pore-lining glutamate residues (Glu-343 and Glu-519) with essential roles in hCNT3 Na+/nucleoside and H+/nucleoside cotransport. Mutation of Glu-343 and Glu-519 to aspartate, glutamine, and cysteine severely compromised hCNT3 transport function, and changes included altered nucleoside and cation activation kinetics (all mutants), loss or impairment of H+ dependence (all mutants), shift in Na+:nucleoside stoichiometry from 2:1 to 1:1 (E519C), complete loss of catalytic activity (E519Q) and, similar to the corresponding mutant in Na+-specific hCNT1, uncoupled Na+ currents (E343Q). Consistent with close-proximity integration of cation/solute-binding sites within a common cation/permeant translocation pore, mutation of Glu-343 and Glu-519 also altered hCNT3 nucleoside transport selectivity. Both residues were accessible to the external medium and inhibited by p-chloromercuribenzene sulfonate when converted to cysteine.Physiologic nucleosides and the majority of synthetic nucleoside analogs with antineoplastic and/or antiviral activity are hydrophilic molecules that require specialized plasma membrane nucleoside transporter (NT)3 proteins for transport into or out of cells (14). NT-mediated transport is required for nucleoside metabolism by salvage pathways and is a critical determinant of the pharmacologic actions of nucleoside drugs (36). By regulating adenosine availability to purinoreceptors, NTs also modulate a diverse array of physiological processes, including neurotransmission, immune responses, platelet aggregation, renal function, and coronary vasodilation (4, 6, 7). Two structurally unrelated NT families of integral membrane proteins exist in human and other mammalian cells and tissues as follows: the SLC28 concentrative nucleoside transporter (CNT) family and the SLC29 equilibrative nucleoside transporter (ENT) family (3, 4, 6, 8, 9). ENTs are normally present in most, possibly all, cell types (4, 6, 8). CNTs, in contrast, are found predominantly in intestinal and renal epithelia and other specialized cell types, where they have important roles in absorption, secretion, distribution, and elimination of nucleosides and nucleoside drugs (13, 5, 6, 9).The CNT protein family in humans is represented by three members, hCNT1, hCNT2, and hCNT3. Belonging to a CNT subfamily phylogenetically distinct from hCNT1/2, hCNT3 utilizes electrochemical gradients of both Na+ and H+ to accumulate a broad range of pyrimidine and purine nucleosides and nucleoside drugs within cells (10, 11). hCNT1 and hCNT2, in contrast, are Na+-specific and transport pyrimidine and purine nucleosides, respectively (1113). Together, hCNT1–3 account for the three major concentrative nucleoside transport processes of human and other mammalian cells. Nonmammalian members of the CNT protein family that have been characterized functionally include hfCNT, a second member of the CNT3 subfamily from the ancient marine prevertebrate the Pacific hagfish Eptatretus stouti (14), CeCNT3 from Caenorhabditis elegans (15), CaCNT from Candida albicans (16), and the bacterial nucleoside transporter NupC from Escherichia coli (17). hfCNT is Na+- but not H+-coupled, whereas CeCNT3, CaCNT, and NupC are exclusively H+-coupled. Na+:nucleoside coupling stoichiometries are 1:1 for hCNT1 and hCNT2 and 2:1 for hCNT3 and hfCNT3 (11, 14). H+:nucleoside coupling ratios for hCNT3 and CaCNT are 1:1 (11, 16).Although much progress has been made in molecular studies of ENT proteins (4, 6, 8), studies of structurally and functionally important regions and residues within the CNT protein family are still at an early stage. Topological investigations suggest that hCNT1–3 and other eukaryote CNT family members have a 13 (or possibly 15)-transmembrane helix (TM) architecture, and multiple alignments reveal strong sequence similarities within the C-terminal half of the proteins (18). Prokaryotic CNTs lack the first three TMs of their eukaryotic counterparts, and functional expression of N-terminally truncated human and rat CNT1 in Xenopus oocytes has established that these three TMs are not required for Na+-dependent uridine transport activity (18). Consistent with this finding, chimeric studies involving hCNT1 and hfCNT (14) and hCNT1 and hCNT3 (19) have demonstrated that residues involved in Na+- and H+-coupling reside in the C-terminal half of the protein. Present in this region of the transporter, but of unknown function, is a highly conserved (G/A)XKX3NEFVA(Y/M/F) motif common to all eukaryote and prokaryote CNTs.By virtue of their negative charge and consequent ability to interact directly with coupling cations and/or participate in cation-induced and other protein conformational transitions, glutamate and aspartate residues play key functional and structural roles in a broad spectrum of mammalian and bacterial cation-coupled transporters (2030). Little, however, is known about their role in CNTs. This study builds upon a recent mutagenesis study of conserved glutamate and aspartate residues in hCNT1 (31) to undertake a parallel in depth investigation of corresponding residues in hCNT3. By employing the multifunctional capability of hCNT3 as a template for these studies, this study provides novel mechanistic insights into the molecular mechanism(s) of CNT-mediated cation/nucleoside cotransport, including the role of the (G/A)XKX3NEFVA(Y/M/F) motif.  相似文献   

9.
The salt stress-induced SALT-OVERLY-SENSITIVE (SOS) pathway in Arabidopsis (Arabidopsis thaliana) involves the perception of a calcium signal by the SOS3 and SOS3-like CALCIUM-BINDING PROTEIN8 (SCaBP8) calcium sensors, which then interact with and activate the SOS2 protein kinase, forming a complex at the plasma membrane that activates the SOS1 Na+/H+ exchanger. It has recently been reported that phosphorylation of SCaBP proteins by SOS2-like protein kinases (PKSs) stabilizes the interaction between the two proteins as part of a regulatory mechanism that was thought to be common to all SCaBP and PKS proteins. Here, we report the calcium-independent activation of PKS24 by SCaBP1 and show that activation is dependent on interaction of PKS24 with the C-terminal tail of SCaBP1. However, unlike what has been found for other PKS-SCaBP pairs, multiple amino acids in SCaBP1 are phosphorylated by PKS24, and this phosphorylation is dependent on the interaction of the proteins through the PKS24 FISL motif and on the efficient activation of PKS24 by the C-terminal tail of SCaBP1. In addition, we show that Thr-211 and Thr-212, which are not common phosphorylation sites in the conserved PFPF motif found in most SCaBP proteins, are important for this activation. Finally, we also found that SCaBP1-regulated PKS24 kinase activity is important for inactivating the Arabidopsis plasma membrane proton-translocating adenosine triphosphatase. Together, these results suggest the existence of a novel SCaBP-PKS regulatory mechanism in plants.Calcium is a ubiquitous second messenger that plays an important role in the regulation of plant growth and development. Many different types of calcium-binding proteins have been identified in plants (Harper et al., 2004), including the SALT-OVERLY-SENSITIVE3 (SOS3)-LIKE CALCIUM BINDING PROTEINS (SCaBPs; Liu and Zhu, 1998; Gong et al., 2004). Because the calcium-binding domain of these proteins shares sequence similarity with the yeast calcineurin B subunit, they have also been called CALCINEURIN B-LIKE PROTEINS (CBLs; Kudla et al., 1999; Luan et al., 2002). The founding member of this gene family, SOS3, was identified in a genetic screen from a salt-sensitive Arabidopsis (Arabidopsis thaliana) mutant (Liu and Zhu, 1998). SCaBP/CBL proteins interact with the SOS2-LIKE PROTEIN KINASES (PKSs)/CBL-INTERACTING PROTEIN KINASES (CIPKs; Shi et al., 1999; Halfter et al., 2000; Guo et al., 2001). The genetic linkage between these two families was established after identification of SOS2 from a genetic screen similar to the one that identified the sos3 mutant (Liu et al., 2000). SOS3 interacts with SOS2 in vivo and in vitro and activates SOS2 in a calcium-dependent manner in vitro (Halfter et al., 2000). The SOS3-SOS2 complex further activates SOS1, a plasma membrane (PM) Na+/H+ antiporter, by directly phosphorylating the SOS1 C terminus (Shi et al., 2000; Qiu et al., 2002; Quintero et al., 2002, 2011; Yu et al., 2010).In addition to the calcium-dependent activation of PKSs by SCaBP calcium sensors, two other regulatory mechanisms have been identified for these protein families. First, PKSs have a conserved 21-amino acid peptide (FISL motif) in their regulatory domain that is necessary for efficient interaction with the SCaBP calcium sensors (Guo et al., 2001; Albrecht et al., 2001; Gong et al., 2004). The PKS regulatory domain interacts with its kinase domain via the FISL motif to repress PKS activity; interaction of SCaBP with the PKS FISL motif releases the kinase domain inhibition allowing for kinase activity (Guo et al., 2001; Gong et al., 2004). Second, the PKSs phosphorylate a Ser residue in the conserved C-terminal PFPF motif of the SCaBP proteins. This phosphorylation enhances the interaction between the two proteins and fully activates the complex (Lin et al., 2009; Du et al., 2011; Hashimoto et al., 2012).In this study, we identified a novel PKS activation mechanism involving the calcium-independent activation of PKS24 by SCaBP1 and show that it requires binding of SCaBP1 to the FISL motif of PKS24 and the involvement of two Thr residues in the SCaBP1 C-terminal tail.  相似文献   

10.
A role for SR proteins in plant stress responses   总被引:1,自引:0,他引:1  
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11.
NHE5 is a brain-enriched Na+/H+ exchanger that dynamically shuttles between the plasma membrane and recycling endosomes, serving as a mechanism that acutely controls the local pH environment. In the current study we show that secretory carrier membrane proteins (SCAMPs), a group of tetraspanning integral membrane proteins that reside in multiple secretory and endocytic organelles, bind to NHE5 and co-localize predominantly in the recycling endosomes. In vitro protein-protein interaction assays revealed that NHE5 directly binds to the N- and C-terminal cytosolic extensions of SCAMP2. Heterologous expression of SCAMP2 but not SCAMP5 increased cell-surface abundance as well as transporter activity of NHE5 across the plasma membrane. Expression of a deletion mutant lacking the SCAMP2-specific N-terminal cytosolic domain, and a mini-gene encoding the N-terminal extension, reduced the transporter activity. Although both Arf6 and Rab11 positively regulate NHE5 cell-surface targeting and NHE5 activity across the plasma membrane, SCAMP2-mediated surface targeting of NHE5 was reversed by dominant-negative Arf6 but not by dominant-negative Rab11. Together, these results suggest that SCAMP2 regulates NHE5 transit through recycling endosomes and promotes its surface targeting in an Arf6-dependent manner.Neurons and glial cells in the central and peripheral nervous systems are especially sensitive to perturbations of pH (1). Many voltage- and ligand-gated ion channels that control membrane excitability are sensitive to changes in cellular pH (1-3). Neurotransmitter release and uptake are also influenced by cellular and organellar pH (4, 5). Moreover, the intra- and extracellular pH of both neurons and glia are modulated in a highly transient and localized manner by neuronal activity (6, 7). Thus, neurons and glia require sophisticated mechanisms to finely tune ion and pH homeostasis to maintain their normal functions.Na+/H+ exchangers (NHEs)3 were originally identified as a class of plasma membrane-bound ion transporters that exchange extracellular Na+ for intracellular H+, and thereby regulate cellular pH and volume. Since the discovery of NHE1 as the first mammalian NHE (8), eight additional isoforms (NHE2-9) that share 25-70% amino acid identity have been isolated in mammals (9, 10). NHE1-5 commonly exhibit transporter activity across the plasma membrane, whereas NHE6-9 are mostly found in organelle membranes and are believed to regulate organellar pH in most cell types at steady state (11). More recently, NHE10 was identified in human and mouse osteoclasts (12, 13). However, the cDNA encoding NHE10 shares only a low degree of sequence similarity with other known members of the NHE gene family, raising the possibility that this sodium-proton exchanger may belong to a separate gene family distantly related to NHE1-9 (see Ref. 9).NHE gene family members contain 12 putative transmembrane domains at the N terminus followed by a C-terminal cytosolic extension that plays a role in regulation of the transporter activity by protein-protein interactions and phosphorylation. NHEs have been shown to regulate the pH environment of synaptic nerve terminals and to regulate the release of neurotransmitters from multiple neuronal populations (14-16). The importance of NHEs in brain function is further exemplified by the findings that spontaneous or directed mutations of the ubiquitously expressed NHE1 gene lead to the progression of epileptic seizures, ataxia, and increased mortality in mice (17, 18). The progression of the disease phenotype is associated with loss of specific neuron populations and increased neuronal excitability. However, NHE1-null mice appear to develop normally until 2 weeks after birth when symptoms begin to appear. Therefore, other mechanisms may compensate for the loss of NHE1 during early development and play a protective role in the surviving neurons after the onset of the disease phenotype.NHE5 was identified as a unique member of the NHE gene family whose mRNA is expressed almost exclusively in the brain (19, 20), although more recent studies have suggested that NHE5 might be functional in other cell types such as sperm (21, 22) and osteosarcoma cells (23). Curiously, mutations found in several forms of congenital neurological disorders such as spinocerebellar ataxia type 4 (24-26) and autosomal dominant cerebellar ataxia (27-29) have been mapped to chromosome 16q22.1, a region containing NHE5. However, much remains unknown as to the molecular regulation of NHE5 and its role in brain function.Very few if any proteins work in isolation. Therefore identification and characterization of binding proteins often reveal novel functions and regulation mechanisms of the protein of interest. To begin to elucidate the biological role of NHE5, we have started to explore NHE5-binding proteins. Previously, β-arrestins, multifunctional scaffold proteins that play a key role in desensitization of G-protein-coupled receptors, were shown to directly bind to NHE5 and promote its endocytosis (30). This study demonstrated that NHE5 trafficking between endosomes and the plasma membrane is regulated by protein-protein interactions with scaffold proteins. More recently, we demonstrated that receptor for activated C-kinase 1 (RACK1), a scaffold protein that links signaling molecules such as activated protein kinase C, integrins, and Src kinase (31), directly interacts with and activates NHE5 via integrin-dependent and independent pathways (32). These results further indicate that NHE5 is partly associated with focal adhesions and that its targeting to the specialized microdomain of the plasma membrane may be regulated by various signaling pathways.Secretory carrier membrane proteins (SCAMPs) are a family of evolutionarily conserved tetra-spanning integral membrane proteins. SCAMPs are found in multiple organelles such as the Golgi apparatus, trans-Golgi network, recycling endosomes, synaptic vesicles, and the plasma membrane (33, 34) and have been shown to play a role in exocytosis (35-38) and endocytosis (39). Currently, five isoforms of SCAMP have been identified in mammals. The extended N terminus of SCAMP1-3 contain multiple Asn-Pro-Phe (NPF) repeats, which may allow these isoforms to participate in clathrin coat assembly and vesicle budding by binding to Eps15 homology (EH)-domain proteins (40, 41). Further, SCAMP2 was shown recently to bind to the small GTPase Arf6 (38), which is believed to participate in traffic between the recycling endosomes and the cell surface (42, 43). More recent studies have suggested that SCAMPs bind to organellar membrane type NHE7 (44) and the serotonin transporter SERT (45) and facilitate targeting of these integral membrane proteins to specific intracellular compartments. We show in the current study that SCAMP2 binds to NHE5, facilitates the cell-surface targeting of NHE5, and elevates Na+/H+ exchange activity at the plasma membrane, whereas expression of a SCAMP2 deletion mutant lacking the N-terminal domain containing the NPF repeats suppresses the effect. Further we show that this activity of SCAMP2 requires an active form of a small GTPase Arf6, but not Rab11. We propose a model in which SCAMPs bind to NHE5 in the endosomal compartment and control its cell-surface abundance via an Arf6-dependent pathway.  相似文献   

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13.
14.
Organelle movement in plants is dependent on actin filaments with most of the organelles being transported along the actin cables by class XI myosins. Although chloroplast movement is also actin filament-dependent, a potential role of myosin motors in this process is poorly understood. Interestingly, chloroplasts can move in any direction and change the direction within short time periods, suggesting that chloroplasts use the newly formed actin filaments rather than preexisting actin cables. Furthermore, the data on myosin gene knockouts and knockdowns in Arabidopsis and tobacco do not support myosins'' XI role in chloroplast movement. Our recent studies revealed that chloroplast movement and positioning are mediated by the short actin filaments localized at chloroplast periphery (cp-actin filaments) rather than cytoplasmic actin cables. The accumulation of cp-actin filaments depends on kinesin-like proteins, KAC1 and KAC2, as well as on a chloroplast outer membrane protein CHUP1. We propose that plants evolved a myosin XI-independent mechanism of the actin-based chloroplast movement that is distinct from the mechanism used by other organelles.Key words: actin, Arabidopsis, blue light, kinesin, myosin, organelle movement, phototropinOrganelle movement and positioning are pivotal aspects of the intracellular dynamics in most eukaryotes. Although plants are sessile organisms, their organelles are quickly repositioned in response to fluctuating environmental conditions and certain endogenous signals. By and large, plant organelle movements and positioning are dependent on actin filaments, although microtubules play certain accessory roles in organelle dynamics.1,2 Actin inhibitors effectively retard the movements of mitochondria,36 peroxisomes,5,711 Golgi stacks,12,13 endoplasmic reticulum (ER),14,15 and nuclei.1618 These organelles are co-aligned and associated with actin filaments.5,7,8,1012,15,18 Recent progress in this field started to reveal the molecular motility system responsible for the organelle transport in plants.19Chloroplast movement is among the most fascinating models of organelle movement in plants because it is precisely controlled by ambient light conditions.20,21 Weak light induces chloroplast accumulation response so that chloroplasts can capture photosynthetic light efficiently (Fig. 1A). Strong light induces chloroplast avoidance response to escape from photodamage (Fig. 1B).22 The blue light-induced chloroplast movement is mediated by the blue light receptor phototropin (phot). In some cryptogam plants, the red light-induced chloroplast movement is regulated by a chimeric phytochrome/phototropin photoreceptor neochrome.2325 In a model plant Arabidopsis, phot1 and phot2 function redundantly to regulate the accumulation response,26 whereas phot2 alone is essential for the avoidance response.27,28 Several additional factors regulating chloroplast movement were identified by analyses of Arabidopsis mutants deficient in chloroplast photorelocation.2932 In particular, identification of CHUP1 (chloroplast unusual positioning 1) revealed the connection between chloroplasts and actin filaments at the molecular level.29 CHUP1 is a chloroplast outer membrane protein capable of interacting with F-actin, G-actin and profilin in vitro.29,33,34 The chup1 mutant plants are defective in both the chloroplast movement and chloroplast anchorage to the plasma membrane,22,29,33 suggesting that CHUP1 plays an important role in linking chloroplasts to the plasma membrane through the actin filaments. However, how chloroplasts move using the actin filaments and whether chloroplast movement utilizes the actin-based motility system similar to other organelle movements remained to be determined.Open in a separate windowFigure 1Schematic distribution patterns of chloroplasts in a palisade cell under different light conditions, weak (A) and strong (B) lights. Shown as a side view of mid-part of the cell and a top view with three different levels (i.e., top, middle and bottom of the cell). The cell was irradiated from the leaf surface shown as arrows. Weak light induces chloroplast accumulation response (A) and strong light induces the avoidance response (B).Here, we review the recent findings pointing to existence of a novel actin-based mechanisms for chloroplast movement and discuss the differences between the mechanism responsible for movement of chloroplasts and other organelles.  相似文献   

15.
Fetal cells migrate into the mother during pregnancy. Fetomaternal transfer probably occurs in all pregnancies and in humans the fetal cells can persist for decades. Microchimeric fetal cells are found in various maternal tissues and organs including blood, bone marrow, skin and liver. In mice, fetal cells have also been found in the brain. The fetal cells also appear to target sites of injury. Fetomaternal microchimerism may have important implications for the immune status of women, influencing autoimmunity and tolerance to transplants. Further understanding of the ability of fetal cells to cross both the placental and blood-brain barriers, to migrate into diverse tissues, and to differentiate into multiple cell types may also advance strategies for intravenous transplantation of stem cells for cytotherapeutic repair. Here we discuss hypotheses for how fetal cells cross the placental and blood-brain barriers and the persistence and distribution of fetal cells in the mother.Key Words: fetomaternal microchimerism, stem cells, progenitor cells, placental barrier, blood-brain barrier, adhesion, migrationMicrochimerism is the presence of a small population of genetically distinct and separately derived cells within an individual. This commonly occurs following transfusion or transplantation.13 Microchimerism can also occur between mother and fetus. Small numbers of cells traffic across the placenta during pregnancy. This exchange occurs both from the fetus to the mother (fetomaternal)47 and from the mother to the fetus.810 Similar exchange may also occur between monochorionic twins in utero.1113 There is increasing evidence that fetomaternal microchimerism persists lifelong in many child-bearing women.7,14 The significance of fetomaternal microchimerism remains unclear. It could be that fetomaternal microchimerism is an epiphenomenon of pregnancy. Alternatively, it could be a mechanism by which the fetus ensures maternal fitness in order to enhance its own chances of survival. In either case, the occurrence of pregnancy-acquired microchimerism in women may have implications for graft survival and autoimmunity. More detailed understanding of the biology of microchimeric fetal cells may also advance progress towards cytotherapeutic repair via intravenous transplantation of stem or progenitor cells.Trophoblasts were the first zygote-derived cell type found to cross into the mother. In 1893, Schmorl reported the appearance of trophoblasts in the maternal pulmonary vasculature.15 Later, trophoblasts were also observed in the maternal circulation.1620 Subsequently various other fetal cell types derived from fetal blood were also found in the maternal circulation.21,22 These fetal cell types included lymphocytes,23 erythroblasts or nucleated red blood cells,24,25 haematopoietic progenitors7,26,27 and putative mesenchymal progenitors.14,28 While it has been suggested that small numbers of fetal cells traffic across the placenta in every human pregnancy,2931 trophoblast release does not appear to occur in all pregnancies.32 Likewise, in mice, fetal cells have also been reported in maternal blood.33,34 In the mouse, fetomaternal transfer also appears to occur during all pregnancies.35  相似文献   

16.
During excitation, muscle cells gain Na+ and lose K+, leading to a rise in extracellular K+ ([K+]o), depolarization, and loss of excitability. Recent studies support the idea that these events are important causes of muscle fatigue and that full use of the Na+,K+-ATPase (also known as the Na+,K+ pump) is often essential for adequate clearance of extracellular K+. As a result of their electrogenic action, Na+,K+ pumps also help reverse depolarization arising during excitation, hyperkalemia, and anoxia, or from cell damage resulting from exercise, rhabdomyolysis, or muscle diseases. The ability to evaluate Na+,K+-pump function and the capacity of the Na+,K+ pumps to fill these needs require quantification of the total content of Na+,K+ pumps in skeletal muscle. Inhibition of Na+,K+-pump activity, or a decrease in their content, reduces muscle contractility. Conversely, stimulation of the Na+,K+-pump transport rate or increasing the content of Na+,K+ pumps enhances muscle excitability and contractility. Measurements of [3H]ouabain binding to skeletal muscle in vivo or in vitro have enabled the reproducible quantification of the total content of Na+,K+ pumps in molar units in various animal species, and in both healthy people and individuals with various diseases. In contrast, measurements of 3-O-methylfluorescein phosphatase activity associated with the Na+,K+-ATPase may show inconsistent results. Measurements of Na+ and K+ fluxes in intact isolated muscles show that, after Na+ loading or intense excitation, all the Na+,K+ pumps are functional, allowing calculation of the maximum Na+,K+-pumping capacity, expressed in molar units/g muscle/min. The activity and content of Na+,K+ pumps are regulated by exercise, inactivity, K+ deficiency, fasting, age, and several hormones and pharmaceuticals. Studies on the α-subunit isoforms of the Na+,K+-ATPase have detected a relative increase in their number in response to exercise and the glucocorticoid dexamethasone but have not involved their quantification in molar units. Determination of ATPase activity in homogenates and plasma membranes obtained from muscle has shown ouabain-suppressible stimulatory effects of Na+ and K+.

Introduction: Transport and content of Na+ and K+ in skeletal muscle

The Na+,K+-ATPase (also known as the Na+,K+ pump) is the major translator of metabolic energy in the form of ATP to electrical and chemical gradients for the two most common ions in the body. These gradients enable the generation of action potentials, which are essential for muscle cell function. Evaluation of the physiological and clinical significance of the Na+,K+ pumps requires measuring the transmembrane fluxes of Na+ and K+ in intact muscles or cultured muscle cells. The simplest approach involves incubating intact muscles isolated from small animals in temperature-controlled and oxygenated buffers with electrolyte and glucose concentration comparable to that normally present in blood plasma. Initial studies used cut hemi- or quarter-diaphragm muscles from rats, mice, or guinea pigs for incubation because these muscles were considered thin enough to allow adequate oxygenation under these conditions (Gemmill, 1940). However, such preparations have numerous cut muscle ends, allowing large passive movements of Na+ and K+ and free access of Ca2+ to the cell interior. This unavoidably boosts the energy required for active transport of Na+, K+, and Ca2+, and leads to impaired cell survival. Thus, in cut muscles, the components of O2 consumption and 42K uptake attributable to the Na+,K+ pump (i.e., the fraction suppressible by the cardiac glycoside ouabain, which binds to and inhibits the Na+,K+-ATPase) have been severely overestimated. (The ouabain-suppressible components of O2 consumption or 42K uptake are measured in isolated muscles incubated without or with ouabain and calculated as the difference.) Such overestimation led to the assumption that in skeletal muscle, the Na+,K+ pumps mediate a large fraction of total energy turnover, suggesting that a major part of the thermogenic action of thyroid hormone is caused by an increased rate of active Na+,K+ transport (Asano et al., 1976). In contrast, in intact resting muscle preparations, only 2–10% of the total energy turnover is used for active Na+,K+ transport (Creese, 1968; for details, see Clausen et al., 1991). Even during maximum contractile work in human muscles, only a small fraction (2%) (Medbø and Sejersted, 1990) of total energy release is used for the Na+,K+ pumps. Thus, in skeletal muscle, the thermogenic action of the Na+,K+ pumps is modest.For the analysis of Na+,K+ transport in skeletal muscle, isolated intact limb muscles are used, primarily mammalian soleus, extensor digitorum longus (EDL), extensor digitorum brevis, or epitrochlearis muscles. These preparations can survive during incubation for many hours and can undergo repeated excitation. More recently, the isolated rat sternohyoid muscle, which also offers thin dimensions, cellular integrity, and simple handling, has been introduced (Mu et al., 2011).Measurement of muscle Na+ and K+ content requires extraction. In the past, this was done by digesting the muscle preparation in nitric acid, a sometimes risky procedure. More recently, this approach has been superseded by homogenization of the tissue in 0.3 M trichloroacetic acid, followed by centrifugation to sediment the proteins (Kohn and Clausen, 1971). The clear supernatant may then be diluted for flame photometric determination of Na+ and K+ or counting of the isotopes 22Na or 42K. Because 42K has a short half-life (12.5 h) and is of limited availability, the K+ analogue 86Rb is often used as a tracer for K+. For the quantification of Na+,K+ pump–mediated (i.e., ouabain-suppressible) transport of K+, 86Rb gives the same results as 42K (Clausen et al., 1987; Dørup and Clausen, 1994). For other flux measurements, however, the results obtained with 86Rb differ appreciably from those obtained with 42K. For example, the fractional loss of 86Rb from intact resting rat soleus muscles is only 45% of that measured using 42K. Moreover, two agents shown to stimulate the Na+,K+ pumps in isolated rat soleus muscle, salbutamol (Clausen and Flatman, 1977) and rat calcitonin gene–related peptide (CGRP) (Andersen and Clausen, 1993), both induce a highly significant stimulation of 86Rb efflux from the same muscle (Dørup and Clausen, 1994). In contrast, these same agents induced a rapid but transient (20-min duration) inhibition of the fractional loss of 42K, indicating that a large fraction of the 42K lost from the cells is reaccumulated as a result of stimulation of the Na+,K+ pumps (Andersen and Clausen, 1993; Dørup and Clausen, 1994). Finally, in skeletal muscle, bumetanide, an inhibitor of the NaK2Cl cotransporter, produces no inhibition of 42K uptake but clear-cut inhibition of 86Rb uptake (see and22 in Dørup and Clausen, 1994). Thus, results obtained with 86Rb must be verified with 42K or flame photometric measurements of changes in intracellular Na+ and K+ content. The activity of the Na+,K+ pump depends on ATP supplied by glycolysis or oxidative phosphorylation. Excitability of isolated rat soleus or EDL muscles can be maintained in the presence of the electron transport inhibitor cyanide or during anoxia (Murphy and Clausen, 2007; Fredsted et al., 2012), whereas contractions are markedly suppressed by 2-deoxyglucose, which interferes with the production of glycolytic ATP. Thus, in keeping with the studies of Glitsch (2001), these data suggest that glycolytic ATP is a primary source of energy for the Na+,K+ pumps (see also Okamoto et al., 2001). More focused studies showed that Na+,K+-pump function is not adequately supported when cytoplasmic ATP is at the normal resting concentration of ∼8 mM, but that the addition of as little as 1 mM phosphoenol pyruvate produces a marked increase in Na+,K+-pump function that is supported by endogenous pyruvate kinase bound within the t-tubular triad (Dutka and Lamb, 2007). In anoxic rat EDL muscles, contractility could be restored by stimulating the Na+,K+ pumps with the β2 agonists salbutamol or terbutaline, effects that were abolished by ouabain or 2-deoxyglucose (Fredsted et al., 2012). Glycolytic ATP furnishes energy for contractile activity under the critical condition of anoxia. Because the Na+,K+ pumps are essential for the maintenance of excitability and contractions, it would not be surprising if they were also kept going on glycolytic ATP.

Table 1.

Acute stimulation of the Na+,K+ pumps in skeletal muscle
Stimulating hormones, agents, and conditionsMechanisms of action
Epinephrine, norepinephrineStimulate generation of cAMP, which in turn activates the Na+,K+ pumps via PKA, increasing the affinity of the Na+K+ pumps for Na+
Isoproterenol, salbutamol, salmeterol
Other β2 agonists
β3 agonists
CalcitoninsStimulate generation of cAMP
CGRP
Amylin
cAMP, dibutyryl cAMPActivates Na+,K+ pumps via PKA
TheophyllineInhibits phosphodiesterase A, which degrades cAMP. This leads to intracellular accumulation of cAMP. Theophylline is a degradation product of caffeine.
Insulin, insulin-like growth factor IBoth act via the insulin receptors, increasing the affinity of Na+,K+ pumps for Na+
MonensinA Na+ ionophore that increases [Na+]i, which directly stimulates the Na+,K+ pumps
VeratridineAugments the Na+ influx per action potential, thereby increasing [Na+]i
ExcitationAugments [Na+] influx, thereby increasing [Na+]i
Increasing temperatureWhen temperature increases 10°C, the rate of Na+,K+ pumping increases 2.3-fold
ATP, ADPStimulate the Na+,K+ pumps via purinergic receptors
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Table 2.

Relative changes in [3H]ouabain binding and the α2 subunit of Na+,K+-ATPase in skeletal muscle
References, muscle preparation, treatment[3H]Ouabain bindingα2-subunit abundance
Thompson et al., 2001, rats treated for 14 d with dexamethasone+22–48% (P < 0.05)+53% (P < 0.05)
Juel et al, 2001, 1 h treadmill running 150-200 g rats, giant vesicles+29% (P < 0.05)+32% (P < 0.05)
Green et al., 2004, 6 d submaximal cycling+13% (P < 0.05)+9% (P < 0.05)
Sandiford et al., 2005, electrical stimulation of rat soleus+16–21% (P < 0.05)+38% (P < 0.05)
Green et al., 2007, 16 h heavy intermittent cycling+8% (P < 0.05)+26% (P < 0.05)
Green et al., 2008, 3 d submaximal cycling+12% (P < 0.05)+42% (P < 0.05)
Green et al., 2009, chronic obstructive lung disease−6% (NS)+12% (P < 0.05)
McKenna et al., 2012, young compared to aged subjects−0.7% (NS)−24% (P < 0.05)
Boon et al., 2012, spinal cord injury in human subject−47% (P < 0.05)−52% (P < 0.05)
Chibalin et al., 2012, nicotine pretreatment in 190 g rats0% (NS)−25% (P < 0.05)
Open in a separate windowData were obtained from 10 different publications listed in the references of this paper. Each of them was selected for reporting the results of measurements of the content of [3H]ouabain-binding sites as well as α2-subunit abundance in the same muscle. Experimental details are given in the cited articles. The relative changes induced by the listed factors are given in percentages. Biopsies from human subjects were taken from the vastus lateralis muscle. The rat muscles were obtained from the hind limbs.

The Na+,K+-ATPase and why it should be quantified

The Na+,K+ pump, which is identical to the membrane-bound Na+,K+-ATPase discovered by Skou (1965), has been found in most skeletal muscle types from many species. As illustrated in Fig. 1, the Na+,K+-pump molecule comprises a catalytic α subunit, a β subunit involved in its translocation to the plasma membrane, and a regulatory subunit (FXYD; in skeletal muscle named FXYD1 or phospholemman). The Na+,K+ pumps in skeletal muscle are subject to acute activation or inhibition of their transport rate as well as long-term regulation of their content (expressed in molar units, usually as pmol per gram wet weight) (Clausen, 2003). In human skeletal muscle, the content of Na+,K+ pumps measured using [3H]ouabain binding is ∼300 pmol/g wet wt (Clausen, 2003). In an adult human subject weighing 70 kg, the muscles will weigh around 28 kg and therefore contain a total of 28,000 × 300 pmol = 8.4 µmoles of Na+,K+ pumps. Muscles represent the largest single pool of cells in the human body, containing the largest pool of K+ (2,600 mmol; Clausen, 2010) and one of the largest pools of Na+,K+ pumps (Clausen, 1998). Many diseases are associated with anomalies in the content of Na+,K+ pumps in skeletal muscle (Clausen, 1998, 2003), and during the last decades, the number of published articles on Na+,K+ pumps in skeletal muscle has increased appreciably. The capacity of the Na+,K+ pumps in muscles is crucial for the clearance of extracellular K+ at rest or during exercise, and details of the transport capacity are required for clinical evaluation of the risk of developing hypo- or hyperkalemia (for clinical examples and details, see Clausen, 1998, 2003, 2010; Sejersted and Sjøgaard, 2000). Moreover, this information is key to understanding the functional significance of physiological changes or pathological anomalies in the content of Na+,K+ pumps in skeletal muscle. The Na+,K+-pump capacity may be calculated by multiplying the number of Na+,K+ pumps by their turnover number (determined with the Na+,K+-ATPase from ox brain at 37°C as 8,000 molecules of ATP split per Na+,K+-ATPase molecule per min; Plesner and Plesner, 1981) and by the number of K+ ions transported per cycle, which is normally 2. From this information it can be calculated that the total transport capacity for K+ of the pool of Na+,K+ pumps in human skeletal muscle amounts to 2 × 8,000 × 8.4 µmoles/min = 134 mmoles/min. Provided that all the Na+,K+ pumps in all muscles are operating at maximum speed (which rarely happens), they could clear all extracellular K+ in the human body (56 mmoles, calculated by multiplying the extracellular water space [14 liters] by the content of K+ [4 mmol/liter] in 25 s; Clausen, 2010). The present Review was prompted by the ongoing problems of Na+,K+-pump quantification and recovery, how they can be solved, and what insight may be gained by using more specific methods and adequate quantitative analysis. The early development of the field has been discussed previously (Ewart and Klip, 1995; Sejersted and Sjøgaard, 2000; Clausen, 2003). This Review focuses on the most relevant publications appearing during the last 10 years; to cover the background, it includes references back to the discovery of the Na,K pump in 1957 (Skou, 1957).Open in a separate windowFigure 1.The molecular structure of Na+,K+-ATPase. The figure is based upon the crystal structure of the homologous Na+,K+-ATPase in the E2P2K conformation (Shinoda et al., 2009) and drawn by Flemming Cornelius (Aarhus University, Aarhus, Denmark). The Na+,K+ pump comprises an α and a β subunit, a glycoprotein that participates in the translocation of the molecule from the cell interior to its correct position in the lipid bilayer of the plasma membrane. A regulatory subunit (FXYD) is also shown. During each transport cycle of the Na+,K+ pump, one ATP molecule is bound to the cytoplasmic site of the α subunit; its hydrolysis provides energy for the active transport of Na+ and K+. The transmembrane domain consists of 10 transmembrane helices and contains the binding sites for three Na+ or two K+ ions, respectively, which pass sequentially through the same cavity in the molecule during each transport cycle.

Problems in assessing Na+,K+-ATPase activity and recovery.

When the classical assay for membrane-bound ATPase activity stimulated by Na+ and K+ (Skou, 1965) and blocked by ouabain is used with crude muscle homogenates, there is an overwhelming background of other ATPases. Therefore, during the early phase of Na+,K+-pump investigation (1957–1977), many research groups were primarily interested in the detection and purification of the Na+,K+-ATPase. The differential centrifugation procedures developed during that period often discarded a major part of the enzyme activity present in homogenates of the intact tissue to eliminate other ATP-splitting cellular components, such as myosin, Ca2+-activated ATPase, and unspecified Mg2+-activated ATPase, and obtain a pure enzyme. Therefore, accurate information about Na+,K+-ATPase recovery was not always available, and these methods were not suitable for quantification of the total content of Na+,K+ pumps in the intact tissue. Moreover, the low recovery of Na+,K+-ATPase activity often obtained raised doubts as to whether the Na+,K+-ATPase in the samples tested was representative of the entire population of enzyme molecules present in the starting tissue. These problems were analyzed in a review, which found that, among 12 papers, only 3 obtained a recovery >5% of the Na+,K+-ATPase present in the starting material and, in 9 of these papers, recovery was in the range of 0.2 to 3.5% (Hansen and Clausen, 1988). To obtain better purification of the Na+,K+-ATPase in sarcolemma, giant vesicles were later produced from the sarcolemmal membranes of rat hind-limb muscles. However, they only contained 0.3% of the total content of Na+,K+ pumps present per gram of muscle (Juel et al., 2001). Thus, it is difficult to evaluate to what extent down-regulation of the content of Na+,K+ pumps might reduce Na+,K+ pump–mediated K+ uptake in skeletal muscle and thereby impair the clearance of K+ from extracellular space and plasma.The Na+,K+-ATPase activity has been measured in plasma membranes prepared from various rat muscles. In total membranes, Na+ (0–80 mM) and K+ (0–10 mM) were shown to activate ouabain-suppressible ATPase (Juel, 2009). Surprisingly, Vmax for Na+ given as µmoles of ATP split per milligram protein per hour was higher in total membranes than in the sarcolemmal membranes, which might be expected to show a higher value, because of partial purification of the Na+,K+-ATPase. In total membranes, as well as in plasma membranes, 30 min of running gave only a minor increase in Vmax, which was only significant in a few instances, suggesting only modest translocation of the Na+,K+ pumps from an intracellular pool of endosomal membranes to the plasma membrane (see the section below, Translocation of Na+,K+ pumps compared…, for alternatives to translocation) (Juel, 2009). Because of a lack of accurate information about plasma membrane recovery, however, these observations could not readily be translated to pmol of Na+,K+ pumps per gram wet weight.

3-O-methylfluorescein phosphatase (3-O-MFPase) assay.

Using 3-O-methylfluorescein phosphate as a substrate, K+-dependent and ouabain-suppressible phosphatase activity has been measured in a fluorimetric assay for the Na+,K+-ATPase in skeletal muscle (Nørgaard et al., 1984). Early studies on rat skeletal muscle showed good agreement between the 3-O-MFPase activity of crude homogenates and the number of [3H]ouabain-binding sites measured in the same intact muscles or biopsies thereof (Hansen and Clausen, 1988), suggesting that this assay provided a reliable measure of Na+,K+-ATPase activity. Moreover, the 3-O-MFPase activity showed similar decreases with aging or K+ depletion of the rats as the [3H]ouabain-binding capacity. However, studies on biopsies of human vastus lateralis muscle showed that exercise to fatigue reduced 3-O-MFPase activity by 13% (P < 0.001), whereas [3H]ouabain binding remained constant (Leppik et al., 2004). Several other studies have also reported activity-dependent decreases in 3-O-MFPase activity in muscle: Another study from the same laboratory showed that acute exercise induced a 22% drop in the 3-O-MFPase activity of human vastus lateralis muscle (McKenna et al., 2006). In human vastus lateralis muscle, 16 h of heavy intermittent cycle exercise induced a 15% reduction in 3-O-MFPase (Green et al., 2007). In rats, running exercise induced a 12% decrease in 3-O-MFPase in all muscles (Fowles et al., 2002). However, other studies have not. For instance, electrical stimulation of the isolated rat soleus muscle for 15 or 90 min induced a 40 or 53% increase in 3-O-MFPase activity of crude homogenate, respectively (Sandiford et al., 2005). In keeping with this, giant vesicles representing sarcolemmal membrane obtained from rat muscles after 3 min of running exercise showed a 37% increase in 3-O-MFPase activity (Kristensen et al., 2008). Moreover, a full Na+,K+-ATPase assay showed that 30 min of treadmill running increased Vmax by 12 to 39% in sarcolemmal membranes from rat hind-limb muscles (Juel, 2009). In contrast, high frequency stimulation of isolated rat EDL muscles caused no significant change in the content of 3-O-MFPase (Goodman et al., 2009). These discrepancies indicate that measurements of 3-O-MFPase provide inconsistent information about the content of Na+,K+-ATPase in skeletal muscle. Moreover, as pointed out by Juel (2009), the 3-O-MFPase cannot be used to measure Na+-dependent activation. Therefore, further use of the 3-O-MFPase assay requires closer analysis of the causes of these discrepancies.

[3H]Ouabain binding.

[3H]Ouabain and other labeled cardiac glycosides bind stoichiometrically to the extracellular surface of the α subunit of the Na+,K+ pump (one drug molecule per Na+,K+-ATPase molecule). Therefore, incubation of tissues, cells, or plasma membranes with [3H]ouabain enables the quantification of the content of Na+,K+-ATPase in molar units by liquid scintillation counting of extracts of the tissue, cells, or membranes (Clausen and Hansen, 1974, 1977; Hansen and Clausen, 1988). Because K+ interferes markedly with the binding of cardiac glycosides, studies of [3H]ouabain binding to intact rat, mouse, or guinea pig muscles are performed using K+-free (KR) buffer (Clausen and Hansen, 1974). [3H]Ouabain binding is saturable and reversible without showing [3H]ouabain penetration into the intracellular space. In isolated soleus muscle obtained from 4-wk-old rats, [3H]ouabain binding amounts to 720 pmol/g wet wt, corresponding to 3,350 molecules of Na+,K+-ATPase per µ2 of sarcolemma (not including t-tubules). When injected intraperitoneally, [3H]ouabain binds rapidly to the outer surface of the muscle cells, showing saturation and the same content of [3H]ouabain-binding sites per gram tissue wet weight as measured in intact muscles incubated with [3H]ouabain in vitro (Clausen et al., 1982; Murphy et al., 2008). The [3H]ouabain binding in vivo is faster than in vitro, reaching saturation in 20 min, partly because of the higher temperature and better access to the muscle cells via the capillaries. When bound to the outer surface of intact muscles, [3H]ouabain is rapidly displaced by the addition of an excess of unlabeled ouabain during a subsequent wash performed both after binding in vitro and in vivo, indicating that the [3H]ouabain is not internalized into the cytoplasm (Clausen and Hansen, 1974; Clausen et al., 1982).It has been estimated that, in rat skeletal muscle, most of the Na+,K+ pumps (around 80%) are the α2-subunit isoform, which has high affinity for [3H]ouabain. A minor fraction (around 20%) is the α1 isoform, which has a lower affinity for [3H]ouabain (Hansen, 2001) and therefore may not be detected at the concentration of [3H]ouabain used in the standard assay for quantification of [3H]ouabain-binding sites (10−6 to 5 × 10−6 M). However, these values for α1 and α2 isoforms depend on the antibody used and its interaction with newly synthesized α subunits and are therefore inconclusive (for detailed discussion, see Clausen, 2003). Another study in which relative subunit composition was determined with antibodies indicated that in mouse EDL muscle, the α2 subunit accounted for 87% of the total amount of the α subunit and the α1 subunit for 13% (He et al., 2001). More importantly, measurements of the maximum rate of ouabain-suppressible active 86Rb uptake in Na+-loaded rat soleus muscles have given values in good agreement with those obtained in the standard [3H]ouabain-binding assay, indicating that this assay quantifiably measures the majority of the total content of functional Na+,K+ pumps (Clausen et al., 1987).The phosphate analogue vanadate (VO4) binds to the intracellular surface of the Na+,K+ pumps and thereby facilitates [3H]ouabain binding to the outer surface of the Na+,K+ pumps in the plasma membranes (Hansen and Clausen, 1988). This enabled development of an assay in which cut muscle tissue segments are incubated with [3H]ouabain in a Tris buffer–containing Tris vanadate (Nørgaard et al., 1983). Because the muscle segments are cut, vanadate gains ready access to the cytoplasm and can bind to the inner surface of the Na+,K+-ATPase. This assay gives the same values for [3H]ouabain binding as measurements of [3H]ouabain binding in intact muscles incubated in K+-free KR. A comparison of the binding kinetics of [3H]ouabain obtained using the vanadate-facilitated assay showed that the α1-, α2-, and α3-subunit isoforms of the pumps in human tissues have similar affinity for [3H]ouabain (Wang et al., 2001). This indicates that the vanadate-facilitated [3H]ouabain-binding assay quantifies the sum of the three Na+,K+-pump isoforms present in human muscle biopsies (for details, see Clausen, 2003). Another major advantage of the vanadate-facilitated [3H]ouabain-binding assay with small (2–5-mg) cut muscle specimens is that it can be used to measure Na+,K+ pumps in frozen muscle samples. Storage in the freezer for at least 4 yr causes no change in the content of [3H]ouabain-binding sites of human muscle samples, and samples may be sent on dry ice by air freight. The assay is rapid, efficient, and inexpensive, and the results are reproducible. 17 studies on human skeletal muscle biopsies performed in seven different laboratories obtained similar values for the content of [3H]ouabain-binding sites of human skeletal muscle samples (in the range of 243 to 425 pmol/g wet wt; see Clausen, 2003). More recent measurements of [3H]ouabain-binding sites in human vastus lateralis muscle performed in four different laboratories showed results in the same range (326 ± 30 pmol/g wet wt [Nordsborg et al., 2005]; 272 ± 10 pmol/g wet wt [Green et al., 2004]; 240 ± 10 pmol/g wet wt [Boon et al., 2012]; 350 ± 108 pmol/g wet wt [McKenna et al., 2012]). [3H]ouabain binds to both the outer surface of sarcolemma and the inner surface of the t-tubular lumen. Lau et al. (1979) showed that t-tubules from rabbit muscle bind [3H]ouabain with the same affinity (dissociation constant around 5 × 10−8 M) and capacity (700 pmol/g wet wt) as the intact rat soleus muscle (Clausen and Hansen, 1974).The rate of [3H]ouabain binding depends on the rate of active Na+,K+ transport (Clausen and Hansen, 1977; Andersen and Clausen, 1993; Clausen, 2000, 2003). [3H]Ouabain binding requires a certain configuration of the binding site on the extracellular portion of the α subunit that occurs only once during each Na+,K+-pumping cycle (Schwartz et al., 1975; Clausen and Hansen, 1977). Hence, an increased rate of cycling augments the chances for the [3H]ouabain molecule to bind. Therefore, an increase in the rate of [3H]ouabain binding likely indicates that the Na+,K+-pumping rate is augmented.

Regulation of the Na+,K+ pumps in skeletal muscle

Regulation of the Na+,K+ pumps evaluated by measurement of Na+ and K+ fluxes and [3H]ouabain binding. Short-term regulation of Na+,K+-pump activity.

The Na+ ionophore monensin (10−5 M) induces a graded increase in Na+ influx in skeletal muscle, enabling the evaluation of the effects of increased intracellular Na+ on the rate of active Na+,K+ transport (measured as ouabain-suppressible 42K or 86Rb influx) and of possible changes in pump affinity for intracellular Na+ (Buchanan et al., 2002). The Na+,K+ pumps undergo short-term and long-term regulation, often defined as acute changes in transport activity (e.g., µmoles of Na+ extruded per gram wet weight per minute) or slower and more long-lasting changes in the content of Na+,K+ pumps (expressed as pmol of [3H]ouabain bound per gram wet weight), respectively. In skeletal muscle, the most common cause of an acute increase in Na+,K+-pump activity is excitation-induced rise in intracellular Na+ caused by increased Na+ influx. This and around 20 other conditions or agents shown to stimulate the Na+,K+ pump in skeletal muscle are listed in Hodgkin and Horowicz, 1959). In close agreement with this value, intact rat EDL muscle stimulated for 5 s at 90 Hz to produce isometric tetanic contraction showed a Na+ influx of 12 nmol/g wet wt/action potential and a K+ efflux of 10 nmol/g wet wt/action potential, close to the 1:1 exchange of Na+ and K+ causing the action potential (Clausen et al., 2004). The excitation-induced increase in intracellular Na+ primarily takes place at the inner surface of the plasma membrane, which is close enough to the Na+,K+ pumps to induce prompt activation of their transport activity. An early increase in Na+,K+-pump activity may also involve an excitation-dependent increase in Na+,K+-pump affinity for [Na+]i that is independent of the rise in intracellular Na+ per se (Buchanan et al., 2002). Thus, as shown in Fig. 2, electrical stimulation induces a leftward shift of the curve relating intracellular Na+ to Na+,K+ pump–mediated 86Rb uptake. Electrical stimulation of rat soleus for 10 s at 60 Hz at 30°C induced an immediate 58% increase in intracellular Na+ content. During subsequent rest at 30°C, reextrusion of Na+ was complete in 2 min, and this was followed by a statistically significant undershoot in [Na+]i (P < 0.001) (Everts and Clausen, 1994). During the rapid early decrease in Na+ content, it can be assumed that activity of the Na+,K+ pumps is stimulated 15-fold, even though [Na+]i has decreased from its peak value down to the range found in resting muscle. The excitation-induced increase in Na+ affinity was also detected by a highly significant (P < 0.001–0.05) decrease in intracellular Na+ of ∼25% that lasted up to 30 min after 60 s of electrical stimulation of isolated rat soleus muscles. Similar poststimulatory undershoots in [Na+]i was seen in rat EDL muscle. It was blocked by ouabain or cooling to 0°C and was assumed to be mediated by activation of the Na+,K+ pumps by CGRP released from nerves in the muscles. Thus, reducing CGRP content by capsaicin or by prior denervation of the muscles prevented both excitation-induced force recovery and the drop in intracellular Na+ (Nielsen and Clausen, 1997). This provides further evidence that activity-dependent stimulation of the Na+,K+ pump in muscle is not solely a function of the rise in [Na+]i but might be caused by a mechanism similar to that in the electric organ of Narcine brasiliensis, where electrical stimulation markedly augments the activity of the Na+,K+-ATPase within fractions of a second despite a minimal change in intracellular Na+ (Blum et al., 1990).Open in a separate windowFigure 2.Electrical stimulation affects the relationship between [Na+]i and 86Rb+ uptake rate. Rat soleus muscles were mounted isometrically on electrodes and were stimulated for 10 s at 60 Hz. After a 2-min rest, they were transferred into solutions containing 86Rb+, incubated for 2 min before being washed to remove extracellular 86Rb and Na+, and blotted, and intracellular 86Rb+ and [Na+] were determined. [Na+]i was manipulated by preincubating in buffer in which the Na+ content had been reduced or in standard KR buffer containing the Na+ ionophore monensin. •, resting muscles; ○, muscles stimulated. Each point represents the mean ± SEM of measurements performed on three muscles. The curves were fitted using a computer program. Reprinted with permission from The Journal of Physiology (Buchanan et al., 2002).The slowly hydrolyzed cholinergic antagonist carbachol induces a long-lasting activation of the nicotinic acetylcholine receptors. Our experiments showed that in isolated rat soleus muscle, carbachol augments Na+ influx leading to depolarization, increased intracellular Na+, and loss of force (Macdonald et al., 2005). This reduction in force is significantly (P < 0.05) restored by stimulating the Na+,K+ pumps with epinephrine, salbutamol, or CGRP. All these effects are likely to be indirect, mediated by cAMP generated by stimulation of the adenylate cyclase. CGRP is found in sensory nerve endings from which it may be released by capsaicin or electrical stimulation to stimulate the Na+,K+ pumps in the muscle cells (Nielsen et al., 1998). Thus, CGRP release and the ensuing stimulation of pump activity may contribute to the above-mentioned long-lasting drop in intracellular Na+ caused by electrical stimulation. Because the excitation-induced force recovery seen at increased [K+]o is suppressed by the CGRP analogue CGRP-(8–37), which acts as a competitive CGRP antagonist, it is likely mediated by local release of CGRP from nerve endings in the muscle (Macdonald et al., 2008). In the isolated rat soleus muscle, repeated electrical stimulation caused hyperpolarization (11 mV) and increased amplitude of the action potentials. These effects were abolished by ouabain, cooling, or omission of K+ from the buffer, suggesting that they resulted from of the electrogenic Na+,K+ pumps (Hicks and McComas, 1989) (see below in The electrogenic action of the Na+,K+ pumps).As listed in Clausen and Flatman, 1977). Like electrical stimulation (Fig. 2), salbutamol causes a leftward shift of the curve relating intracellular Na+ to Na+,K+ pump–mediated uptake of 86Rb in rat soleus muscle (Buchanan et al., 2002). The rapid stimulating effect of the above-mentioned agents on the Na+,K+ pumps, their mechanisms, and the physiological importance have been described in detail (Clausen, 2003). The stimulatory effects of these agents have been exploited in the treatment of hyperkalemia and the ensuing muscular weakness or paralysis. Thus, salbutamol was introduced for the treatment of paralytic attacks in patients with hyperkalemic periodic paralysis (Wang and Clausen, 1976). In muscles isolated from knock-in mice with the same genetic anomaly, stimulation of the Na+,K+ pumps with salbutamol or CGRP restored muscle force (Clausen et al., 2011). These observations suggest that the beneficial effect of mild exercise on severe weakness seen during attacks of hyperkalemic periodic paralysis is likely related to the stimulatory effect of locally released CGRP on the Na+,K+ pumps.In rat soleus muscle, ATP induces a twofold increase in Na+,K+ pump–mediated 86Rb uptake (Broch-Lips et al., 2010). ATP induces a marked recovery of force and M-waves (the sum of action potentials as recorded from the surface of the muscle) in muscles inhibited by increasing [K+]o, indicating that ATP-induced stimulation of the Na+,K+ pumps enhances excitability. Similar effects are exerted by ADP and are mediated by purinergic receptors. These effects are of particular interest during intense exercise, where local release of ATP or ADP from the muscle cells may reduce the inhibitory effect on excitability of the concomitant increase in [K+ ]o (Broch-Lips et al., 2010). The importance of the electrogenic action of the Na+,K+ pumps is also evident from the observation that at increased [K+]o, where M-wave amplitude and area are decreased, stimulation with salbutamol or ATP restores the M-waves (Overgaard et al., 1999; Broch-Lips et al., 2010). In conclusion, the short-term increase in Na+,K+-pump activity in response to excitation has at least two components: (1) a direct stimulatory effect of [Na+]i on the Na+,K+ pump; and (2) an increase in the affinity of the Na+,K+ pump for Na+, potentiating the stimulating effect of [Na+]i. This seems to be mediated by cAMP, protein kinases, or ATP, and may involve release of a local hormone (CGRP). For more details, see below in Molecular mechanisms of pump regulation.

Long-term regulation of Na+,K+-pump content.

Long-term increases in the muscle content of Na+,K+ pumps is most commonly seen after training. As measured using [3H]ouabain binding, such an increase has been observed in nine different species and in numerous studies on human and rat muscle (see Clausen, 2003). The relative increase observed in the different studies depends on the duration and intensity of the training and ranges from 14 to 46%. There is a great deal of evidence for such changes in Na+,K+-pump content, which can also be induced by electrical stimulation. For instance, the content of [3H]ouabain-binding sites in the tibialis anterior muscle of the rabbit was more than doubled after 20 d of electrical stimulation in vivo. Furthermore, it was correlated to the amplitude of M-waves (P < 0.01; r = 0.8) (Hicks et al., 1997). In pigs in which shivering is induced by lowered temperature, [3H]ouabain binding is increased by 58–84% after weeks (for details, see Clausen, 2003). Running 100 km in 10.7 h produced a significant (P < 0.05) 13% increase in the content of [3H]ouabain-binding sites in human vastus lateralis muscle (Clausen, 2003). Cycling exercise for 3 d increased the content of [3H]ouabain-binding sites in human vastus lateralis muscle by 8% (P < 0.05) (Green et al., 2004). A recent study showed that rats performing voluntary free wheel running covered a distance of 13 km/day. After 8 wk, their soleus muscles contained 22% more [3H]ouabain-binding sites than those from untrained controls. Moreover, the soleus muscle of the trained rats showed considerably better contractile tolerance to increased [K+]o (9 mM) than those from untrained controls (Broch-Lips et al., 2011). In rats exposed to hypoxia for 6 wk (causing chronic stimulation of respiration), the content of [3H]ouabain-binding sites in the diaphragm was increased by 24% and the contractile endurance of the muscle was significantly augmented (McMorrow et al., 2011).Conversely, a decrease in the muscle content of Na+,K+ pumps is seen during reduced activity or immobilization. In rats, guinea pigs, sheep, and humans, immobilization reduced the content of [3H]ouabain-binding sites by 20–27%, changes that were reversible after mobilization (Clausen, 2003). Thus, after 2 wk of decreased mobility, the content of [3H]ouabain-binding sites in guinea pig gastrocnemius muscles was 258 ± 13 pmol/g wet wt (25% below that of the contra-lateral freely mobile muscle; P < 0.02). After 3 wk of reduced mobility and 3 wk of training by running, the content of [3H]ouabain-binding sites in the gastrocnemius muscle of these animals increased by 57%, whereas those that had not been immobilized reached 498 ± 25 pmol/g wet wt, which is 93% higher than that of the muscles with 2 wk of reduced mobility (Leivseth et al., 1992). This indicates that the regulatory range of Na+,K+-pump content in muscle represents roughly a doubling from the immobilized to the trained muscle. These results are particularly notable because, compared with rats or mice, all the Na+,K+ pumps in guinea pig muscle have high affinity for [3H]ouabain, indicating complete occupancy of the [3H]ouabain-binding sites.Immobilization induced by denervation, a plaster cast, or tenotomy (tendon release) reduced the contents of [3H]ouabain-binding sites in the skeletal muscles of mice, rats, guinea pigs, and sheep (Clausen, 2003; Clausen et al., 1982). In keeping with this, in human subjects complete spinal cord injury and partial denervation caused 58% reduction in the content of [3H]ouabain-binding sites in the vastus lateralis muscle (Ditor et al., 2004). A more recent study confirmed this observation, showing ∼50% reduction in the content of [3H]ouabain-binding sites in skeletal muscles of patients with complete cervical spinal cord injury (Boon et al., 2012). The mechanisms of exercise-related increases or decreases in the content of Na+,K+ pumps was explored in chick embryo leg muscle using monoclonal antibodies for estimating the relative changes in the number of Na+,K+ pumps. Increased intracellular Na+ induced by activating the Na+ channels with veratridine induced a 60–100% increase in the content of Na+,K+ pumps. Conversely, inhibition of the Na+ channels with tetrodotoxin blocked this effect and induced a rapid decrease (Fambrough et al., 1987).

Hormonal regulation of Na+,K+-pump content.

Thyroid hormone exerts the most potent hormonal stimulation of the synthesis of Na+,K+ pumps in skeletal muscle (Asano et al., 1976; for details, see Clausen, 2003). In rats, daily subcutaneous injection of thyroid hormone increases the content of [3H]ouabain-binding sites by 75 pmol/g muscle wet wt/d (Everts and Clausen, 1988). The content of [3H]ouabain-binding sites in the human vastus lateralis muscle varied from 100 pmol/g wet wt to 550 pmol/g wet wt in hypothyroid, euthyroid, and hyperthyroid subjects, with close linear correlation to free T4 index (r = 0.87; P < 0.001) (Clausen, 2003). [3H]ouabain binding in biopsies from vastus lateralis muscle of nine hyperthyroid patients was 89% higher value than in those form euthyroid controls. This increase in [3H]ouabain binding, the concomitant increase in energy expenditure, and plasma thyroid hormone concentration were all restored to the control levels by the standard treatment of hyperthyroidism with the anti-thyroid drug methimazole, which inhibits the synthesis of thyroid hormone (Riis et al., 2005).During fasting, the plasma concentration of thyroid hormones decreases (Spencer et al., 1983). In rats, total fasting for 72 h led to an ∼50% decrease in the content of [3H]ouabain-binding sites in plasma membranes obtained from soleus muscle (Swann, 1984). 3 wk on reduced caloric intake caused a 45–53% decrease in rat plasma thyroid hormone concentration and a 25% decrease in the content of [3H]ouabain-binding sites in the skeletal muscles (see Clausen, 2003). Because reduced caloric intake also decreases muscle mass, K+ clearance from the extracellular space is likely to be further impaired. Worldwide, a starvation-induced decrease in Na+,K+-pump content is probably the most common muscle Na+,K+-pump disorder in human subjects, causing reduced tolerance to the hyperkalemia arising during K+ ingestion and intensive work, leading to impaired physical performance. However, there is no information available about the effects of starvation on the content of Na+,K+ pumps in human skeletal muscle. The increase in Na+,K+-pump content in rat skeletal muscle induced by injection of thyroid hormone is preceded by an increase in the content of Na+ channels in the muscles measured using 3H-labeled saxitoxin as well as increased intracellular Na+ measured by flame photometry (Clausen, 2003). Increased Na+ influx in resting soleus muscle also induces an increase in the Na+,K+-pump content in knock-in mice with hyperkalemic periodic paralysis (Clausen et al., 2011). These observations are in keeping with the stimulatory effect of increased intracellular Na+ on the synthesis of Na+,K+ pumps in cultured muscle cells (Fambrough et al., 1987). Adrenal steroids also influence the content of Na+,K+ pumps in skeletal muscle. Thus, in 36 patients treated for chronic obstructive lung disease with the glucocorticoid dexamethasone, the content of [3H]ouabain-binding sites in needle biopsies of the vastus lateralis muscle was 31% higher than in 23 age- and sex-matched control subjects (P < 0.001) (Ravn and Dørup, 1997). In rats, 7–14 d of continuous infusion of dexamethasone via osmotic mini-pumps induced a 27–42% increase (P < 0.001– 0.01) in the content of [3H]ouabain-binding sites in soleus, EDL, gastrocnemius, and diaphragm muscles. In contrast, acute stimulation of Na+,K+-pump activity by infusion of the β2 agonist terbutaline produced no significant change in [3H]ouabain binding. A more detailed study (Dørup and Clausen, 1997) showed that the up-regulation of Na+,K+ pumps induced by dexamethasone could not be attributed to a mineralocorticoid action. Indeed, the mineralocorticoid aldosterone induced a decrease in the content of [3H]ouabain-binding sites in rat skeletal muscle, which was closely correlated to a concomitant reduction in the content of K+ in the muscles, induced by aldosterone stimulation of renal K+ excretion This is in keeping with the down-regulation of Na+,K+ pumps in skeletal muscle observed in rats maintained on K+-deficient fodder and in patients developing K+ deficiency during treatment with diuretics (Clausen, 1998). In rat skeletal muscles, dexamethasone infusion for 14 d induced a relative rise in Na+,K+-ATPase α2-subunit isoform of 53–78%, and in mRNA for the α2 subunit a 6.5-fold increase was found (Thompson et al., 2001). In young human subjects, the ingestion of dexamethasone (2 mg twice daily for 5 d) increased the content of [3H]ouabain-binding sites by 18% in the vastus lateralis muscle (P < 0.001) and by 24% in the deltoid muscle (P < 0.01). In the same subjects, the content of 3-O-MFPase in vastus lateralis and deltoid muscles increased by 14 and 18%, respectively (P < 0.05) (Nordsborg et al., 2005). Another study by the same group showed that the same dose of dexamethasone induced 17% relative increase in α1- and α2-subunit expression (P < 0.05) in vastus lateralis muscle, a significantly lower exercise-induced net K+ release from the thigh muscles and a borderline significant prolongation of time to exhaustion (P = 0.07) (Nordsborg et al., 2008).In rats made diabetic by streptozotocin pretreatment, the content of [3H]ouabain-binding sites in skeletal muscle was reduced by 24% in soleus and 48% in EDL (Schmidt et al., 1994). These changes were completely restored by insulin treatment (Kjeldsen et al., 1987).

Translocation of Na+,K+ pumps compared with other types of activation.

In skeletal muscle, the Na+,K+-pump molecules are located in the sarcolemma (primarily the α1-subunit isoform) and in the t-tubular membranes (primarily the α2-subunit isoform) (Marette et al., 1993; Williams et al., 2001; Cougnon et al., 2002; Radzyukevich et al., 2013). This localization seems to be strategic, reflecting a particular need for transport of Na+ and K+ across the t-tubular membranes during and after work. Thus, a large fraction of the Na+,K+ exchange takes place via the t-tubular membranes, and because of the small volume of the t-tubules, the intra-tubular concentration of K+ is likely to reach high levels (36.6 mM in 1 s of stimulation at 100 Hz, sufficient to cause severe interference with excitability; Kirsch et al., 1977). A recent simulation study on single rat EDL muscle fibers showed that stimulation at 30 Hz increased the K+ concentration in the t-tubules to a plateau of 14 mM within 1 s (Fraser et al., 2011). Therefore, during intense work, there is an urgent need for increased active transport of K+ out of the t-tubular lumen and into the cytoplasm. The most efficient way of meeting this demand would be augmented affinity of the Na+,K+ pumps for Na+ and/or K+. Alternatively, the entire Na+,K+-pump molecule might move to positions in the plasma membrane where there is more ready access to Na+ or K+. It has long been known that insulin induces a 1.7-fold increase in the binding of [3H]ouabain in frog sartorius muscle (Erlij and Grinstein, 1976) that was proposed to reflect an “unmasking” of an intracellular pool of inactive Na+,K+ pumps. Furthermore, Tsakiridis et al. (1996) showed that in rats, 60 min of treadmill running induced a significant relative increase (43–94%) in the α2 polypeptide of the Na+,K+-ATPase (now termed the α2-subunit isoform) detected by immunoblotting of plasma membranes prepared from hind-limb muscles. This was interpreted as indicating that exercise might induce translocation from an intracellular pool of endosomal membranes to the plasma membrane. Surprisingly, however, there was no concomitant decrease in the α2 polypeptide detectable in the intracellular pool, and there was no information about the recovery of the Na+,K+ pumps in the plasma membranes (Tsakiridis et al., 1996). A later study used differential centrifugation of a whole homogenate to separate sarcolemmal membranes from endosomal membranes (Sandiford et al., 2005). After electrical stimulation in vivo via the nerves, the maximum 3-O-MFPase activity in the sarcolemmal fraction had increased by ∼40%. In the same fraction, the α2 subunit had increased by 38–40%, and in the endosomal fraction, the α2 subunit had decreased by 42%, in keeping with a translocation of Na+,K+ pumps from the endosomal membranes to the sarcolemmal membranes. However, in a more recent study, Na+,K+ pumps could barely be detected in the intracellular pool of membranes (the putative source for the translocation of Na+,K+ pumps to the plasma membrane) (Zheng et al., 2008). In rats, 60 min of treadmill running induced a 29% increase in [3H]ouabain labeling of sarcolemmal giant vesicles obtained from mixed hind-limb muscles, as well as 19–32% increases in the contents of the α1-2 and β1-2 subunits (Juel et al., 2001). The increase in subunits was reversible with a half-life of 20 min, but there is no information about the reversibility of the [3H]ouabain binding. However, as mentioned in the paper (Juel et al., 2001), the content of Na+,K+ pumps in the vesicular membranes measured as [3H]ouabain-binding sites was only 0.3% of the total content of Na+,K+ pumps in rat hind-limb muscles. Therefore, it could not be concluded that the samples of Na+,K+ pumps in the giant vesicles were representative of the entire pool of Na+,K+ pumps in the exercising muscles, raising doubts as to whether translocation of Na+,K+ pumps had taken place.Virtually all the evidence for translocation of Na+,K+ pumps has been obtained using membrane fractions isolated from muscle homogenates. In contrast, studies on intact muscles or cut muscle specimens in vitro or intact muscles in vivo have consistently failed to detect translocation. Thus, measurements of [3H]ouabain binding to intact rat soleus muscles performed under equilibrium conditions showed no effect of insulin or other conditions known to stimulate the activity of the Na+,K+ pumps (electrical stimulation, epinephrine, insulin-like growth factor I, and amylin; see also Clausen and Hansen, 1977; Dørup and Clausen, 1995; Clausen, 2003; McKenna et al., 2003; Murphy et al., 2008). It cannot be excluded, therefore, that the 70% “unmasking” or “translocation” of Na+,K+ pumps by insulin (Erlij and Grinstein, 1976) reflects the increase in the [3H]ouabain binding taking place before binding equilibrium has been reached (see the section above, [3H]Ouabain binding). This is usually caused by inadequate saturation of the ouabain-binding sites with [3H]ouabain (Clausen and Hansen, 1977). Translocation has often been proposed as the cause of Na+,K+-pump stimulation induced by electrical stimulation or exercise. This hypothesis was tested by comparing effects of electrical stimulation on Na+ extrusion and [3H]ouabain binding in isolated rat soleus. Electrical stimulation in vitro at 120 Hz for 10 s caused a rapid 70% rise in intracellular Na+, followed by a subsequent 18-fold increase in net Na+ extrusion from rat soleus muscle but no significant change in the content of [3H]ouabain-binding sites (McKenna et al., 2003). A later study showed that after 10–60 min of running exercise, there were highly significant increases (18–80%; P < 0.001– 0.01) in the Na+ content of intact rat soleus muscles in vivo but no significant change in [3H]ouabain binding (Murphy et al., 2008). In isolated rat EDL muscles, 15 s of 60-Hz stimulation caused a 7.3-fold increase in net Na+ extrusion (Clausen, 2011). Another study on rat EDL showed that a 10-s stimulation at 60 Hz induced a fivefold increase in Na+,K+-pump activity (Nielsen and Clausen, 1997). It seems unlikely that such pronounced increases in Na+ extrusion mediated by the Na+,K+ pumps could be accounted for by the relatively modest translocation of Na+,K+ pumps or binding of [3H]ouabain (no change detectable) described above.Either in vivo or in vitro administration of salbutamol augments K+ content and reduces Na+ content in rat soleus muscle (Murphy et al., 2008), reflecting increased affinity for intracellular Na+ (Buchanan et al., 2002) probably caused by phosphorylation of FXYD1. Conversely, inhibition of the Na+,K+ pumps with ouabain reduces intracellular K+ and increases intracellular Na+ in vivo (Murphy et al., 2008).Measurement of maximum binding capacity for [3H]ouabain in isolated rat soleus (0.72 nmol/g wet wt) would predict that if all Na+,K+ pumps were operating at full speed, the theoretical maximum K+ uptake should reach 0.72 nmol × 8,900 = 6,408 nmol/g/min at 30°C, which is corrected for temperature using the observed Q10 of 2.3 for the rate of Na+,K+ pumping (Clausen and Kohn, 1977). Are such high values seen in the intact soleus muscle? When isolated rat soleus muscles are loaded with Na+ by preincubation in K+-free KR buffer without Ca2+ or Mg2+, intracellular Na+ reaches 126 mM (Clausen et al., 1987). When these muscles are subsequently incubated for 3 min in KR buffer containing 42K or 86Rb and between 5 and 130 mM K+, the ouabain-suppressible rates of 42K or 86Rb uptake can be measured and the results plotted in an Eadie–Hofstee plot (Fig. 4 in Clausen et al., 1987). The maximum rates of 42K and 86Rb uptake determined from this plot reach 6,150 nmol/g wet wt/min, corresponding to 96% of the above-mentioned theoretical maximum K+ uptake at 30°C. When the same Na+-loading procedure was used, the maximum rate of ouabain-suppressible 86Rb uptake was correlated (r = 0.95; P < 0.001) to the content of [3H]ouabain-binding sites over a wide range of values obtained by varying thyroid status, age, or K+ depletion (Fig. 5 in the present paper). These high 86Rb uptake values were not suppressed by Ba2+ or the local anesthetic tetracaine, indicating that they were mediated by the Na+,K+ pumps and not by K+ channels (Clausen et al., 1987).Open in a separate windowFigure 4.Effects of salbutamol, rat CGRP, dbcAMP, and ouabain on the response of tetanic force to electroporation (EP). Force was measured in rat soleus muscles mounted for isometric contractions in KR buffer at 30°C using 2-s pulse trains of 60 Hz. Trains were repeated three times to obtain an initial determination of tetanic force, and data are presented as a percentage of this value. After transfer to an electroporation cuvette, muscles either underwent electroporation (eight pulses of 500 V/cm and 0.1-ms duration) or were left without electroporation. Force was subsequently recorded at the indicated intervals in buffer without additions (EP controls) or in buffer containing salbutamol (10−5 M), rat CGRP (10−7 M), dbcAMP (1 mM), or salbutamol (10−5 M) plus ouabain (10−3 M). Each point represents the mean of observations on 4–13 muscles, with bars denoting SEM. Reprinted with permission from Acta Physiologica (Clausen and Gissel, 2005).Open in a separate windowFigure 5.Correlation between ouabain-suppressible 86Rb uptake and [3H]ouabain-binding site content in soleus muscles of rats with K+ deficiency and varying age or thyroid status. Ouabain-suppressible 86Rb uptake was measured as described in Clausen et al. (1987). [3H]Ouabain-binding site content was determined on muscle samples using the vanadate-facilitated assay. After correction for nonspecific uptake, [3H]ouabain binding was corrected for radioisotopic purity, incomplete saturation, and loss of specifically bound [3H]ouabain. The line was constructed using linear-regression analysis of unweighted values (r = 0.95; P < 0.001). Reprinted with permission from The Journal of Physiology (Clausen et al., 1987).In conclusion, translocation of Na+,K+ pumps happens in skeletal muscle, but it is difficult to define and quantify. As a mechanism for stimulation of the Na+,K+ pumps it is too slow to explain the documented evidence for the acute Na+,K+-pump activation, which may reach the theoretical maximum in less than 1 min.

Subunit isoforms of the Na+,K+-ATPase

In the mammalian brain, the Na+,K+-ATPase exists in two distinct molecular forms that differ with respect to affinity for ouabain (Sweadner, 1979). Similarly, two subunit isoforms of the enzyme, α1 and α2, were detected in rat skeletal muscle (Tsakiridis et al., 1996). The functional Na+,K+ pump is composed of one of four catalytic α subunits (α1–α4) and one of three structural β subunits (β1–β3). In skeletal muscle, a third subunit, FXYD1, is coexpressed with the α subunits and involved in the regulation of Na+,K+-pump activity (Fig. 3).Open in a separate windowFigure 3.Hypothetical relationship between phospholemman (PLM or FXYD1) and the Na+/K+ATPase α subunit indicating possible effects of PLM phosphorylation on their interaction. The PLM cytoplasmic tail can be phosphorylated at Ser63, Ser68, and Thr69 (Ser69 in mouse in panel a). Phosphorylation at any or all of these sites (or, for example, at the site depicted as undergoing phosphorylation by PKA in panel b) may alter orientation of the cytoplasmic tail, thereby affecting its interaction with the Na+/K+ ATPase α subunit to increase ion transport. Reprinted with permission from Current Opinion in Pharmacology (Shattock, 2009).At present, the subunit isoforms of the Na+,K+-ATPase as detected by immunoblotting cannot be quantified and given in molar units for the contents per gram muscle wet weight (Tsakiridis et al., 1996; Clausen, 2003). A recent study (McKenna et al., 2012) indicates that in human vastus lateralis muscle, there is a relative decrease of −24% in the abundance of the α2 subunit from 24 to 67 yr of age (P < 0.05). This decrease was associated with a 36% reduction in muscle strength and peak O2 consumption. However, the same study showed no significant age-dependent change in the content of [3H]ouabain-binding sites (Bangsbo et al., 2009). More recent studies on human vastus lateralis show that 10 d of training increases the abundance of α1 and α2 subunit by 113 and 49%, respectively (Benziane et al., 2011). In rat hind-limb muscle, 60 min of treadmill running (20 m/min) induces a 55–64% increase in the abundance of the Na+,K+-ATPase α1 subunit and a 43–94% increase in that of the α2 subunit (Tsakiridis et al., 1996).In the isolated rat EDL muscle, the mechanisms for the excitation-induced up-regulation of mRNA for the α1-3 subunits were examined. Three bouts of electrical stimulation (60 Hz for10 s) had no immediate effect on the abundance of the mRNA-encoding α1–α3-subunit isoforms (Murphy et al., 2006). However, after 3 h of poststimulatory rest, there were 223, 621, and 892% increases in the abundance of the mRNA-encoding subunits α1, α2, and α3, respectively. However, in rat EDL muscle, even massive increases (598–769%) in intracellular Na+ induced by pretreatment with ouabain, veratridine, or monensin produced no significant change in the mRNA encoding any of the subunit isoforms. Increasing intracellular Ca2+ by preincubation with the Ca2+ ionophore A23187 produced no increase in the mRNA for the two major subunit isoforms (α1 and α2). Thus, a combination of multiple signals seems to be required to recapitulate the increase in abundance of the mRNAs that trigger the increase in synthesis of Na+,K+ pumps induced by exercise. In conclusion, the abundance of the α1 and α2 subunits seems to respond to training and age, but the observed relative changes show considerable variation and cannot yet be “translated” to molar units for Na+,K+ pumps per gram muscle tissue. As shown in Radzyukevich et al., 2013). These defects could be reproduced in isolated muscles from control animals by selectively inhibiting the α2 subunit with 5 µM ouabain.

Molecular mechanisms of pump regulation. Acute regulation of Na+,K+-ATPase by hormones.

Insulin has a hypokalemic action, prompting its use in the therapy of hyperkalemia (Harrop and Benedict, 1924) and inspiring studies with isolated rat muscles that showed that insulin increases the uptake of K+ and the extrusion of Na+ (Creese, 1968), leading to reduced intracellular Na+ and hyperpolarization (Zierler and Rabinowitz, 1964). Because the effects of insulin on Na+,K+ fluxes and membrane potential are suppressed by ouabain, they are likely caused by stimulation of the Na+,K+ pumps (Clausen and Kohn, 1977, Clausen, 2003). The mechanism seems to reflect increased affinity for Na+ on the inner surface of the Na+,K+ pumps (Clausen, 2003). As discussed in the section above on translocation (Translocation of Na+,K+ pumps compared…), the stimulating action of insulin on the Na+,K+ pumps may not be attributed to translocation of Na+,K+ pumps. Studies on cultured rat muscle cells indicate that the stimulatory effect of insulin on 86Rb uptake is abolished by ouabain and reduced by phorbol ester, indicating that it is mediated by Na+,K+ pumps and caused by activation of PKC (Sampson et al., 1994).Similarly, the hypokalemic action of catecholamines inspired studies with isolated muscles demonstrating stimulatory effects of epinephrine and norepinephrine on the uptake of K+ and the net extrusion of Na+ (Clausen and Flatman, 1977). Because of the 3:2 exchange of Na+ versus K+, the Na+,K+ pump is electrogenic and its stimulation leads to hyperpolarization. Detailed studies showed that this stimulation depended on a β2 adrenoceptor–mediated activation of adenylate cyclase increasing the cytoplasmic concentration of cAMP (Clausen and Flatman, 1977; Clausen, 2003). cAMP, via PKA, induces phosphorylation of FXYD1serine68, causing increased Na+ affinity of the Na+,K+ pumps (Shattock, 2009) and ensuing reduction of intracellular Na+. A similar sequence of events explain the cAMP-mediated, Na+,K+ pump–stimulatory action of other hormones and pharmaceuticals (including calcitonin, CGRP, amylin, isoprenaline, and theophylline; see Clausen, 1998, and Fig. 6 in Clausen, 2003). More detailed molecular insight is discussed by Shattock (2009) and in this paper.

Na+,K+-ATPase regulation by auxiliary proteins.

The Na+,K+ pumps are subject to regulation by a series of proteins. The β subunit (Fig. 1) is a glycoprotein required for the transfer of the entire Na,K-ATPase enzyme molecule from its site of synthesis in the endoplasmic reticulum to its site of insertion in the plasma membrane. The γ subunit, originally identified in the kidney, augments the affinity of the myocardial Na+,K+-ATPase for Na+ (Bibert et al., 2008). In mammals, there is a family of at least seven regulatory proteins termed FXYD (“fix-it”) that are associated with the Na+,K+-ATPase in a tissue-specific way (Mahmmoud et al., 2000; Cornelius and Mahmmoud, 2003). The first FXYD identified, called FXYD1 and also known as phospholemman, was originally found in myocardial cells (Palmer et al., 1991). FXYD1 is a major substrate for PKA and PKC and was later found to regulate the Na+,K+-ATPase in heart and skeletal muscle (Shattock, 2009). Increased cAMP activates cAMP-dependent kinase (PKA), which, in turn, phosphorylates serine-68 on FXYD1 (Fig. 3). This disinhibits the Na+,K+ pumps and stimulates their activity by raising Vmax and the sensitivity of the pumps to intracellular Na+. Phosphorylation of FXYD increases the maximal Na+,K+-pump current of α2/β isozymes (Bibert et al., 2008). cAMP seems to function as a common signaling molecule acting via PKA to augment the affinity of the Na+,K+ pump for [Na+]i in muscle (Shattock, 2009). In addition to Ser68, FXYD can also be phosphorylated at Ser63 (by PKC) and at Thr69 (by an unidentified kinase).In the human vastus lateralis muscle, 30 s of intense exercise clearly increased phosphorylation of FXYD (Thomassen et al., 2011). Another study demonstrated that 60 min of acute exercise increases phosphorylation of FXYD from human vastus lateralis by 75% (Benziane et al., 2011). In soleus muscle of wild-type mice, 10 min of electrical stimulation caused a 59% increase in phosphorylation of FXYD. This phosphorylation was not seen in PKCα knockout mice and therefore seems to depend on PKC (Thomassen et al., 2011).

Physiology and pathophysiology of the Na+,K+-ATPase in skeletal muscle

The electrogenic action of the Na+,K+ pumps.

Because each Na+,K+-pump molecule in the plasma membrane exchanges three Na+ ions for two K+ ions in each cycle, each cycle leads to a net extrusion of one positive charge. Measurements on isolated rat soleus muscle show that the resting ouabain-suppressible efflux of 22Na at 30°C is 0.287 µmol/g wet wt/min, and the concomitant influx of 42K is 0.196 µmol/g wet wt/min (Clausen and Kohn, 1977). Thus, the ratio between Na+,K+ pump–mediated Na+ efflux and K+ influx is 0.287/0.196 = 1.46, close to the 3:2 ratio. This electrogenic action of the Na+,K+ pumps is consistent with the observation that, in rat soleus muscle, blocking the Na+,K+ pumps with ouabain causes a depolarization of ∼10 mV in 10 min (Clausen and Flatman, 1977). Conversely, acute stimulation of the rate of active Na+,K+ transport by epinephrine in isolated rat soleus muscle causes a hyperpolarization of ∼8 mV, which is abolished by ouabain. The β2 agonist salbutamol induces a similar effect in vitro, and when given intravenously, it causes 15-mV hyperpolarization in rat soleus (Clausen and Flatman, 1980). The following agents, which stimulate active Na+,K+ transport, all induce significant hyperpolarization in rat, mouse, or guinea pig soleus: epinephrine, norepinephrine, isoprenaline, salbutamol, dbcAMP, phenylephrine, rat, human or salmon calcitonin, CGRP, and insulin (Clausen and Flatman, 1977, 1987; Andersen and Clausen, 1993). Rat soleus muscles can be loaded with Na+ by a 90-min preincubation in K+-free KR. When subsequently transferred to KR with normal K+, the rate of 22Na efflux is more than doubled. This effect is blocked by ouabain and associated with an ∼10-mV hyperpolarization (Clausen and Flatman, 1987). These observations indicate that the electrogenic action of hormonal Na+,K+-pump stimulation can be mimicked by Na+ loading.

The Na+,K+ pumps compensate functional defects caused by plasma membrane leakage

Apart from the channel-mediated selective Na+ and K+ leaks that take place during excitation, the plasma membrane may develop nonspecific leaks caused by loss of integrity during intense work, anoxia, physical damage, electroporation, excessive swelling, rhabdomyolysis, or various muscle diseases. The response to such leaks and the possible role of the Na+,K+ pumps in compensating the ensuing functional disorders have been examined using electroporation of isolated rat soleus and EDL muscles (Clausen and Gissel, 2005). In these studies, muscles were mounted in an electroporation cuvette and exposed to short-lasting (0.1-ms) pulses of an electrical field of 100–800 V/cm across the muscles. This induces rapid formation of pores in the plasma membrane, increasing its permeability, but not in the membranes of intracellular organelles. This allows reversible influx of Na+, loss of K+ and excitability, release of intracellular proteins, and penetration of extracellular markers into the cytoplasm (Clausen and Gissel, 2005). As shown in Fig. 4, eight electroporation pulses of 500 V/cm induces immediate complete loss of tetanic force, which in 200 min is followed by spontaneous recovery to around 30% of the force measured before electroporation. The initial phase of this recovery is considerably (183–433%) enhanced by stimulating the Na+,K+ pumps with salbutamol, rat CGRP, or dibutyryl cAMP. Both the spontaneous 30% force recovery and the 50% force recovery induced by salbutamol were abolished by ouabain, indicating that they were caused by Na+,K+-pump stimulation. The electroporation caused a depolarization from −70 to around −20 mV, followed by a partial spontaneous recovery. Salbutamol (10−5 M) further improved the repolarization by 15 mV (Clausen and Gissel, 2005). In isolated rat EDL muscles, 30–60 min of intermittent stimulation caused a drop in tetanic force to 12% of the initial level, followed by slow spontaneous recovery to 20–25% of the initial force level. This was associated with 11–15-mV depolarization and marked loss of the intracellular protein lactic acid dehydrogenase. Subsequent stimulation of the Na+,K+ pumps with salbutamol restored membrane potential to normal level. Salbutamol, epinephrine, rat CGRP, and dibutyryl cAMP all induced a significant increase (40–90%) in the force recovery after intermittent stimulation (Mikkelsen et al., 2006). In EDL muscle, anoxia caused massive reductions in the content of phosphocreatine and ATP and a marked loss of force, which was clearly reduced by the two β2 agonists salbutamol and terbutaline. The force recovery seen after reoxygenation was markedly improved by salbutamol, the long-acting β2 agonist salmeterol, theophylline (a phosphodiesterase inhibitor that inhibits the breakdown of cAMP), and dibutyryl cAMP (causing the following relative increases in force: 55–262%). The salbutamol-induced increase in force recovery could be related to partial restoration of excitability. In anoxic muscles, salbutamol had no effect on phosphocreatine or ATP but decreased intracellular Na+ concentration and increased 86Rb uptake and K+ content, indicating that the mechanism is cAMP-mediated stimulation of the electrogenic Na+,K+ pumps (Fredsted et al., 2012) and cannot be related to recovery of energy status. Thus, the Na+,K+ pumps may restore excitability even when energy status is low. This interpretation is in keeping with a detailed analysis in isolated mouse soleus and EDL muscles, suggesting that impaired excitability is the main contributor to severe fatigue, discounting anoxia as the major contributor to fatigue in isolated muscles (Cairns et al., 2009).

Functional significance of Na+,K+ pumps.

The evidence described above indicates that the content of Na+,K+ pumps measured using [3H]ouabain binding represents Na+,K+ pumps, which may all become operational when activated by intracellular Na+ loading and increased extracellular K+, by hormones or excitation. Is it possible to induce a rapid activation of all the Na+,K+ pumps in the intact muscle? In rat soleus muscle, electrical stimulation at 120 Hz for 10 s increases intracellular Na+ to ∼50 mM (Nielsen and Clausen, 1997). When the muscles were subsequently allowed to rest in standard KR at 30°C, the net efflux of Na+ recorded over the first 30 s was shown to reach 9,000 nmol/g wet wt/min, 97% of the theoretical maximum Na+ transport rate of 9,300 nmol/g wet wt/min (Fig. 5 in Nielsen and Clausen, 1997). This indicates that during intense exercise, most likely to produce a high degree of Na+,K+-pump use, a 97% utilization has been documented. Apparently, it requires the combination of large increases in intracellular Na+ and extracellular K+, which, as described above, can be mimicked by Na+ loading and subsequent incubation at high [K+]o (Clausen et al., 1987). In rat soleus, 60-Hz stimulation induces a total efflux of K+ of 5,580 nmol/g wet wt/min (Clausen et al., 2004). Only full activation of all Na+,K+ pumps would enable K+ reuptake sufficient to allow extracellular K+ clearance to keep pace with excitation-induced loss of K+. As already noted, the theoretical maximum Na+,K+ pump–mediated K+ uptake in rat soleus should reach 6,408 nmol K+/g wet wt/min, slightly exceeding the above-mentioned total loss of K+. Such large increases in active Na+,K+ transport would appear energetically unlikely. However, as stated previously, both in resting and working muscles, the energy requirement of the Na+,K+ pumps only amounts to 2–10% of the total energy turnover. This evaluation provides an example of the importance of accurate quantification of the Na+,K+ pumps and their energy requirements.This information raises the question of whether such combinations of high [Na+]i and [K+]o occur in working intact muscle in vitro or in vivo. Several studies indicate that isolated muscles and muscle fibers, when stimulated at resting length, take up substantial amounts of Na+ and release nearly equimolar amounts of K+ (Hodgkin and Horowicz, 1959; Clausen, 2008b, 2011, 2013; Clausen et al., 2004). In vitro experiments show that when the amount of K+ released from the cells (µmoles/gram wet weight as measured by flame photometry) is divided by the extracellular space (milliliter/gram wet weight), it can be calculated that in isolated soleus and EDL muscles, [K+]o reaches levels of 20 or 50 mM, respectively, sufficient to suppress excitability (Clausen, 2008a). More detailed studies showed that in isolated EDL muscles, 60 s of stimulation at 20 Hz increases [K+]o to around 45 mM, leading to loss of excitability (Clausen, 2011). Recent in vitro and in vivo studies show that during direct or indirect electrical stimulation (at 5 Hz for 300 s or 60 Hz for 60 s) of rat EDL muscles, the mean extracellular K+ in the stimulated muscles may reach up to 50–70 mM (Clausen, 2013). This was combined with an appreciable decrease in [Na+]o (46 mM), causing further loss of force. This provides new evidence that in the intact organism, excitation-induced rise in [K+]o and decrease in [Na+]o can be major causes of muscle fatigue. Moreover, as described above, the Na+,K+ pumps available in the muscles may fully use their maximum transport capacity to counterbalance the excitation-induced rise in [K+]o. An important function of the Na+,K+ pumps is to protect muscle excitability against increases in [K+]o. When exposed to 10–15 mM K+, contractility of rat soleus or EDL muscles is reduced or lost. Force may rapidly be restored by stimulating the Na+,K+ pumps with salbutamol or insulin (Clausen, 2003; Clausen and Nielsen, 2007).The excitation-induced rise in [K+]o depends on muscle fiber type (slow-twitch or fast-twitch fibers) and the frequency of contractions. In isolated rat EDL muscle (predominantly fast-twitch fibers), the excitation-induced influx of Na+ and release of K+ per action potential is, respectively, 6.5- and 6.6-fold larger than in soleus (predominantly slow-twitch fibers) (Clausen et al., 2004). When stimulated continuously at 60 Hz, the rate of force decline over the first 20 s is 5.9-fold faster in EDL than in soleus muscle. When tested using continuous stimulation in the frequency range of 10 to 200 Hz, the rates of force decline in rat soleus and EDL muscles are closely correlated (r2 = 0.93; P < 0.002 and 0.99; P < 0.01, respectively) to the excitation-induced measured increase in [K+]o (Clausen, 2008b). These observations indicate that the faster rate of force decline and fatigability seen in fast-twitch muscles as compared with slow-twitch muscles is caused by the much larger excitation-induced passive Na+,K+ fluxes. In view of this difference, leading to a larger work-induced increase in intracellular Na+, it would be expected that the content of Na+,K+ pumps in EDL would undergo long-term up-regulation to a level considerably over and above that seen in soleus. However, measurements of [3H]ouabain-binding sites in vitro and in vivo show that rat EDL only contains 20–30% more Na+,K+ pumps per gram wet weight than soleus (Clausen et al., 1982; Murphy et al., 2008).Control soleus muscle, ouabain-pretreated soleus, or soleus from K+-deficient rats show a graded reduction in functional Na+,K+ pumps from 756 pmol/g wet wt down to 110 pmol/g wet wt. The content of [3H]ouabain-binding sites is significantly correlated (r2 = 0.88; P < 0.013) to the rate of force decline developing over 30 s of continuous stimulation at 60 Hz (Nielsen and Clausen, 1996). This illustrates the crucial role of the Na+,K+-pump capacity in maintaining contractile endurance. In keeping with this, K+ deficiency in human subjects, which reduces the content of Na+,K+ pumps, causes reduced grip strength and fatigue (Clausen, 1998, 2003). Conversely, 19 studies have documented that exercise training leads to an increase in the content of Na+,K+ pumps measured as [3H]ouabain binding (Clausen, 2003). This up-regulation improved the clearance of plasma K+ during exercise, a mechanism for reducing fatigue (McKenna et al., 1993). During contractile activity, the K+ released from the muscle cells is normally cleared via the circulation. When the blood vessels are compressed because of static contractions when carrying heavy burdens (Barcroft and Millen, 1939), this clearance is reduced or abolished. Therefore, extracellular K+ may undergo further increase, and its clearance will depend on the transport capacity and activity of the Na+,K+ pumps. Through a similar mechanism, deficient circulation may lead to reduction of contractile force.

Conclusions

Recent studies indicate that in skeletal muscle, excitation-induced net gain of Na+ and loss of K+ are considerably larger than reported previously and represent important causes of fatigue. This implies that to counterbalance these rapid passive Na+,K+ fluxes, working muscles have to make full use of the transport capacity of their Na+,K+ pumps. This capacity can be quantified using [3H]ouabain binding and expressed in picomoles of Na+,K+ pumps per gram muscle wet weight, offering values that can be confirmed by measurements of maximum ouabain-suppressible fluxes of Na+ and K+. This allows quantification of the capacity to clear extracellular K+ in patients with reduced content of Na+,K+ pumps in their muscles. Numerous studies have shown that the content of Na+,K+ pumps in skeletal muscles can be quantified, documenting the up-regulation of the content of Na+,K+ pumps induced by exercise and hormones as well as the down-regulation seen in various diseases associated with reduced physical performance or fatigue. Assays based on immunoblotting provide relative changes in abundance that cannot yet be translated into molar units. The activity of the Na+,K+ pumps may increase rapidly (by electrical stimulation up to 20-fold in 10 s) or by Na+ loading, insulin, catecholamines, calcitonins, amylin, and theophylline.  相似文献   

17.
Na+/H+ antiporters influence proton or sodium motive force across the membrane. Synechocystis sp. PCC 6803 has six genes encoding Na+/H+ antiporters, nhaS1–5 and sll0556. In this study, the function of NhaS3 was examined. NhaS3 was essential for growth of Synechocystis, and loss of nhaS3 was not complemented by expression of the Escherichia coli Na+/H+ antiporter NhaA. Membrane fractionation followed by immunoblotting as well as immunogold labeling revealed that NhaS3 was localized in the thylakoid membrane of Synechocystis. NhaS3 was shown to be functional over a pH range from pH 6.5 to 9.0 when expressed in E. coli. A reduction in the copy number of nhaS3 in the Synechocystis genome rendered the cells more sensitive to high Na+ concentrations. NhaS3 had no K+/H+ exchange activity itself but enhanced K+ uptake from the medium when expressed in an E. coli potassium uptake mutant. Expression of nhaS3 increased after shifting from low CO2 to high CO2 conditions. Expression of nhaS3 was also found to be controlled by the circadian rhythm. Gene expression peaked at the beginning of subjective night. This coincided with the time of the lowest rate of CO2 consumption caused by the ceasing of O2-evolving photosynthesis. This is the first report of a Na+/H+ antiporter localized in thylakoid membrane. Our results suggested a role of NhaS3 in the maintenance of ion homeostasis of H+, Na+, and K+ in supporting the conversion of photosynthetic products and in the supply of energy in the dark.Na+/H+ antiporters are integral membrane proteins that transport Na+ and H+ in opposite directions across the membrane and that occur in virtually all cell types. These transporters play an important role in the regulation of cytosolic pH and Na+ concentrations and influence proton or sodium motive force across the membrane (1, 2). In Escherichia coli, three Na+/H+ antiporters (NhaA, NhaB, and ChaA) have been described in detail. Of these, NhaA is the functionally best characterized transporter. The crystal structure of NhaA has been resolved (3). In addition, mutants of nhaA, nhaB, and chaA as well as the triple mutant have been generated (4). The triple mutant was shown to be hypersensitive to extracellular Na+. The genome of the cyanobacterium Synechocystis sp. PCC 6803 contains six genes encoding Na+/H+ antiporters (NhaS1–5 and sll0556). NhaS1 (slr1727) has also been designated SynNhaP (5, 6). Null mutants of nhaS1, nhaS2, nhaS4, and nhaS5 have been generated; however, a null mutant of nhaS3 could not be obtained, indicating that it is an essential gene (68). By heterologous expression in E. coli, Na+/H+ exchange activities could be shown for NhaS1–5 (5, 6). Inactivation of nhaS1 and nhaS2 results in retardation of growth of Synechocystis (5, 6). It has been reported that in these mutants the concentration of Na+ in cytosol and intrathylakoid space (lumen) increases and impairs the photosynthetic and/or respiratory activity of the cell (9, 10). Therefore the Na+ extrusion by Synechocystis Na+/H+ antiporters similar to E. coli NhaA, NhaB, and ChaA is essential for the adaptation to salinity stress.In contrast to the case in E. coli, Na+ is an essential element for the growth of some cyanobacteria (11, 12). Interestingly, the Na+/H+ antiporter homolog NhaS4 was identified as an uptake system for Na+ from the medium during a screen for mutations in Synechocystis that result in lack of growth at low Na+ concentrations (7). The requirement of a Na+ uptake antiporter for cell growth is consistent with the physiology of Synechocystis. Specifically, photoautotrophic bacteria like cyanobacteria share some components (plastoquinone, cytochrome b6f, and c6) of the thylakoid membrane for electron transport for both photophosphorylation and respiratory oxidative phosphorylation. Na+/H+ antiporters therefore may coordinate both H+ and Na+ gradients across the plasma and thylakoid membranes to adapt to daily environmental changes (11). It remains to be determined whether the six Na+/H+ antiporters are localized to the plasma membrane or to the thylakoid membrane in Synechocystis. Information on the membrane localization will also provide information on the physiological role in Synechocystis. In this study, we explored the membrane localization of NhaS3, the role of specific amino acid residues for its function, and the effect of CO2 concentration and circadian rhythms on the expression pattern of nhaS3 to gain insight into the physiological role of NhaS3 in Synechocystis.  相似文献   

18.
Under abiotic stress conditions, rapid increases in reactive oxygen species (ROS) levels occurs within plant cells. Although their role as a major signalling agent in plants is now acknowledged, elevated ROS levels can result in an impairment of membrane integrity. Similar to our previous findings on imposition of salt stress, application of the hydroxyl radical (OH) to Arabidopsis roots results in a massive efflux of K+ from epidermal cells. This is likely to cause significant damage to cell metabolism. Since K+ loss also occurs after salt application and salt stress leads to increased cellular ROS levels, we suggest that at least some of the detrimental effects of salinity is due to damage by its resulting ROS on K+ homeostasis. We also observed a comparative reduction in K+ efflux by compatible solutes after both oxidative and salt stress. Thus, we propose that under saline conditions, compatible solutes mitigate the oxidative stress damage to membrane transporters. Whether this amelioration is due to free-radical scavenging or by direct protection of transporter systems, warrants further investigation.Key words: compatible solutes, hydroxyl radical, potassium efflux, reactive oxygen species, salt stressReactive oxygen species (ROS) are continuously produced as by-products of various metabolic pathways.1 Under unstressed steady-state conditions, cellular ROS levels are kept in check by the sophisticated antioxidant defence system.2 However, under adverse environmental conditions, the balance between ROS production and its subsequent scavenging may be perturbed, leading to a rapid increase in ROS levels.3 Although significant progress has been made in defining ROS as a major signalling agent in plants,3 ROS can react with a large variety of biomolecules, causing lipid peroxidation and impairing membrane integrity.4,5 One such abiotic stress is salt stress,6 with ROS generation occurring within minutes of salt application.7 Alleviation of oxidative damage may be, therefore, an important strategy of plant salt tolerance.8One of the earliest measurable responses to salt stress is a massive K+ efflux from plant roots.9,10 Such K+ efflux is initiated within seconds of acute salt stress and may last for several hours11,12 reducing the intracellular K+ pool13,14 and significantly impairing cell metabolism. Consistent with the key role of K+ homeostasis in salt tolerance mechanisms15 a reduction of K+ efflux correlates with increased salt tolerance.11,12We have previously reported that hydroxyl radical (OH) application to Arabidopsis roots also results in a rapid efflux of K+ from the epidermis.16 In this report, we find a similar K+ efflux response.17 As is the case for salt stress,9 we found that membrane depolarisation could be responsible for a substantial part of this efflux. However, an observed discrepancy between the membrane depolarisation and the pattern of K+ efflux indicates that voltage-dependence is not the only factor influencing K+ loss from the root cells after oxidative stress. Demidchik et al.16 demonstrated that stress-induced K+ efflux could be mediated by activation of K+ outward rectifying channels directly by OH. This direct effect on K+ transporters could also account for our observed delay before the peak efflux of K+ is measured, indicating that a certain amount of time is required before maximal direct damage by OH to transporters occurs. Because both K+ channel blockers and non-selective cation channel blockers reduce this efflux, it indicates non-specificity in OH attack. Furthermore, combinations of these channel blockers were effective in reducing K+ efflux implying that, at least in the short term, the damaging effects of OH is due to compromising the transporter systems as opposed to lipid peroxidation. Certainly, K+ channels harbour reactive groups, thus are expected to be sensitive to ROS.18We have previously shown that the exogenous application of low concentrations of a variety of compatible solutes reduces the salt-induced K+ efflux.19,20 Plants, when confronted with a saline environment, respond with a significant elevation in their compatible solute levels. This ameliorates the detrimental effects of salinity.21 However, their original proposed role in cellular osmoregulation is under question: their concentration in transgenic plants overexpressing osmolyte biosynthetic genes is not significant for osmotic adjustment, despite showing improved salt tolerance.8 Furthermore, one hallmark of the detoxification effect is its lack of specificity, that is, transgenic plants have increased tolerance not only to high salt, but also to drought, cold and heat shock,22,23 stresses that also result in ROS production.3 Certainly, ecotopic expression studies suggest that compatible solutes increase stress tolerance by protection of membranes and proteins against ROS.6We show that in this work that exogenous application of low concentrations of a range of compatible solutes significantly reduces OH-induced K+ efflux,17 a similar effect to that we reported after salt application to barley roots19 and also observed in Arabidopsis (Fig. 1). Interestingly, we found that not only known free-radical scavenging osmolytes,24 but also glycine betaine, previously found to be non-effective in ROS scavenging,24 were effective in reducing OH-induced K+ efflux. Indeed, glycine betaine showed a greater mitigation of OH-induced K+ efflux compared to that induced by 50 mM NaCl (Fig. 1). However, it is open to speculation as to whether this mitigation is via direct channel blocking, a direct protection of ion channel proteins or by some other protective mechanism.Open in a separate windowFigure 1Effects of exogenous supply of compatible solutes on net peak K+ efflux after application of either 1 mM Cu/a or 50 mM NaCl. Roots were preincubated for 1 h in 5 mM concentration of a number of compatible solutes prior to treatment, Mean ± SE (n = 6-8).In our further investigations we have found that salt-tolerant barley show a reduced ROS-induced K+ efflux compared to sensitive varieties.25 This superior ability of salt-tolerant barley cultivars of preventing K+ loss further indicates a possible causal link between salt and oxidative stress tolerance. We propose that upon the imposition of salt stress, the instantaneously resulting membrane depolarisation9 results in activation of depolarisation activated K+ outward-rectifying channels, leading to the initial massive K+ efflux. Over the longer term, ROS levels within the plant cell increase,7 resulting in direct damage to K+ transporters and the longer-term sustained loss of K+ from the cell. Due to mitigation of both NaCl- and OH-induced K+ efflux by compatible solutes, we propose that one of their primary amelioratory effects is through reducing the damaging effects of salt-produced ROS on K+ transporter, and by this means, reducing the effects of stress damage. Whether this amelioration is achieved through free-radical scavenging or due to a direct protection of membrane transports warrants further investigation.  相似文献   

19.
20.
The molecular mechanisms of K+ homeostasis are only poorly understood for protozoan parasites. Trypanosoma brucei subsp. parasites, the causative agents of human sleeping sickness and nagana, are strictly extracellular and need to actively concentrate K+ from their hosts’ body fluids. The T. brucei genome contains two putative K+ channel genes, yet the trypanosomes are insensitive to K+ antagonists and K+ channel-blocking agents, and they do not spontaneously depolarize in response to high extracellular K+ concentrations. However, the trypanosomes are extremely sensitive to K+ ionophores such as valinomycin. Surprisingly, T. brucei possesses a member of the Trk/HKT superfamily of monovalent cation permeases which so far had only been known from bacteria, archaea, fungi, and plants. The protein was named TbHKT1 and functions as a Na+-independent K+ transporter when expressed in Escherichia coli, Saccharomyces cerevisiae, or Xenopus laevis oocytes. In trypanosomes, TbHKT1 is expressed in both the mammalian bloodstream stage and the Tsetse fly midgut stage; however, RNA interference (RNAi)-mediated silencing of TbHKT1 expression did not produce a growth phenotype in either stage. The presence of HKT genes in trypanosomatids adds a further piece to the enigmatic phylogeny of the Trk/HKT superfamily of K+ transporters. Parsimonial analysis suggests that the transporters were present in the first eukaryotes but subsequently lost in several of the major eukaryotic lineages, in at least four independent events.Potassium (K+) is the most abundant cation in the cytosol of any cell and hence an essential macronutrient for life on earth. Concentrative K+ uptake across the plasma membrane is energized directly by ATPases and indirectly by the negative membrane potential or by coupling, via symport or antiport, to other transport processes such as H+ flux. The ancestry of K+ transporters renders them ideal subjects for phylogenetic comparisons. Indeed, the different kinds of known K+ transporters—pumps, channels, permeases, symporters, and antiporters—are all found in bacteria (43). Eukaryotes do not appear to have invented further mechanisms of K+ transport; on the contrary, some families of K+ transporters were lost over the course of eukaryote evolution, particularly among the metazoa (53).The Trk/HKT superfamily (TC transporter classification 2.A.38 [43]) consists of bacterial TrkH and KtrB, plant HKT, and fungal Trk transporters (15). These proteins share a topology with 8 transmembrane (TM) domains and, sandwiched between odd- and even-numbered TM domains, 4 shorter hydrophobic helices that resemble the P-loops of K+ channels (14, 27, 55). In the K+ channel, these pore-forming loops end in the filter residues glycine-tyrosine-glycine, which coordinate K+ by means of their backbones’ carbonyl oxygens (13). The P-loop-like helices of Trk/HKT transporters end in a single conserved glycine (48), and these glycines have been shown to determine K+ selectivity over Na+ of the transporters (34, 49). Thus, a Trk/HKT monomer with 8 TM domains and 4 P-loops is thought to have a similar pore architecture to a K+ channel tetramer with two TM domains and one P-loop per subunit. The Trk/HKT transporters are important for cellular K+ acquisition in microorganisms, since trk null mutant yeast or bacteria exhibit growth phenotypes on media containing low K+ concentrations (20, 46). The roles of the Trk/HKT transporters in plants are more diverse, including Na+ distribution (10, 33, 47), osmoregulation (32), and salt tolerance (39). So far, no HKT/Trk transporter has been described from the metazoa or protista.Trypanosoma brucei subsp. parasites comprise the causative agents of human and livestock trypanosomosis: sleeping sickness and nagana, respectively. The distribution of the parasites is restricted by that of their vector, the blood-sucking tsetse fly (Glossina spp.), to the so-called tsetse belt comprising 36 countries between the Sahara desert and the Kalahari (3, 8). African trypanosomes proliferate extracellularly in the blood, evading the mammalian immune response by antigenic variation. Untreated sleeping sickness is fatal. There is an urgent need for new and better drugs since the current ones, the arsenical melarsoprol in particular, suffer from severe side effects (31). In the mammalian bloodstream, the parasites encounter a rich and steady supply of nutrients, readily imported by specific permeases or endocytosed via receptors (7). Research on trypanosomal nutrient uptake has so far concentrated on transporters of organic substrates: nucleobases, nucleosides, sugars, and amino acids (4, 12, 26, 30, 35, 56). Little is known about how the parasites import inorganic nutrients. The malaria parasite Plasmodium falciparum possesses two putative K+ channel subunits with 6 TM domains and one P-loop (19, 52). Disruption of an orthologous gene in Plasmodium berghei strongly impaired the development of the malaria parasites in the mosquito (18). However, these putative channels have not yet been proven to be permeable to K+. The T. brucei genome (6) is annotated to contain two putative K+ channels; in addition, a putative ATPase has been identified resembling fungal Na+/K+ efflux ATPases (5, 45). None of these has been addressed experimentally. Here we present the identification and characterization of TbHKT1 (Tb10.70.2940), a Trk/HKT-type K+ transporter from Trypanosoma brucei and representative of a new clade of Trk/HKT genes from kinetoplastid parasites.  相似文献   

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