Sodium Absorption, Volume Control and Potassium Channels: In Tribute to a Great Biologist

It is well established, for all Na-absorbing epithelia, that an increase in the rate of transcellular Na+ absorption is accompanied by an increase in the conductance of the basolateral membrane to K+. For the case of small intestinal epithelial cells from the salamander Necturus maculosus, where the rate of transcellular Na+ absorption can be increased manyfold by the addition of sugars or amino acids to the luminal bathing solution, it appears that this parallelism between Na-K pump rate and basolateral membrane K+ conductance is closely related to volume regulation by the enterocyte. Recent studies have disclosed the presence of stretch-activated K+ channels, in a highly enriched basolateral membrane fraction isolated from these epithelial cells, whose activity is increased by an increase in vesicle volume and inhibited by a decrease in vesicle volume or ATP. The activity of this channel also appears to be regulated by the degree of organization of the cortical actin cytoskeleton; activity is increased by depolymerization of the actin cytoskeleton and decreased by repolymerization of that structure. We postulate that the inhibitory effect of ATP is related to its role in promoting the polymerization of G-actin to form F-actin. We propose that enterocyte swelling that results from the intracellular accumulation of sugars or amino acids in osmotically active forms brings about disorganization of the cortical actin cytoskeleton and activates these channels and is, at least in part, responsible for the "pump-leak parallelism" in this amphibian.     

p11, an annexin II subunit,an auxiliary protein associated with the background K+ channel,TASK-1

TASK-1 belongs to the 2P domain K+ channel family and is the prototype of background K+ channels that set the resting membrane potential and tune action potential duration. Its activity is highly regulated by hormones and neurotransmitters. Although numerous auxiliary proteins have been described to modify biophysical, pharmacological and expression properties of different voltage- and Ca2+-sensitive K+ channels, none of them is known to modulate 2P domain K+ channel activity. We show here that p11 interacts specifically with the TASK-1 K+ channel. p11 is a subunit of annexin II, a cytoplasmic protein thought to bind and organize specialized membrane cytoskeleton compartments. This association with p11 requires the integrity of the last three C-terminal amino acids, Ser-Ser-Val, in TASK-1. Using series of C-terminal TASK-1 deletion mutants and several TASK-1-GFP chimeras, we demonstrate that association with p11 is essential for trafficking of TASK-1 to the plasma membrane. p11 association with the TASK-1 channel masks an endoplasmic reticulum retention signal identified as Lys-Arg-Arg that precedes the Ser-Ser-Val sequence.     

Site-specific mutations in a minimal voltage-dependent K+ channel alter ion selectivity and open-channel block

MinK is a small membrane protein of 130 amino acids with a single potential membrane-spanning alpha-helical domain. Its expression in Xenopus oocytes induces voltage-dependent, K(+)-selective channels. Using site-directed mutagenesis of a synthetic gene, we have identified residues in the hydrophobic region of minK that influence both ion selectivity and open-channel block. Single amino acid changes increase the channel's relative permeability for NH4+ and Cs+ without affecting its ability to exclude Na+ and Li+. Blockade by two common K+ channel pore blockers, tetraethylammonium and Cs+, was also modified. These results suggest that an ion selectivity region and binding sites for the pore blockers within the conduction pathway have been modified. We conclude that the gene encoding minK is a structural gene for a K+ channel protein.     

Topology of the P segments in the sodium channel pore revealed by cysteine mutagenesis.

The P segments of the voltage-dependent Na+ channel line the outer mouth and selectivity filter of the pore. The residues that form the cytoplasmic mouth of the pore of the channel have not been identified. To study the structure of the inner pore mouth, the presumed selectivity filter residues (D400, E755, K1237, and A1529), and three amino acids just amino-terminal to each of these residues in the rat skeletal muscle Na+ channel, were mutated to cysteine and expressed in tsA 201 cells. These amino acids are predicted (by analogy to K+ channels) to be on the cytoplasmic side of the putative selectivity filter residues. Inward and outward Na+ currents were measured with the whole-cell configuration of the patch-clamp technique. Cysteinyl side-chain accessibility was gauged by sensitivity to Cd2+ block and by reactivity with methanethiosulfonate (MTS) reagents applied to both the inside and the outside of the cell. Outward currents through the wild-type and all of the mutant channels were unaffected by internal Cd2+ (100 microM). Similarly, 1 mM methanethiosulfonate ethylammonium (MTSEA) applied to the inside of the membrane did not affect wild-type or mutant outward currents. However, two mutants amino-terminal to the selectivity position in domain III (F1236C and T1235C) and one in domain IV (S1528C) were blocked with high affinity by external Cd2+. The Na+ current through F1236C and S1528C channels was inhibited by MTSEA applied to the outside of the cell. The accessibility of these mutants to externally applied cysteinyl ligands indicates that the side chains of the mutated residues face outward rather than inward. The K+ channel model of the P segments as protein loops that span the selectivity region is not applicable to the Na+ channel.     

Role of microtubules in polarized delivery of apical membrane proteins to the brush border of the intestinal epithelium

Colchicine- and vinblastine-induced depolymerization of microtubules (MTs) in the intestinal epithelium of rats and mice resulted in significant delivery of three apical membrane proteins (alkaline phosphatase, sucrase-isomaltase, and aminopeptidase N) to the basolateral membrane domain. In addition, typical brush borders (BBs) occurred at the basolateral cell surface, consisting of numerous microvilli that contained the four major components of the cytoskeleton of apical microvilli (actin, villin, fimbrin, and the 110-kD protein). Formation of basolateral microvilli required polymerization of actin and proceeded at glycocalyx-studded plaques that resembled the dense plaques located at the tips of apical microvilli. BBs from the basolateral membrane became internalized into BB-containing vacuoles which served as recipient organelles for newly synthesized apical membrane proteins. The BB vacuoles fused with each other and finally were inserted into the apical BB. Polarized distribution of Na+,K+- ATPase, a basolateral membrane protein, was not affected by drug- induced depolymerization of MTs. These observations indicate that Golgi- derived carrier vesicles (CVs) containing apical membrane proteins are vectorially guided to the apical cell surface by a retrograde transport along MTs. MTs are uniformly oriented towards a narrow space underneath the apical terminal web (termed subterminal space) that contains MT- organizing properties and controls polarized alignment of MTs. In contrast to apical CVs, targeting of basolateral CVs appears to be independent of MTs but demands a barrier at the apical membrane domain that prevents basolateral CVs from apical fusion (transport barrier hypothesis).     

A novel two-pore domain K+ channel,TRESK, is localized in the spinal cord

To find a novel human ion channel gene we have executed an extensive search by using a human genome draft sequencing data base. Here we report a novel two-pore domain K+ channel, TRESK (TWIK-related spinal cord K+ channel). TRESK is coded by 385 amino acids and shows low homology (19%) with previously characterized two-pore domain K+ channels. However, the most similar channel is TREK-2 (two-pore domain K+ channel), and TRESK also has two pore-forming domains and four transmembrane domains that are evolutionarily conserved in the two-pore domain K+ channel family. Moreover, we confirmed that TRESK is expressed in the spinal cord. Electrophysiological analysis demonstrated that TRESK induced outward rectification and functioned as a background K+ channel. Pharmacological analysis showed TRESK to be inhibited by previously reported K+ channel inhibitors Ba2+, propafenone, glyburide, lidocaine, quinine, quinidine, and triethanolamine. Functional analysis demonstrated TRESK to be inhibited by unsaturated free fatty acids such as arachidonic acid and docosahexaenoic acid. TRESK is also sensitive to extreme changes in extracellular and intracellular pH. These results indicate that TRESK is a novel two-pore domain K+ channel that may set the resting membrane potential of cells in the spinal cord.     

Src Family Protein Tyrosine Kinase Regulates the Basolateral K Channel in the Distal Convoluted Tubule (DCT) by Phosphorylation of KCNJ10 Protein

The loss of function of the basolateral K channels in the distal nephron causes electrolyte imbalance. The aim of this study is to examine the role of Src family protein tyrosine kinase (SFK) in regulating K channels in the basolateral membrane of the mouse initial distal convoluted tubule (DCT1). Single-channel recordings confirmed that the 40-picosiemen (pS) K channel was the only type of K channel in the basolateral membrane of DCT1. The suppression of SFK reversibly inhibited the basolateral 40-pS K channel activity in cell-attached patches and decreased the Ba²⁺-sensitive whole-cell K currents in DCT1. Inhibition of SFK also shifted the K reversal potential from −65 to −43 mV, suggesting a role of SFK in determining the membrane potential in DCT1. Western blot analysis showed that KCNJ10 (Kir4.1), a key component of the basolateral 40-pS K channel in DCT1, was a tyrosine-phosphorylated protein. LC/MS analysis further confirmed that SFK phosphorylated KCNJ10 at Tyr⁸ and Tyr⁹. The single-channel recording detected the activity of a 19-pS K channel in KCNJ10-transfected HEK293T cells and a 40-pS K channel in the cells transfected with KCNJ10+KCNJ16 (Kir.5.1) that form a heterotetramer in the basolateral membrane of the DCT. Mutation of Tyr⁹ did not alter the channel conductance of the homotetramer and heterotetramer. However, it decreased the whole-cell K currents, the probability of finding K channels, and surface expression of KCNJ10 in comparison to WT KCNJ10. We conclude that SFK stimulates the basolateral K channel activity in DCT1, at least partially, by phosphorylating Tyr⁹ on KCNJ10. We speculate that the modulation of tyrosine phosphorylation of KCNJ10 should play a role in regulating membrane transport function in DCT1.     

Prolactin, an activator of epithelial Na+ channel, inhibits basolateral K+ channels in adult tree frog skin

Adult amphibian skin actively transports Na+ from its apical to basolateral side while in turn, K+ is recycled through Na+, K+-ATPase and K+ channels located in the basolateral membrane. We previously found that PRL stimulates Na+ transport in the skin of the adult tree frog (Hyla arborea japonica) via an increase in the open-channel density of the epithelial Na+ channel (ENaC). If PRL also activates basolateral K+ channels, this activation would help to stimulate Na+ transport, too. Whether PRL does indeed stimulate basolateral K+ channels in the adult tree frog was examined by measuring the short-circuit current across nystatin-treated skin. Both tolbutamide, a K(ATP) channel blocker, and tetrapentylammonium (TPA), a KCa channel blocker, blocked the current, the effect of TPA being more powerful than that of tolbutamide. Contrary to expectation, PRL inhibited the basolateral K+ channels in this skin. In the presence of basolateral amiloride, PRL still inhibited the basolateral K+ current, suggesting that the (Na+)-H+ exchanger located in the basolateral membrane does not mediate the inhibitory effect of PRL on the basolateral K+ channels in Hyla.     

Phosphatidylinositol 3-phosphate indirectly activates KCa3.1 via 14 amino acids in the carboxy terminus of KCa3.1

KCa3.1 is an intermediate conductance Ca2+-activated K+ channel that is expressed predominantly in hematopoietic cells, smooth muscle cells, and epithelia where it functions to regulate membrane potential, Ca2+ influx, cell volume, and chloride secretion. We recently found that the KCa3.1 channel also specifically requires phosphatidylinositol-3 phosphate [PI(3)P] for channel activity and is inhibited by myotubularin-related protein 6 (MTMR6), a PI(3)P phosphatase. We now show that PI(3)P indirectly activates KCa3.1. Unlike KCa3.1 channels, the related KCa2.1, KCa2.2, or KCa2.3 channels do not require PI(3)P for activity, suggesting that the KCa3.1 channel has evolved a unique means of regulation that is critical for its biological function. By making chimeric channels between KCa3.1 and KCa2.3, we identified a stretch of 14 amino acids in the carboxy-terminal calmodulin binding domain of KCa3.1 that is sufficient to confer regulation of KCa2.3 by PI(3)P. However, mutation of a single potential phosphorylation site in these 14 amino acids did not affect channel activity. These data together suggest that PI(3)P and these 14 amino acids regulate KCa3.1 channel activity by recruiting an as yet to be defined regulatory subunit that is required for Ca2+ gating of KCa3.1.     

Characterization and cellular localization of the epithelial Na+ channel. Studies using an anti-Na+ channel antibody raised by an antiidiotypic route.

Amiloride-sensitive Na+ channels are expressed at the apical membrane of high resistance, Na+-transporting epithelial. The specific interaction of amiloride with this transport protein suggested the feasibility of raising anti-Na+ channel antibodies by an antiidiotypic approach designed to generate antibodies directed against the amiloride-binding domain on the channel. Antiidiotypic monoclonal antibody RA6.3 mimicked the effect of amiloride by inhibiting Na+ transport across A6 cell monolayers when applied to the apical cell surface. Inhibition of transport required pretreatment of the apical cell surface with trypsin in the presence of amiloride in order to enhance accessibility of the antibody to the amiloride-binding site. This antibody specifically immunoprecipitated a large 750,000-700,000 Da protein from [35S]methionine-labeled A6 cell cultures, which was resolved further under reducing conditions as a set of polypeptides with apparent molecular masses of 260,000-230,000, 180,000, 140,000-110,000, and 70,000 Da. The antibody recognized the 140,000-Da subunit, known to contain the amiloride-binding domain, on immunoblots of purified A6 cell Na+ channel. Immunoprecipitation of apical or basolateral plasma membrane proteins selectively labeled with 125I demonstrated that expression of the oligomeric Na+ channel was restricted to the apical plasma membrane. Immunocytochemical localization in A6 cultures revealed apical membrane as well as cytosolic immunoreactive sites. Immunostaining was also observed at or near the basolateral plasma membrane.     

A PDZ-containing scaffold related to the dystrophin complex at the basolateral membrane of epithelial cells

Membrane scaffolding complexes are key features of many cell types, serving as specialized links between the extracellular matrix and the actin cytoskeleton. An important scaffold in skeletal muscle is the dystrophin-associated protein complex. One of the proteins bound directly to dystrophin is syntrophin, a modular protein comprised entirely of interaction motifs, including PDZ (protein domain named for PSD-95, discs large, ZO-1) and pleckstrin homology (PH) domains. In skeletal muscle, the syntrophin PDZ domain recruits sodium channels and signaling molecules, such as neuronal nitric oxide synthase, to the dystrophin complex. In epithelia, we identified a variation of the dystrophin complex, in which syntrophin, and the dystrophin homologues, utrophin and dystrobrevin, are restricted to the basolateral membrane. We used exogenously expressed green fluorescent protein (GFP)-tagged fusion proteins to determine which domains of syntrophin are responsible for its polarized localization. GFP-tagged full-length syntrophin targeted to the basolateral membrane, but individual domains remained in the cytoplasm. In contrast, the second PH domain tandemly linked to a highly conserved, COOH-terminal region was sufficient for basolateral membrane targeting and association with utrophin. The results suggest an interaction between syntrophin and utrophin that leaves the PDZ domain of syntrophin available to recruit additional proteins to the epithelial basolateral membrane. The assembly of multiprotein signaling complexes at sites of membrane specialization may be a widespread function of dystrophin-related protein complexes.     

Interaction of integrin-linked kinase with the kidney chloride/bicarbonate exchanger, kAE1

Kidney anion exchanger 1 (kAE1) mediates chloride/bicarbonate exchange at the basolateral membrane of kidney alpha-intercalated cells, thereby facilitating bicarbonate reabsorption into the blood. Human kAE1 lacks the N-terminal 65 residues of the erythroid form (AE1, band 3), which are essential for binding of cytoskeletal and cytosolic proteins. Yeast two-hybrid screening identified integrin-linked kinase (ILK), a serine/threonine kinase, and an actin-binding protein as an interacting partner with the N-terminal domain of kAE1. Interaction between kAE1 and ILK was confirmed in co-expression experiments in HEK 293 cells and is mediated by a previously unidentified calponin homology domain in the kAE1 N-terminal region. The calponin homology domain of kAE1 binds the C-terminal catalytic domain of ILK to enhance association of kAE1 with the actin cytoskeleton. Overexpression of ILK increased kAE1 levels at the cell surface as shown by flow cytometry, cell surface biotinylation, and anion transport activity assays. Pulse-chase experiments revealed that ILK associates with kAE1 early in biosynthesis, likely in the endoplasmic reticulum. ILK co-localized with kAE1 at the basolateral membrane of polarized Madin-Darby canine kidney cells and in alpha-intercalated cells of human kidneys. Taken together these results suggest that ILK and kAE1 traffic together from the endoplasmic reticulum to the basolateral membrane. ILK may provide a linkage between kAE1 and the underlying actin cytoskeleton to stabilize kAE1 at the basolateral membrane, resulting in higher levels of cell surface expression.     

Regulation of Na,K-ATPase by PLMS,the phospholemman-like protein from shark: molecular cloning,sequence, expression,cellular distribution,and functional effects of PLMS

In Na,K-ATPase membrane preparations from shark rectal glands, we have previously identified an FXYD domain-containing protein, phospholemman-like protein from shark, PLMS. This protein was shown to associate and modulate shark Na,K-ATPase activity in vitro. Here we describe the complete coding sequence, expression, and cellular localization of PLMS in the rectal gland of the shark Squalus acanthias. The mature protein contained 74 amino acids, including the N-terminal FXYD motif and a C-terminal protein kinase multisite phosphorylation motif. The sequence is preceded by a 20 amino acid candidate cleavable signal sequence. Immunogold labeling of the Na,K-ATPase alpha-subunit and PLMS showed the presence of alpha and PLMS in the basolateral membranes of the rectal gland cells and suggested their partial colocalization. Furthermore, through controlled proteolysis, the C terminus of PLMS containing the protein kinase phosphorylation domain can be specifically cleaved. Removal of this domain resulted in stimulation of maximal Na,K-ATPase activity, as well as several partial reactions. Both the E1 approximately P --> E2-P reaction, which is partially rate-limiting in shark, and the K+ deocclusion reaction, E2(K) --> E1, are accelerated. The latter may explain the finding that the apparent Na+ affinity was increased by the specific C-terminal PLMS truncation. Thus, these data are consistent with a model where interaction of the phosphorylation domain of PLMS with the Na,K-ATPase alpha-subunit is important for the modulation of shark Na,K-ATPase activity.     

Kidney Na+,K(+)-ATPase is associated with moesin

Na+,K(+)-ATPase is a ubiquitous plasmalemmal membrane protein essential for generation and maintenance of transmembrane Na+ and K+ gradients in virtually all animal cell types. Activity and polarized distribution of renal Na+,(+)-ATPase appears to depend on connection of ankyrin to the spectrin-based membrane cytoskeleton as well as on association with actin filaments. In a previous study we showed copurification and codistribution of renal Na+,K(+)-ATPase not only with ankyrin, spectrin and actin, but also with two further peripheral membrane proteins, pasin 1 and pasin 2. In this paper we show by sequence analysis through mass spectrometry as well as by immunoblotting that pasin 2 is identical to moesin, a member of the FERM (protein 4.1, ezrin, radixin, moesin) protein family, all members of which have been shown to serve as cytoskeletal adaptor molecules. Moreover, we show that recombinant full-length moesin as well as its FERM domain bind to Na+,K(+)-ATPase and that this binding can be inhibited by an antibody specific for the ATPase activity-containing cytoplasmic loop (domain 3) of the Na+,K(+)-ATPase alpha-subunit. This loop has been previously shown to be a site essential for ankyrin binding. These observations indicate that moesin might not only serve as direct linker molecule of Na+,K(+)-ATPase to actin filaments but also modify ankyrin binding at domain 3 of Na+,K(+)-ATPase in a way similar to protein 4.1 modifying the binding of ankyrin to the cytoplasmic domain of the erythrocyte anion exchanger (AE1).     

The lymphocyte-specific protein LSP1 binds to F-actin and to the cytoskeleton through its COOH-terminal basic domain

The lymphocyte-specific phosphoprotein LSP1 associates with the cytoplasmic face of the plasma membrane and with the cytoskeleton. Mouse LSP1 protein contains 330 amino acids and contains an NH2-terminal acidic domain of approximately 177 amino acids. The COOH-terminal half of the LSP1 protein is rich in basic residues. In this paper we show that LSP1 protein which is immunoprecipitated with anti-LSP1 antibodies from NP-40-soluble lysates of the mouse B-lymphoma cell line BAL17 is associated with actin. In vitro binding experiments using recombinant LSP1 (rLSP1) protein and rabbit skeletal muscle actin show that LSP1 binds along the sides of F-actin but does not bind to G-actin. rLSP1 does not alter the initial polymerization kinetics of actin. The highly conserved COOH-terminal basic domains of mouse and human LSP1 share a significant homology with the 20-kD COOH-terminal F-actin binding fragment of caldesmon. A truncated rLSP1 protein containing the entire COOH-terminal basic domain from residue 179 to 330, but not the NH2-terminal acidic domain binds to F-actin at least as well as rLSP1. When LSP1/CAT fusion proteins are expressed in a LSP1-negative T-lymphoma cell line, only fusion proteins containing the basic COOH-terminal domain associate with the NP-40-insoluble cytoskeleton. These data show that LSP1 binds F-actin through its COOH-terminal basic domain and strongly suggest that LSP1 interacts with the cytoskeleton by direct binding to F-actin. We propose that LSP1 plays a role in mediating cytoskeleton driven responses in lymphocytes such as receptor capping, cell motility, or cell-cell interactions.     

Association of kidney and parotid Na+, K(+)-ATPase microsomes with actin and analogs of spectrin and ankyrin

Kidney Na+,K(+)-ATPase has been recently shown to bind erythroid ankyrin and to colocalize with ankyrin at the basolateral cell surface of kidney epithelial cells. These observations suggest that Na+,K(+)-ATPase is linked via ankyrin to the spectrin/actin-based membrane cytoskeleton. In the present study we show that Na+,K(+)-ATPase and analogs of spectrin, ankyrin and actin copurify from detergent extracts of pig kidney and parotid gland membranes. Actin, spectrin and ankyrin were extracted from purified Na+,K(+)-ATPase microsomes at virtually identical conditions as their counterparts from the erythrocyte membrane, i.e., 1 mM EDTA (spectrin, actin) and 1 M KCl (ankyrin). Visualization of the stripped proteins by rotary shadowing revealed numerous elongated spectrin-like dimers (100 nm) and tetramers (215 nm), a fraction of which (17%) was associated with globular (10 nm) ankyrin-like particles. Like erythrocyte ankyrin, kidney ankyrin was cleaved into a soluble 72 kDa fragment and a membrane-bound 90 kDa fragment. Consistent with our previous immunocytochemical findings on the pig kidney, Na+,K(+)-ATPase and ankyrin were found to be colocalized at the basolateral plasma membrane of striated ducts and acini of the pig parotid gland. The present findings confirm and extend the recently proposed concept that in polarized epithelial cells Na+,K(+)-ATPase may serve as major attachment site for the spectrin-based membrane cytoskeleton to the basolateral cell domain. Connections of integral membrane proteins to the cytoskeleton may help to place these proteins at specialized domains of the cell surface and to prevent them from endocytosis.     

Odd behaviour of Li+ in frog skin ion transport

1. Frog skin epithelium has basolateral K+ channels that normally define the basolateral membrane potential between 80 and 100 mV. 2. The membrane mentioned also has almost silent chloride channels and a [Na+, K+, 2Cl-] cotransport, the latter probably maintains the high Cl- in the capital (also called syncytium) cells. 3. If the K+ channels are blocked by Ba2+ (or Li+) it is possible to demonstrate potential gating of the chloride channels of the basolateral membrane. 4. When the normal K+ channels are blocked, a potential-dependent K+ conductance slowly emerges. 5. If Li+ is substituted for outside Na+ the skin shows potential oscillations of about 40 mV at a frequency of about six per hour. 6. The anion channel inhibitor Indacrinone stops these oscillations. 7. The role of Cl- and K+ channels in these oscillations is discussed. 8. The transepithelial inward transport of Li+ requires the presence of Na+ and seems to be due to exchange of cellular Li+ against inside Na+ via the basolateral Na+/H+ exchanger.     

Rapid responses to aldosterone in human distal colon.

Aldosterone at normal physiological levels induces rapid increases in intracellular calcium and pH in human distal colon. The end target of these rapid signaling responses are basolateral K+ channels. Using spectrofluorescence microscopy and Ussing chamber techniques, we have shown that aldosterone activates basolateral Na/H exchange via a protein kinase C and calcium-dependent signaling pathway. The resultant intracellular alkalinization up-regulates an adenosine triphosphate (ATP)-dependent K+ channel (K(ATP)) and inhibits a Ca2+ -dependent K+ channel (K(Ca)). In Ussing chamber experiments, we have shown that the K(ATP) channel is required to drive sodium absorption, whereas the K(Ca) channel is necessary for both cyclic adenosine monophosphate and calcium-dependent chloride secretion. The rapid effects of aldosterone on intracellular calcium, pH, protein kinase C and K(ATP), K(Ca) channels are insensitive to cycloheximide, actinomycin D, and spironalactone, indicating a nongenomic mechanism of action. We propose that the physiological role for the rapid nongenomic effect of aldosterone is to prime pluripotential epithelia for absorption by simultaneously up-regulating K(ATP) channels to drive absorption through surface cells and down-regulating the secretory capacity by inhibiting K(Ca) channels involved in secretion through crypt cells.     

Calmodulin regulates assembly and trafficking of SK4/IK1 Ca2+-activated K+ channels

Calmodulin (CaM) regulates gating of several types of ion channels but has not been implicated in channel assembly or trafficking. For the SK4/IK1 K+ channel, CaM bound to the proximal C terminus ("Ct1 " domain) acts as the Ca2+ sensor. We now show that CaM interacting with the C terminus of SK4 also controls channel assembly and surface expression. In transfected cells, removing free CaM by overexpressing the CaM-binding domain, Ct1, redistributed full-length SK4 protein from the plasma membrane to the cytoplasm and decreased whole-cell currents. Making more CaM protein available by overexpressing the CaM gene abrogated the dominant-negative effect of Ct1 and restored both surface expression of SK4 protein and whole-cell currents. The distal C-terminal domain ("Ct2") also plays a role in assembly, but is not CaM-dependent. Co-immunoprecipitation experiments demonstrated that multimerization of SK4 subunits was enhanced by CaM and inhibited by removal of CaM, indicating that CaM regulates trafficking of SK4 by affecting the assembly of channels. Our results support a model in which CaM-dependent association of SK4 monomers at their Ct1 domains regulates channel assembly and surface expression. This appears to represent a novel mechanism for controlling ion channels, and consequently, the cellular functions that depend on them.