Clathrin-mediated endocytosis of Na+,K+-ATPase in response to parathyroid hormone requires ERK-dependent phosphorylation of Ser-11 within the alpha1-subunit

Parathyroid hormone (PTH) inhibits Na(+),K(+)-ATPase activity through protein kinase C- (PKC) and extracellular signal-regulated kinase- (ERK) dependent pathways and increases serine phosphorylation of the alpha(1)-subunit. To determine whether specific serine phosphorylation sites within the Na(+),K(+)-ATPase alpha(1)-subunit are involved in the Na(+),K(+)-ATPase responses to PTH, we examined the effect of PTH in opossum kidney cells stably transfected with wild type rat Na(+),K(+)-ATPase alpha(1)-subunit (WT), serine 11 to alanine mutant alpha(1)-subunit (S11A), or serine 18 to alanine mutant alpha(1)-subunit (S18A). PTH increased phosphorylation and endocytosis of the Na(+),K(+)-ATPase alpha(1)-subunit into clathrin-coated vesicles in cells transfected with WT and S18A rat Na(+),K(+)-ATPase alpha(1)-subunits. PTH did not increase the level of phosphorylation or stimulate translocation of Na(+),K(+)-ATPase alpha(1)-subunits into clathrin-coated vesicles in cells transfected with the S11A mutant. PTH inhibited ouabain-sensitive (86)Rb uptake and Na(+),K(+)-ATPase activity (ouabain-sensitive ATP hydrolysis) in WT- and S18A-transfected opossum kidney cells but not in S11A-transfected cells. Pretreatment of the cells with the PKC inhibitors and ERK inhibitor blocked PTH inhibition of (86)Rb uptake, Na(+),K(+)-ATPase activity, alpha(1)-subunit phosphorylation, and endocytosis in WT and S18A cells. Consistent with the notion that ERK phosphorylates Na(+),K(+)-ATPase alpha(1)-subunit, ERK was shown to be capable of causing phosphorylation of Na(+),K(+)-ATPase alpha(1)-subunit immunoprecipitated from WT and S18A but not from S11A-transfected cells. These results suggest that PTH regulates Na(+),K(+)-ATPase by PKC and ERK-dependent alpha(1)-subunit phosphorylation and that the phosphorylation requires the expression of a serine at the 11 position of the Na(+),K(+)-ATPase alpha(1)-subunit.     

Relevance of dopamine signals anchoring dynamin-2 to the plasma membrane during Na+,K+-ATPase endocytosis

Clathrin-dependent endocytosis of Na(+),K(+)-ATPase in response to dopamine regulates its catalytic activity in intact cells. Because fission of clathrin-coated pits requires dynamin, we examined the mechanisms by which dopamine receptor signals promote dynamin-2 recruitment and assembly at the site of Na(+),K(+)-ATPase endocytosis. Western blotting revealed that dopamine increased the association of dynamin-2 with the plasma membrane and with phosphatidylinositol 3-kinase. Dopamine inhibited Na(+),K(+)-ATPase activity in OK cells and in those overexpressing wild type dynamin-2 but not in cells expressing a dominant-negative mutant. Dephosphorylation of dynamin is important for its assembly. Dopamine increased protein phosphatase 2A activity and dephosphorylated dynamin-2. In cells expressing a dominant-negative mutant of protein phosphatase 2A, dopamine failed to dephosphorylate dynamin-2 and to reduce Na(+),K(+)-ATPase activity. Dynamin-2 is phosphorylated at Ser(848), and expression of the S848A mutant significantly blocked the inhibitory effect of dopamine. These results demonstrate a distinct signaling network originating from the dopamine receptor that regulates the state of dynamin-2 phosphorylation and that promotes its location (by interaction with phosphatidylinositol 3-kinase) at the site of Na(+),K(+)-ATPase endocytosis.     

Upregulation of apical NHE3 in renal OK cells overexpressing the rodent alpha(1)-subunit of the Na(+) pump

Vectorial Na(+) reabsorption across the proximal tubule is mediated by apical entry of Na(+), primarily via Na(+)/H(+) exchanger isoform 3 (NHE3), and basolateral extrusion via the Na(+) pump (Na(+)-K(+)-ATPase). We hypothesized that regulation of Na(+) reabsorption should involve not only the activity of the basolateral Na(+)-K(+)-ATPase, but also the apical NHE3, in a concerted manner. To generate a cell line that overexpresses Na(+)-K(+)-ATPase, opossum kidney (OK) cells were transfected with the rodent Na(+)-K(+)-ATPase alpha(1)-subunit (pCMV ouabain vector), and native cells were used as a control. The existence of distinct functional classes of Na(+)-K(+)-ATPase in wild-type and transfected cells was confirmed by the inhibition profile of Na(+)-K(+)-ATPase activity by ouabain. In contrast to wild-type cells, transfected cells exhibited two IC(50) values for ouabain: the first value was similar to the IC(50) of control cells, and the second value was 2 log units greater than the first, consistent with the presence of rat and opossum alpha(1)-isozymes. It is shown that transfection of OK cells with Na(+)-K(+)-ATPase increased Na(+)-K(+)-ATPase and NHE3 activities. This was associated with overexpression of the Na(+)-K(+)-ATPase alpha(1)-subunit and NHE3 in transfected OK cells. The abundance of the Na(+)-K(+)-ATPase beta(1)-subunit was slightly lower in transfected OK cells. In conclusion, the increase in expression and function of Na(+)-K(+)-ATPase in cells transfected with the rodent Na(+) pump alpha(1)-subunit cDNA is expected to stimulate apical Na(+) influx into the cells, thereby accounting for the observed stimulation of the apical NHE3 activity.     

Dopamine regulation of Na+-K+-ATPase requires the PDZ-2 domain of sodium hydrogen regulatory factor-1 (NHERF-1) in opossum kidney cells

Na(+)-K(+)-ATPase activity in renal proximal tubule is regulated by several hormones including parathyroid hormone (PTH) and dopamine. The current experiments explore the role of Na(+)/H(+) exchanger regulatory factor 1 (NHERF-1) in dopamine-mediated regulation of Na(+)-K(+)-ATPase. We measured dopamine regulation of ouabain-sensitive (86)Rb uptake and Na(+)-K(+)-ATPase α1 subunit phosphorylation in wild-type opossum kidney (OK) (OK-WT) cells, OKH cells (NHERF-1-deficient), and OKH cells stably transfected with full-length human NHERF-1 (NF) or NHERF-1 constructs with mutated PDZ-1 (Z1) or PDZ-2 (Z2) domains. Treatment with 1 μM dopamine decreased ouabain-sensitive (86)Rb uptake, increased phosphorylation of Na(+)-K(+)-ATPase α1-subunit, and enhanced association of NHERF-1 with D1 receptor in OK-WT cells but not in OKH cells. Transfection with wild-type, full-length, or PDZ-1 domain-mutated NHERF-1 into OKH cells restored dopamine-mediated regulation of Na(+)-K(+)-ATPase and D1-like receptor association with NHERF-1. Dopamine did not regulate Na(+)-K(+)-ATPase or increase D1-like receptor association with NHERF-1 in OKH cells transfected with mutated PDZ-2 domain. Dopamine stimulated association of PKC-ζ with NHERF-1 in OK-WT and OKH cells transfected with full-length or PDZ-1 domain-mutated NHERF-1 but not in PDZ-2 domain-mutated NHERF-1-transfected OKH cells. These results suggest that NHERF-1 mediates Na(+)-K(+)-ATPase regulation by dopamine through its PDZ-2 domain.     

Activation of AMP-activated protein kinase stimulates Na+,K+-ATPase activity in skeletal muscle cells

Contraction stimulates Na(+),K(+)-ATPase and AMP-activated protein kinase (AMPK) activity in skeletal muscle. Whether AMPK activation affects Na(+),K(+)-ATPase activity in skeletal muscle remains to be determined. Short term stimulation of rat L6 myotubes with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), activates AMPK and promotes translocation of the Na(+),K(+)-ATPase α(1)-subunit to the plasma membrane and increases Na(+),K(+)-ATPase activity as assessed by ouabain-sensitive (86)Rb(+) uptake. Cyanide-induced artificial anoxia, as well as a direct AMPK activator (A-769662) also increase AMPK phosphorylation and Na(+),K(+)-ATPase activity. Thus, different stimuli that target AMPK concomitantly increase Na(+),K(+)-ATPase activity. The effect of AICAR on Na(+),K(+)-ATPase in L6 myotubes was attenuated by Compound C, an AMPK inhibitor, as well as siRNA-mediated AMPK silencing. The effects of AICAR on Na(+),K(+)-ATPase were completely abolished in cultured primary mouse muscle cells lacking AMPK α-subunits. AMPK stimulation leads to Na(+),K(+)-ATPase α(1)-subunit dephosphorylation at Ser(18), which may prevent endocytosis of the sodium pump. AICAR stimulation leads to methylation and dephosphorylation of the catalytic subunit of the protein phosphatase (PP) 2A in L6 myotubes. Moreover, AICAR-triggered dephosphorylation of the Na(+),K(+)-ATPase was prevented in L6 myotubes deficient in PP2A-specific protein phosphatase methylesterase-1 (PME-1), indicating a role for the PP2A·PME-1 complex in AMPK-mediated regulation of Na(+),K(+)-ATPase. Thus contrary to the common paradigm, we report AMPK-dependent activation of an energy-consuming ion pumping process. This activation may be a potential mechanism by which exercise and metabolic stress activate the sodium pump in skeletal muscle.     

AS160 associates with the Na+,K+-ATPase and mediates the adenosine monophosphate-stimulated protein kinase-dependent regulation of sodium pump surface expression

The Na(+),K(+)-ATPase is the major active transport protein found in the plasma membranes of most epithelial cell types. The regulation of Na(+),K(+)-ATPase activity involves a variety of mechanisms, including regulated endocytosis and recycling. Our efforts to identify novel Na(+),K(+)-ATPase binding partners revealed a direct association between the Na(+),K(+)-ATPase and AS160, a Rab-GTPase-activating protein. In COS cells, coexpression of AS160 and Na(+),K(+)-ATPase led to the intracellular retention of the sodium pump. We find that AS160 interacts with the large cytoplasmic NP domain of the α-subunit of the Na(+),K(+)-ATPase. Inhibition of the activity of the adenosine monophosphate-stimulated protein kinase (AMPK) in Madin-Darby canine kidney cells through treatment with Compound C induces Na(+),K(+)-ATPase endocytosis. This effect of Compound C is prevented through the short hairpin RNA-mediated knockdown of AS160, demonstrating that AMPK and AS160 participate in a common pathway to modulate the cell surface expression of the Na(+),K(+)-ATPase.     

Simultaneous phosphorylation of Ser11 and Ser18 in the alpha-subunit promotes the recruitment of Na(+),K(+)-ATPase molecules to the plasma membrane

Renal sodium homeostasis is a major determinant of blood pressure and is regulated by several natriuretic and antinatriuretic hormones. These hormones, acting through intracellular second messengers, either activate or inhibit proximal tubule Na(+),K(+)-ATPase. We have shown previously that phorbol ester (PMA) stimulation of endogenous PKC leads to activation of Na(+),K(+)-ATPase in cultured proximal tubule cells (OK cells) expressing the rodent Na(+), K(+)-ATPase alpha-subunit. We have now demonstrated that the treatment with PMA leads to an increased amount of Na(+),K(+)-ATPase molecules in the plasmalemma, which is proportional to the increased enzyme activity. Colchicine, dinitrophenol, and potassium cyanide prevented the PMA-dependent stimulation of activity without affecting the increased level of phosphorylation of the Na(+), K(+)-ATPase alpha-subunit. This suggests that phosphorylation does not directly stimulate Na(+),K(+)-ATPase activity; instead, phosphorylation may be the triggering mechanism for recruitment of Na(+),K(+)-ATPase molecules to the plasma membrane. Transfected cells expressing either an S11A or S18A mutant had the same basal Na(+),K(+)-ATPase activity as cells expressing the wild-type rodent alpha-subunit, but PMA stimulation of Na(+),K(+)-ATPase activity was completely abolished in either mutant. PMA treatment led to phosphorylation of the alpha-subunit by stimulation of PKC-beta, and the extent of this phosphorylation was greatly reduced in the S11A and S18A mutants. These results indicate that both Ser11 and Ser18 of the alpha-subunit are essential for PMA stimulation of Na(+), K(+)-ATPase activity, and that these amino acids are phosphorylated during this process. The results presented here support the hypothesis that PMA regulation of Na(+),K(+)-ATPase is the result of an increased number of Na(+),K(+)-ATPase molecules in the plasma membrane.     

Long-term regulation of Na+,K+-ATPase in opossum kidney cells by ouabain

Na(+),K(+)-ATPase, a basolateral transporter responsible for tubular reabsorption of Na(+) and for providing the driving force for vectorial transport of various solutes and ions, can also act as a signal transducer in response to the interaction with steroid hormones. At nanomolar concentrations ouabain binding to Na(+),K(+)-ATPase activates a signaling cascade that ultimately regulates several membrane transporters including Na(+),K(+)-ATPase. The present study evaluated the long-term effect of ouabain on Na(+),K(+)-ATPase activity (Na(+) transepithelial flux) and expression in opossum kidney (OK) cells with low (40) and high (80) number of passages in culture, which are known to overexpress Na(+),K(+)-ATPase (Silva et al., 2006, J Membr Biol 212, 163-175). Activation of a signal cascade was evaluated by quantification of ERK1/2 phosphorylation by Western blot. Na(+),K(+)-ATPase activity was determined by electrophysiological techniques and expression by Western blot. Incubation of cells with ouabain induced activation of ERK1/2. Long-term incubation with ouabain induced an increase in Na(+) transepithelial flux and Na(+),K(+)-ATPase expression only in OK cells with 80 passages in culture. This increase was prevented by incubation with inhibitors of MEK1/2 and PI-3K. In conclusion, ouabain-activated signaling cascade mediated by both MEK1/2 and PI-3K is responsible for long-term regulation of Na(+) transepithelial flux in epithelial renal cells. OK cell line with high number of passages is suggested to constitute a particular useful model for the understanding of ouabain-mediated regulation of Na(+) transport.     

Analysis of Na+,K+-ATPase motion and incorporation into the plasma membrane in response to G protein-coupled receptor signals in living cells

Dopamine (DA) increases Na(+),K(+)-ATPase activity in lung alveolar epithelial cells. This effect is associated with an increase in Na(+),K(+)-ATPase molecules within the plasma membrane (). Analysis of Na(+),K(+)-ATPase motion was performed in real-time in alveolar cells stably expressing Na(+),K(+)-ATPase molecules carrying a fluorescent tag (green fluorescent protein) in the alpha-subunit. The data demonstrate a distinct (random walk) pattern of basal movement of Na(+),K(+)-ATPase-containing vesicles in nontreated cells. DA increased the directional movement (by 3.5 fold) of the vesicles and an increase in their velocity (by 25%) that consequently promoted the incorporation of vesicles into the plasma membrane. The movement of Na(+),K(+)-ATPase-containing vesicles and incorporation into the plasma membrane were microtubule dependent, and disruption of this network perturbed vesicle motion toward the plasma membrane and prevented the increase in the Na(+),K(+)-ATPase activity induced by DA. Thus, recruitment of new Na(+),K(+)-ATPase molecules into the plasma membrane appears to be a major mechanism by which dopamine increases total cell Na(+),K(+)-ATPase activity.     

Na+,K+-ATPase trafficking in skeletal muscle: insulin stimulates translocation of both alpha 1- and alpha 2-subunit isoforms

We determined insulin-stimulated Na(+),K(+)-ATPase isoform-specific translocation to the skeletal muscle plasma membrane. When rat muscle plasma membrane fractions were isolated by discontinuous sucrose gradients, insulin-stimulated translocation of alpha(2)- but not alpha(1)-subunits was detected. However, using cell surface biotinylation techniques, an insulin-induced membrane translocation of both alpha(1) and alpha(2)-subunits in rat epitrochlearis muscle and cultured human skeletal muscle cells was noted. Na(+),K(+)-ATPase alpha-subunit translocation was abolished by the phosphatidylinositol (PI) 3-kinase inhibitor wortmannin, as well as by the protein kinase C inhibitor GF109203X. Thus, insulin mediates Na(+),K(+)-ATPase alpha(1)- and alpha(2)-subunit translocation to the skeletal muscle plasma membrane via a PI 3-kinase-dependent mechanism.     

ERK1/2 mediates insulin stimulation of Na(+),K(+)-ATPase by phosphorylation of the alpha-subunit in human skeletal muscle cells

Insulin stimulates Na(+),K(+)-ATPase activity and induces translocation of Na(+),K(+)-ATPase molecules to the plasma membrane in skeletal muscle. We determined the molecular mechanism by which insulin regulates Na(+),K(+)-ATPase in differentiated primary human skeletal muscle cells (HSMCs). Insulin action on Na(+),K(+)-ATPase was dependent on ERK1/2 in HSMCs. Sequence analysis of Na(+),K(+)-ATPase alpha-subunits revealed several potential ERK phosphorylation sites. Insulin increased ouabain-sensitive (86)Rb(+) uptake and [(3)H]ouabain binding in intact cells. Insulin also increased phosphorylation and plasma membrane content of the Na(+),K(+)-ATPase alpha(1)- and alpha(2)-subunits. Insulin-stimulated Na(+),K(+)-ATPase activation, phosphorylation, and translocation of alpha-subunits to the plasma membrane were abolished by 20 microm PD98059, which is an inhibitor of MEK1/2, an upstream kinase of ERK1/2. Furthermore, inhibitors of phosphatidylinositol 3-kinase (100 nm wortmannin) and protein kinase C (10 microm GF109203X) had similar effects. Notably, insulin-stimulated ERK1/2 phosphorylation was abolished by wortmannin and GF109203X in HSMCs. Insulin also stimulated phosphorylation of alpha(1)- and alpha(2)-subunits on Thr-Pro amino acid motifs, which form specific ERK substrates. Furthermore, recombinant ERK1 and -2 kinases were able to phosphorylate alpha-subunit of purified human Na(+),K(+)-ATPase in vitro. In conclusion, insulin stimulates Na(+),K(+)-ATPase activity and translocation to plasma membrane in HSMCs via phosphorylation of the alpha-subunits by ERK1/2 mitogen-activated protein kinase.     

Arrestins and spinophilin competitively regulate Na+,K+-ATPase trafficking through association with a large cytoplasmic loop of the Na+,K+-ATPase

The activity and trafficking of the Na(+),K(+)-ATPase are regulated by several hormones, including dopamine, vasopressin, and adrenergic hormones through the action of G-protein-coupled receptors (GPCRs). Arrestins, GPCR kinases (GRKs), 14-3-3 proteins, and spinophilin interact with GPCRs and modulate the duration and magnitude of receptor signaling. We have found that arrestin 2 and 3, GRK 2 and 3, 14-3-3 epsilon, and spinophilin directly associate with the Na(+),K(+)-ATPase and that the associations with arrestins, GRKs, or 14-3-3 epsilon are blocked in the presence of spinophilin. In COS cells that overexpressed arrestin, the Na(+),K(+)-ATPase was redistributed to intracellular compartments. This effect was not seen in mock-transfected cells or in cells expressing spinophilin. Furthermore, expression of spinophilin appeared to slow, whereas overexpression of beta-arrestins accelerated internalization of the Na(+),K(+)-ATPase endocytosis. We also find that GRKs phosphorylate the Na(+),K(+)-ATPase in vitro on its large cytoplasmic loop. Taken together, it appears that association with arrestins, GRKs, 14-3-3 epsilon, and spinophilin may be important modulators of Na(+),K(+)-ATPase trafficking.     

The 14-3-3 protein translates the NA+,K+-ATPase {alpha}1-subunit phosphorylation signal into binding and activation of phosphoinositide 3-kinase during endocytosis

Clathrin-dependent endocytosis of Na(+),K(+)-ATPase molecules in response to G protein-coupled receptor signals is triggered by phosphorylation of the alpha-subunit and the binding of phosphoinositide 3-kinase. In this study, we describe a molecular mechanism linking phosphorylation of Na(+),K(+)-ATPase alpha-subunit to binding and activation of phosphoinositide 3-kinase. Co-immunoprecipitation studies, as well as experiments using confocal microscopy, revealed that dopamine favored the association of 14-3-3 protein with the basolateral plasma membrane and its co-localization with the Na(+),K(+)-ATPase alpha-subunit. The functional relevance of this interaction was established in opossum kidney cells expressing a 14-3-3 dominant negative mutant, where dopamine failed to decrease Na(+),K(+)-ATPase activity and to promote its endocytosis. The phosphorylated Ser-18 residue within the alpha-subunit N terminus is critical for 14-3-3 binding. Activation of phosphoinositide 3-kinase by dopamine during Na(+),K(+)-ATPase endocytosis requires the binding of the kinase to a proline-rich domain within the alpha-subunit, and this effect was blocked by the presence of a 14-3-3 dominant negative mutant. Thus, the 14-3-3 protein represents a critical linking mechanism for recruiting phosphoinositide 3-kinase to the site of Na(+),K(+)-ATPase endocytosis.     

The current produced by the E779A mutant rat Na(+)/K(+) pump alpha1-subunit expressed in HEK 293 cells

The current (I(p)) generated by the wild-type or the glutamate (E) 779 alanine (A) mutant of the rat Na(+)/K(+) pump alpha1-subunit expressed in HEK 293 cells was studied at 35 degrees C by means of whole-cell recording in Na(+)-free and Na(+)-containing solution. Glutamate 779 is located in the fifth transmembrane domain of the alpha-subunit of the Na(+)/K(+)-ATPase. Compared with the wild-type, the E779A mutant exhibited an apparent K(+)(o)-affinity decreased by a factor of 3-4 both in Na(+)-free and in Na(+)-containing media. The competition of Na(+)(o) and K(+)(o) for cation binding sites of the pump remained unchanged. Similarly, in Na(+)-free solution the shape of the I(p)-V curves for various external K(+)-concentrations ([K(+)](o)) was essentially the same. However, in Na(+)-containing solutions the shape of I(p)-V curves from cells expressing the mutant of the rat alpha1-subunit clearly differed from the shape observed in cells expressing the wild-type, but voltage dependence of the pump current persisted. A prominent Na(+)(o)-activated, electrogenic Na(+)-transport mediated by the pump, displaying little voltage dependence in the potential range tested (-80 to +60 mV), was present in the cells expressing the E779A mutant pump. The data suggest that exchanging E779 for A in the rat Na(+)/K(+) pump alpha1-subunit causes a modest decrease in the apparent K(+)(o) affinity and a profound, Na(+)(o)-dependent alteration in the electrogenicity of the mutant pump expressed in HEK 293 cells.     

Mutation of aspartate 804 of Na(+),K(+)-ATPase modifies the cation binding pocket and thereby generates a high Na(+)-ATPase activity

A series of six different mutants (D804A, D804E, D804G, D804N, D804Q, and D804S) of aspartate 804 present in transmembrane segment 6 of the rat Na(+),K(+)-ATPase alpha(1)-subunit were prepared and expressed in Sf9 cells by use of the baculovirus expression system. In contrast to the wild-type enzyme all mutants except D804Q showed a very high Na(+)-ATPase activity, which was hardly further stimulated by the addition of K(+). The ATPase activity of the mutants was already nearly maximal at 10 microM ATP and most of them could be phosphorylated in the absence of Na(+) at pH 6.0 and 21 degrees C, suggesting that they strongly prefer the E(1) over the E(2) conformation. However, Na(+) dose-dependently lowered the steady-state phosphorylation level, as a consequence of the increased affinity for Na(+) in the dephosphorylation reaction of the mutants compared to the wild-type enzyme. Conversely, the affinity for K(+) in the dephosphorylation reaction was decreased for the mutants as compared to that for the wild-type enzyme. When the pH was increased or the temperature was decreased, the phosphorylation level of the mutants decreased and the Na(+) activation in the phosphorylation reaction became apparent. It is concluded that upon mutation of aspartate 804 the affinity of the cation-binding pocket is changed relatively in favor of Na(+) instead of K(+), as a consequence of which the enzyme has obtained a preference for the E(1) conformation.     

A hybrid between Na+,K+-ATPase and H+,K+-ATPase is sensitive to palytoxin, ouabain, and SCH 28080

Na(+),K(+)-ATPase is inhibited by cardiac glycosides such as ouabain, and palytoxin, which do not inhibit gastric H(+),K(+)-ATPase. Gastric H(+),K(+)-ATPase is inhibited by SCH28080, which has no effect on Na(+),K(+)-ATPase. The goal of the current study was to identify amino acid sequences of the gastric proton-potassium pump that are involved in recognition of the pump-specific inhibitor SCH 28080. A chimeric polypeptide consisting of the rat sodium pump alpha3 subunit with the peptide Gln(905)-Val(930) of the gastric proton pump alpha subunit substituted in place of the original Asn(886)-Ala(911) sequence was expressed together with the gastric beta subunit in the yeast Saccharomyces cerevisiae. Yeast cells that express this subunit combination are sensitive to palytoxin, which interacts specifically with the sodium pump, and lose intracellular K(+) ions. The palytoxin-induced K(+) efflux is inhibited by the sodium pump-specific inhibitor ouabain and also by the gastric proton pump-specific inhibitor SCH 28080. The IC(50) for SCH 28080 inhibition of palytoxin-induced K(+) efflux is 14.3 +/- 2.4 microm, which is similar to the K(i) for SCH 28080 inhibition of ATP hydrolysis by the gastric H(+),K(+)-ATPase. In contrast, palytoxin-induced K(+) efflux from cells expressing either the native alpha3 and beta1 subunits of the sodium pump or the alpha3 subunit of the sodium pump together with the beta subunit of the gastric proton pump is inhibited by ouabain but not by SCH 28080. The acquisition of SCH 28080 sensitivity by the chimera indicates that the Gln(905)-Val(930) peptide of the gastric proton pump is likely to be involved in the interactions of the gastric proton-potassium pump with SCH 28080.     

Failure to up-regulate gill Na+,K+-ATPase alpha-subunit isoform alpha1b may limit seawater tolerance of land-locked Arctic char (Salvelinus alpinus)

Many populations of Arctic char (Salvelinus alpinus) are land-locked, physically separated from the ocean by natural barriers and unable to migrate to sea like anadromous populations. Previous studies which experimentally transferred land-locked Arctic char to seawater report high mortality rates due to osmoregulatory failure and an inability to up-regulate gill Na(+),K(+)-ATPase activity. This study examined the mRNA expression of two recently discovered alpha-subunit isoforms of gill Na(+)K(+)-ATPase (alpha1a and alpha1b) during seawater exposure of land-locked Arctic char. mRNA levels of these gill Na(+),K(+)-ATPasealpha-subunit isoforms were compared to Na(+),K(+)-ATPase activity and protein levels and related to osmoregulatory performance. Land-locked Arctic char were unable to regulate plasma osmolality following seawater exposure. Seawater exposure did not induce an increase in gill Na(+),K(+)-ATPase activity or protein levels. Na(+),K(+)-ATPase isoform alpha1a mRNA quickly decreased upon exposure to seawater, while isoform alpha1b levels were unchanged. These results suggest the inability of land-locked Arctic char to acclimate to seawater is due a failure to up-regulate gill Na(+),K(+)-ATPase activity which may be due to their inability to increase Na(+),K(+)-ATPase alpha1b mRNA expression.     

Wild Arctic char (Salvelinus alpinus) upregulate gill Na+,K+-ATPase during freshwater migration

The successful acclimation of eurhyhaline fishes from seawater to freshwater requires the gills to stop actively secreting ions and start actively absorbing ions. Gill Na(+),K(+)-ATPase is known to be an integral part of the active ion secretion model of marine fishes, but its importance in the active ion uptake model of freshwater fishes is less clear. This study, conducted in the high Arctic, examines gill Na(+),K(+)-ATPase regulation in wild anadromous arctic char returning to freshwater from the ocean. Gill Na(+),K(+)-ATPase activity, protein expression, and mRNA expression of Na(+),K(+)-ATPase isoforms alpha 1a and alpha 1b were monitored in arctic char at three points along their migration route to and from Somerset Island, Nunavut, Canada: out at sea (Whaler's Point), in seawater near the river mouth (Nat's Camp), and after entering the Union River. Arctic char collected from the Union River had more than twofold greater gill Na(+),K(+)-ATPase activity. This was associated with a significant increase (threefold) in Na(+),K(+)-ATPase isoform alpha 1a mRNA expression and a significant increase in plasma sodium and osmolality levels compared with seawater char. Compared with char sampled from Whaler's Point, Na(+),K(+)-ATPase isoform alpha 1b mRNA expression was decreased by approximately 50% in char sampled at Nat's Camp and the Union River. These results suggest that the upregulation of gill Na(+),K(+)-ATPase activity is involved in freshwater acclimation of arctic char and implicate a role for Na(+),K(+)-ATPase isoform alpha 1a in this process. In addition, we discuss evidence that arctic char go through a preparatory phase, or "reverse smoltification," before entering freshwater.     

Diet deficient in alpha-linolenic acid alters fatty acid composition and enzymatic properties of Na+, K+-ATPase isoenzymes of brain membranes in the adult rat

The effects of dietary (n-6)/(n-3) polyunsaturated fatty acid balance on fatty acid composition, ouabain inhibition, and Na(+) dependence of Na(+), K(+)-ATPase isoenzymes of whole brain membranes were studied in 60-day-old rats fed over two generations a diet either devoid of alpha-linolenic acid [18:3(n-3)] (sunflower oil diet) or rich in 18:3(n-3) (soybean oil diet). In the brain membranes, the sunflower oil diet led to a dramatic decrease in docosahexaenoic acid [22:6(n-3)] membrane content. The activities of Na(+), K(+)-ATPase isoenzymes were discriminated on the basis of their differential affinities for ouabain and their sensitivity to sodium concentration. The ouabain titration curve of Na(+), K(+)-ATPase activity displayed three inhibitory processes with markedly different affinity [i.e., low (alpha1), high (alpha2), and very high (alpha3)] for brain membranes of rats fed the sunflower oil diet, whereas the brain membranes of rats fed the soybean oil diet exhibited only two inhibitory processes, low (alpha1) and high (alpha2' = alpha2 + alpha3). Regardless of the diet, on the basis of the Na(+) dependence of Na(+), K(+)-ATPase activity, three isoenzymes were found: alpha1 form displaying an affinity 1.5- to 2-fold higher that of than alpha2 and 3-fold higher that of alpha3. In rats fed the sunflower oil diet, alpha2 isoenzyme exhibited higher affinity for sodium (Ka = 8.8 mmol/L) than that of rats fed the soybean oil diet (Ka = 11.7 mmol/L). These results suggest that the membrane lipid environment modulates the functional properties of Na(+), K(+)-ATPase isoenzymes of high ouabain affinity (alpha2).