Alpha and beta subunits of Na,K-ATPase interact with BiP and calnexin.
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Animals↗
Biomedical subjects
K Geering
Publications and source records attributed to K Geering.
Structural domains implicated in ER degradation of alpha subunits of Na,K-ATPase.
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Animals↗
The gamma subunit is a specific component of the Na,K-ATPase and modulates its transport function.
The role of small, hydrophobic peptides that are associated with ion pumps or channels is still poorly understood. By using the Xenopus oocyte as an expression system, we have characterized the structural and functional properties of the gamma peptide which co-purifies with Na,K-ATPase. Immuno-radiolabeling of epitope-tagged gamma subunits in intact oocytes and protease protection assays show that the gamma peptide is a type I membrane protein lacking a signal sequence and exposing the N-terminus to the extracytoplasmic side. Co-expression of the rat or Xenopus gamma subunit with various proteins in the oocyte reveals that it specifically associates only with isozymes of Na,K-ATPase. The gamma peptide does not influence the formation and cell surface expression of functional Na,K-ATPase alpha-beta complexes. On the other hand, the gamma peptide itself needs association with Na,K-ATPase in order to be stably expressed in the oocyte and to be transported efficiently to the plasma membrane. Gamma subunits do not associate with individual alpha or beta subunits but only interact with assembled, transport-competent alpha-beta complexes. Finally, electrophysiological measurements indicate that the gamma peptide modulates the K+ activation of Na,K pumps. These data document for the first time the membrane topology, the specificity of association and a potential functional role for the gamma subunit of Na,K-ATPase.
Amino Acid Sequence↗
Role of glycosylation and disulfide bond formation in the beta subunit in the folding and functional expression of Na,K-ATPase.
Initial folding is a prerequisite for subunit assembly in oligomeric proteins. In this study, we have compared the role of co-translational modifications in the acquisition of an assembly-competent conformation of the beta subunit, the assembly of which is required for the structural and functional maturation of the catalytic Na,K-ATPase alpha subunit. Cysteine or asparagine residues implicated in disulfide bond formation or N-glycosylation, respectively, in the Xenopus beta1 subunit were eliminated by site-directed mutagenesis, and the assembly efficiency of the mutants and the functional expression of Na+,K+ pumps were studied after expression in Xenopus oocytes. Our results show that lack of each one of the two most C-terminal disulfide bonds indeed permits short term but completely abolishes long term assembly of the beta subunit. On the other hand, lack of the most N-terminal disulfide bonds allows the expression of a small number of functional Na+,K+ pumps at the cell surface. Elimination of all three but not of one or two glycosylation sites produces beta subunits that remain stably expressed in the endoplasmic reticulum, in association with binding protein but not as irreversible aggregates. The assembly efficiency of nonglycosylated beta subunits is decreased but a reduced number of functional Na+,K+ pumps is expressed at the cell surface. The lack of sugars does not influence the apparent K+ or ouabain affinity of the Na+,K+ pumps. Thus, these data show that disulfide bond formation and N-glycosylation may play important but qualitatively distinct roles in the initial folding of oligomeric protein subunits. Moreover, the results suggest that an endoplasmic reticulum degradation pathway exists, which is glycosylation-dependent.
Animals↗
Phorbol 12-myristate 13-acetate down-regulates Na,K-ATPase independent of its protein kinase C site: decrease in basolateral cell surface area.
The effect of protein kinase C (PKC) stimulation on the pump current (Ip) generated by the Na,K-ATPase was measured in A6 epithelia apically permeabilized with amphotericin B. Phorbol 12-myristate 13-acetate (PMA) produced a decrease in Ip carried by sodium pumps containing the endogenous Xenopus laevis or transfected Bufo marinus alpha 1 subunits (approximately 30% reduction within 25 min, maximum after 40 min) independent of the PKC phosphorylation site (T15A/S16A). In addition to this major effect of PMA, which was independent of the intracellular sodium concentration and was prevented by the PKC inhibitor bisindolylmaleimide GF 109203X (BIM), another BIM-resistant, PKC site-independent decrease was observed when the Ip was measured at low sodium concentrations (total reduction approximately 50% at 5 mM sodium). Using ouabain binding and cell surface biotinylation, stimulation of PKC was shown to reduce surface Na,K-ATPase by 14 to 20% within 25 min. The same treatment stimulated fluid phase endocytosis sevenfold and decreased by 16.5% the basolateral cell surface area measured by transepithelial capacitance measurements. In conclusion, PKC stimulation produces a decrease in sodium pump function which can be attributed, to a large extent, to a withdrawal of sodium pumps from the basolateral cell surface independent of their PKC site. This reduction of the number of sodium pumps is parallel to a decrease in basolateral membrane area.
Amiloride↗
Na,K-ATPase.
Na,K-ATPase is the driving force for renal Na+ reabsorption and is thus critically implicated in the control of extracellular volume and blood pressure. This review focuses on most recent advances in the elucidation of intrinsic structural features that are important for Na,K-ATPase function and in the identification of regulatory mechanisms that are implicated in the modulation of Na,K-ATPase activity in the kidney under physiological or pathophysiological conditions.
Animals↗
alpha 1 but not alpha 2 or alpha 3 isoforms of Na,K-ATPase are efficiently phosphorylated in a novel protein kinase C motif.
Protein kinase C (PKC) phosphorylates the catalytic alpha 1 subunit of Na,K-ATPase in purified enzyme preparations and in intact cells. Little is known, however, whether all three known alpha isoforms are substrates for PKC and whether direct phosphorylation is implicated in the modulation of the transport activity of the different Na,K-ATPase isozymes. In this study, we investigated the structural requirements for PKC phosphorylation of alpha 1, alpha 2, and alpha 3 isoforms of different species after expression in Xenopus oocytes. By using a combination of site-directed mutagenesis and computer-assisted protein modeling, we characterized a novel Ser-X-His motif which in concert with more distantly located basic residues acts as an efficient substrate for PKC-mediated phosphorylation in the N-terminus of most Na,K-ATPase alpha 1 isoforms. As indicated by controlled proteolysis, alpha 2 isoforms are also phosphorylated in the N-terminus but to a much lower extent than alpha 1 isoforms containing the Ser-X-His motif. Phosphorylation and phosphoamino acid analysis of fusion proteins containing the wild-type or mutant N-terminus of alpha 2 reveal that Thr-Thr-Ser-X-Asn or Thr-Thr-Ala-X-Asn motifs represent weak targets for PKC phosphorylation. Finally, our data suggest that, with the exception of rat alpha 3, all alpha 3 isoforms from other species are not substrates for PKC. On the basis of the phosphorylation efficiency, we may speculate that only alpha 1 but not alpha 2 or alpha 3 isoforms of Na,K-ATPase are likely candidates for regulatory PKC phosphorylation.
Amino Acid Sequence↗
Degradation and endoplasmic reticulum retention of unassembled alpha- and beta-subunits of Na,K-ATPase correlate with interaction of BiP.
Assembly of alpha- and beta-subunits in the endoplasmic reticulum is a prerequisite for the structural and functional maturation of oligomeric P-type ATPases. In Xenopus oocytes, overexpressed, unassembled alpha- and beta-subunits of Xenopus Na,K-ATPase are retained in the endoplasmic reticulum (ER) and are degraded with different kinetics, while unassembled beta-subunits of gastric H, K-ATPase leave the ER. In this study, we have investigated the role of the immunoglobulin-binding protein, BiP, in the folding, assembly, and ER retention of ATPase subunits. We determined the primary sequence of Xenopus BiP and used polyclonal antibodies to examine the interaction with BiP of various wild type and mutant alpha- and beta-subunits overexpressed in Xenopus oocytes. Our results show that ER-retained, unassembled Na,K-ATPase beta-subunits, but not transport-competent H,K-ATPase beta-subunits, efficiently associate with BiP until assembly with alpha-subunits occurs. Furthermore, the kinetics of BiP interaction with unassembled wild type and with mutant Na,K-ATPase beta-subunits parallels their respective stability against cellular degradation. Finally, alpha-subunits that are overexpressed in oocytes and are rapidly degraded and endogenous oocyte alpha-subunits that are stably expressed as individual assembly-competent proteins also interact with oocyte or exogenous BiP, and the interaction time correlates with the protein's stability. These data demonstrate for the first time that BiP might be involved in a long term maturation arrest and/or in the ER quality control of a multimembrane-spanning protein and lend support for a universal chaperone function of BiP.
Animals↗
Oligomerization and maturation of Na,K-ATPase: functional interaction of the cytoplasmic NH2 terminus of the beta subunit with the alpha subunit.
Subunit assembly plays an essential role in the maturation of oligomeric proteins. In this study, we have characterized the main structural and functional consequences of the assembly of alpha and beta subunits of Na,K-ATPase. Xenopus oocytes injected with alpha and/or beta cRNA were treated with brefeldin A, which permitted the accumulation of individual subunits or alpha-beta complexes in the ER. Only alpha subunits that are associated with beta subunits become resistant to trypsin digestion and cellular degradation. Similarly, assembly with beta subunits is necessary and probably sufficient for the catalytic alpha subunit to acquire its main functional properties at the level of the ER, namely the ability to adopt different ligand-dependent conformations and to hydrolyze ATP in an Na(+)- and K(+)-dependent, ouabain-inhibitable fashion. Not only the alpha but also the beta subunit undergoes a structural change after assembly, which results in a global increase in its protease resistance. Furthermore, extensive and controlled proteolysis assays on wild-type and NH2-terminally modified beta subunits revealed a K(+)-dependent interaction of the cytoplasmic NH2 terminus of the beta subunit with the alpha subunit, which is likely to be involved in the modulation of the K(+)-activation of the Na,K-pump transport activity. Thus, we conclude that the ER assembly process not only establishes the basic structural interactions between individual subunits, which are required for the maturation of oligomeric proteins, but also distinct, functional interactions, which are involved in the regulation of functional properties of mature proteins.
Amino Acid Sequence↗
Adrenergic, dopaminergic, and muscarinic receptor stimulation leads to PKA phosphorylation of Na-K-ATPase.
Na-K-adenosinetriphosphatase (Na-K-ATPase) is a potential target for phosphorylation by protein kinase A (PKA) and C (PKC). We have investigated whether the Na-K-ATPase alpha-subunit becomes phosphorylated at its PKA or PKC phosphorylation sites upon stimulation of G protein-coupled receptors primarily linked either to the PKA or the PKC pathway. COS-7 cells, transiently or stably expressing Bufo marinus Na-K-ATPase wild-type alpha- or mutant alpha-subunits affected in its PKA or PKC phosphorylation site, were transfected with recombinant DNA encoding beta 2- or alpha 1-adrenergic (AR), dopaminergic (D1A-R), or muscarinic cholinergic (M1-AChR) receptor subspecies. Agonist stimulation of beta 2-AR or D1A-R led to phosphorylation of the wild-type alpha-subunit, as well as the PKC mutant, but not of the PKA mutant, indicating that these receptors can phosphorylate the Na-K-ATPase via PKA activation. Surprisingly, stimulation of the alpha 1B-AR, alpha 1C-AR, and M1-AChR also increased the phosphorylation of the wild-type alpha-subunit and its PKC mutant but not of its PKA mutant. Thus the phosphorylation induced by these primarily phospholipase C-linked receptors seems mainly mediated by PKA activation. These data indicate that the Na-K-ATPase alpha-subunit can act as an ultimate target for PKA phosphorylation in a cascade starting with agonist-receptor interaction and leading finally to a phosphorylation-mediated regulation of the enzyme.
Animals↗
Improved monitoring of preterm infants by Fuzzy Logic.
Keeping the oxygenation status of newborn infants within physiologic limits is a crucial task in intensive care. For this purpose several vital parameters are supervised routinely by monitors, such as electrocardiograph, transcutaneous partial oxygen pressure monitor and pulse oximeter. Each monitor issues an alarm signal whenever an upper or lower limit of the parameter(s) measured is exceeded. However, in practice it turns out, that a considerable amount of false alarms is generated by artefacts, which are attributed mostly to movements of the infants. Eliminating these false alarms would be of benefit to the staff as well as the patients of the intensive care unit. Accordingly, an automated system based on Fuzzy Logic was developed, which is capable of distinguishing between critical situations and artefacts. The system is based on a Transputer IMS T425 in a PC, which collects the data from the monitors, plots it on a colour screen, saves it to hard disk and analyses it by Fuzzy Logic. Fuzzy algorithms were developed to generate more reliable alarms. All vital parameters of eight infants, who either moved often and/or frequently produced real alarm situations, were recorded. Synchronously the infants' movements and care procedures were video taped. The data and video were analysed off line with the help of an experienced neonatologist. His judgement was compared to the analysis of the Fuzzy Logic system. The results show that it is possible to improve the reliability of the monitored data with the aid of an evaluation strategy based on Fuzzy Logic and hence distinguish between real alarm situations and movement artefacts to the extent that an application in an intensive care unit under routine conditions becomes conceivable.
Algorithms↗
Labeling of a cysteine in the cardiotonic glycoside binding site by the steroid derivative HDMA.
The digoxigenin derivative N-hydroxysuccinimidyl digoxigenin-3-O-methylcarbonyl-epsilon-aminocaproate (HDMA) has been shown to covalently label the ouabain binding site of the Na,K-ATPase epsilon subunit [Antolovic et al. (1995) Eur. J. Biochem. 227, 61-67]. In the present study we observed both, labeling and inactivation of the activity, of wild type Na,K-ATPase overexpressed in Xenopus oocyte. In contrast, no significant inhibition and no labeling could be detected when a Cys-113 of the first transmembrane segment was mutated to serine, although the affinity of this mutant for digoxigenin or HDMA measured in acute inhibition experiments was similar to the wild type. This indicates that after docking of its genin moiety, HDMA can form a thioester bond with Cys-113.
Affinity Labels↗
Primary structure and functional expression of the mouse and frog alpha-subunit of the gastric H(+)-K(+)-ATPase.
The H(+)-K(+)-ATPase of the gastric parietal cells is responsible for the acidification of the stomach lumen. This heterodimeric protein belongs to the family of cation-translocating P-type ATPases, which includes the closely related Na(+)-ATPase. We have cloned the alpha-subunit cDNA of the Xenopus and murine gastric H(+)-K(+)-ATPase (alpha H-K). We have expressed Xenopus and murine alpha H-K along with the previously cloned gastric H(+)-K(+)-ATPase beta-subunit of rabbit (beta H-K) in Xenopus oocytes by cRNA injection. An antibody directed against the beta H-K coimmunoprecipitates under nondenaturing conditions the alpha H-K of both species, demonstrating assembly of the alpha/beta complex. Additionally, we demonstrate the presence of K(+)-transporting H(+)-K(+)-ATPase in the plasma membrane of oocytes by 86Rb- uptake. The H(+)-K(+)-ATPase-mediated K+ uptake was inhibited by the gastric H(+)-K(+)-ATPase inhibitor Sch-28080, but not by ouabain, and shows K(+)-dependent activation (K1/2 approximately 2 mM). Furthermore, H(+)-K(+)-ATPase-expressing oocytes show a Sch-28080 inhibitable proton extrusion. Our data indicate that the expressed H(+)-K(+)-ATPase behaves functionally in oocytes as in the gastric gland.
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Human ATP1AL1 gene encodes a ouabain-sensitive H-K-ATPase.
The cDNA for ATP1AL1, the fifth member of the human Na-K-adenosinetriphosphatase (ATPase)/H-K-ATPase gene family, was recently cloned (A. V. Grishin, V. E. Sverdlov, M. B. Kostina, and N. N. Modyanov. FEBS Lett. 349: 144-150, 1994). The encoded protein (ATP1AL1) has all the primary structural features common to the catalytic alpha-subunit of ion-transporting P-type ATPases and is similar (63-64% identity) to the Na-K-ATPase alpha-subunit isoforms and the gastric H-K-ATPase alpha-subunit. In this study, ATP1AL1 was expressed in Xenopus laevis oocytes in combination with the beta-subunit of rabbit gastric H-K-ATPase. The functional properties of the stable alpha/beta-complex were studied by 86Rb+ uptake and demonstrated that ATP1AL1 is a novel human K(+)-dependent ATPase [apparent half-constant activation/(K1/2) for K+ approximately 375 microM)]. ATP1AL1-mediated inward K+ transport was inhibited by ouabain (inhibition constant approximately 13 microM) and was found to be inhibited by high concentrations of SCH-28080 (approximately 70% at 500 microM). ATP1AL1 expression resulted in the alkalinization of the oocytes' cytoplasm and ouabain-sensitive proton extrusion, as measured with pH-sensitive microelectrodes. These data argue that ATP1AL1 is the catalytic alpha-subunit of a human nongastric P-type ATPase capable of exchanging extracellular potassium for intracellular protons.
Animals↗
Phosphorylation of the Na,K-ATPase alpha-subunit by protein kinase A and C in vitro and in intact cells. Identification of a novel motif for PKC-mediated phosphorylation.
Na,K-ATPase is a potential target for regulatory phosphorylation by protein kinase A and C (PKA and PKC). To identify the phosphorylation sites, we have mutated the alpha 1-subunit of Bufo marinus in a highly conservative PKA and in 20 different PKC consensus sequences. The mutants were expressed in Xenopus oocytes and their phosphorylation capacity tested in homogenates upon stimulation of PKA or PKC. While serine 943 (Ser-943) was identified as a unique target site for PKA, none of the PKC consensus serine or threonine residues are implicated in PKC phosphorylation. Controlled trypsinolysis of phosphorylated alpha-subunits of various purified enzyme preparations and of alpha/beta complexes from oocyte homogenates revealed that PKC phosphorylation was exclusively associated with the N terminus. A fusion protein containing the first 32 amino acids of the Bufo alpha-subunit was phosphorylated in vitro and serine and threonine residues (Thr-15 and Ser-16) in this region were identified by site-directed mutagenesis as the PKC phosphorylation sites. Finally, the Bufo alpha-subunit was phosphorylated by protein kinases in transfected COS-7 cells. In intact cells, PKA stimulation induced phosphorylation exclusively on Ser-943 and PKC stimulation mainly on Thr-15 and Ser-16, which are contained in a novel PKC phosphorylation motif.
Amino Acid Sequence↗
In vivo phosphorylation of the Na,K-ATPase alpha subunit in sciatic nerves of control and diabetic rats: effects of protein kinase modulators.
The phosphorylation state of the Na,K-ATPase alpha subunit has been examined in 32P-labeled sciatic nerves of control and streptozotocin-treated diabetic rats. Intact nerves were challenged with protein kinase (PK) modulators and alpha-subunit 32P labeling was analyzed after immunoprecipitation. In control nerves, the PKC activator phorbol 12-myristate 13-acetate (PMA) had little effect on alpha-subunit 32P labeling. In contrast, staurosporine, a PKC inhibitor, and extracellular calcium omission decreased it. In Ca(2+)-free conditions, PMA restored the labeling to basal levels. The cAMP-raising agent forskolin reduced the 32P labeling of the alpha subunit. The results suggest that nerve Na,K-ATPase is tonically phosphorylated by PKC in a Ca(2+)-dependent manner and that PKA modulates the phosphorylation process. In nerves of diabetic rats, PMA increased 32P labeling of the alpha subunit. In contrast to staurosporine or extracellular calcium omission, the decreased state of phosphorylation seen with forskolin was no longer significant in diabetic nerves. No change in the level of alpha-subunit isoforms (alpha 1 or alpha 2) was detected by Western blot analysis in such nerves. In conclusion, the altered effect of PK activators on Na,K-ATPase phosphorylation state is consistent with the view that a defect in PKC activation exists in diabetic nerves.
Alkaloids↗
Modulation of the Na,K-pump function by beta subunit isoforms.
To study the role of the Na,K-ATPase beta subunit in the ion transport activity, we have coexpressed the Bufo alpha 1 subunit (alpha 1) with three different isotypes of beta subunits, the Bufo Na,K-ATPase beta 1 (beta 1NaK) or beta 3 (beta 3NaK) subunit or the beta subunit of the rabbit gastric H,K-ATPase (beta HK), by cRNA injection in Xenopus oocyte. We studied the K+ activation kinetics by measuring the Na,K-pump current induced by external K+ under voltage clamp conditions. The endogenous oocyte Na,K-ATPase was selectively inhibited, taking advantage of the large difference in ouabain sensitivity between Xenopus and Bufo Na,K pumps. The K+ half-activation constant (K1/2) was higher in the alpha 1 beta 3NaK than in the alpha 1 beta 1NaK groups in the presence of external Na+, but there was no significant difference in the absence of external Na+. Association of alpha 1 and beta HK subunits produced active Na,K pumps with a much lower apparent affinity for K+ both in the presence and in the absence of external Na+. The voltage dependence of the K1/2 for external K+ was similar with the three beta subunits. Our results indicate that the beta subunit has a significant influence on the ion transport activity of the Na,K pump. The small structural differences between the beta 1NaK and beta 3NaK subunits results in a difference of the apparent affinity for K+ that is measurable only in the presence of external Na+, and thus appears not to be directly related to the K+ binding site. In contrast, association of an alpha 1 subunit with a beta HK subunit results in a Na,K pump in which the K+ binding or translocating mechanisms are altered since the apparent affinity for external K+ is affected even in the absence of external Na+.
Animals↗
Polyadenylation of Na(+)-K(+)-ATPase beta 1-subunit during early development of Xenopus laevis.
In fully grown Xenopus oocytes, the synthesis of beta-subunits is limiting for the formation of functional Na(+)-K(+)-adenosinetriphosphatase alpha/beta-complexes (Geering, K. FEBS Lett. 285: 189-193, 1991). In the present study, we show that during oocyte growth (from stage I to stage VI) alpha 1-, but not beta 1- or beta 3-isoform, mRNAs accumulate. In addition, beta-mRNAs are apparently sequestered in an untranslated pool in fully grown oocytes (stage VI). From fertilization to morulation, the total pools of alpha 1-, beta 1-, or beta 3-mRNAs vary little. Whereas polyadenylated [poly(A)+] alpha 1- and beta 3-isoform mRNAs did not change significantly, poly(A)+ beta 1-mRNA abundance increased three- to fourfold at morulation, accompanied by a parallel increase in beta 1-protein synthesis. After midblastula transition (i.e., at early gastrula) and during neurulation, poly(A)+ alpha 1- and beta 3-mRNAs accumulated rapidly, whereas poly(A)+ beta 1-mRNA accumulation was delayed by approximately 2 h, beginning only at early neurula. Our results indicate that 1) the abundance of poly(A)+ beta 1-mRNA is rate limiting during embryonic development for the assembly of alpha 1/beta 1-heterodimers, shown to be involved in the vectorial transport of sodium in kidney cells, and 2) the polyadenylation of beta 1-mRNA is a rate-limiting factor during morulation for the synthesis and assembly of new sodium pumps at the time of blastocoel fluid formation. The 3'-untranslated region of beta 1-mRNA (but not of alpha 1-mRNA) expresses cytoplasmic polyadenylation elements (CPEs) with the consensus sequence AXX-AUUUU(A/U)(A/U)(A/U). A role of CPE in the differential polyadenylation of alpha 1- and beta 1-mRNA is proposed.
Aging↗