Subunit assembly and functional maturation of Na,K-ATPase.
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Animals↗
Biomedical subjects
K Geering
Publications and source records attributed to K Geering.
Cross-reactivity of an antiserum to the alpha-subunit of the Na+, K+-ATPase of toad (Bufo marinus) kidney with basal and apical membranes of transporting epithelia of the rat.
An antibody to the 96 kD alpha-subunit of the Na+, K+-ATPase from Bufo marinus has been used in immunostaining rat kidney and salivary glands. Intense staining was observed on basolateral membranes of distal tubules of the kidney and striated ducts of the three major salivary glands. Less intense staining was seen on the basolateral membranes of parotid acinar cells, but no staining was seen on the acinar cells of submandibular or sublingual glands. These sites of staining have been shown, by other methods, to posses substantial Na+, K+-ATPase, indicating that the antibody recognizes antigenic determinants of the sodium pump highly conserved in the course of evolution. In addition, staining with this antibody was observed at the apical region of cells of the proximal straight tubule and of the papillary collecting duct in the kidney. Absorption studies suggest that the apical antigenic determinants are the same or closely related to each other but are distinct from basolateral antigenic determinants.
Animals↗
Functional expression of the amiloride-sensitive sodium channel in Xenopus oocytes.
Expression of the amiloride-sensitive sodium channel was examined in Xenopus oocytes that were microinjected with A6 cell mRNA. Amiloride-inhibitable 22Na flux could be measured in intact oocytes 2-3 days after injection with 25 ng of poly(A)+ RNA isolated from aldosterone-treated A6 cells. The rate of 22Na uptake was approximately 15-fold greater in oocytes microinjected with 25 ng of poly(A)+ RNA than in water-injected control oocytes. An increase in 22Na uptake by mRNA-injected oocytes occurred whether the mRNA was isolated from A6 cells grown on a porous or nonporous support. In the presence of 4 mM NaCl, amiloride caused dose-dependent inhibition of 22Na uptake in mRNA-injected oocytes, which was half-maximal at 6 x 10(-8) M. Both 1 microM amiloride and 0.1 microM benzamil inhibited 22Na uptake in mRNA-injected oocytes by greater than 95%, whereas less than 50% inhibition occurred with 1 microM 5-(N-ethyl-N-isopropyl)amiloride. When A6 cell mRNA was size fractionated by sucrose density-gradient centrifugation, amiloride-sensitive 22Na uptake was expressed predominantly by oocytes injected with mRNA from two contiguous fractions.
Amiloride↗
Inhibition of N-glycosylation affects transepithelial Na+ but not Na+-K+-ATPase transport.
Tunicamycin (TM) was used in toad urinary bladder (TBM) cells to study the role of N-glycosylation of the beta-subunit of Na+-K+-ATPase. Inhibition of the beta-subunit core glycosylation was dose dependent and coincided with a specific 70% decrease in newly synthesized beta- and alpha-subunits. Na+-K+-ATPase activity paralleled the decrease in the cellular content of the alpha-subunit, although the cellular and cell surface-expressed Na+-K+-ATPase pool was progressively filled up with nonglycosylated beta-subunits. In addition, the decrease in maximal Na+ transport capacity of the Na+-K+-ATPase as assessed by short-circuit current (SCC) measurements in the presence of amphotericin B correlated with the decrease in the total cell surface-expressed beta-subunit population despite the fact that it was composed of 47% nonglycosylated beta-subunits after 42 h of TM treatment. These results are consistent with the interpretation that beta-subunit glycosylation is not important either for the enzyme's intracellular sorting to the plasma membrane or its hydrolytic and transport properties. Finally, TM produced effects on basal SCC and electrical resistance that differed in their times of onset and time periods needed for recovery. Thus, in addition to the Na+-K+-ATPase, other glycoproteins in the apical membrane and the tight junctions must be implicated in the maintenance of transepithelial Na+ transport.
Adenosine Triphosphate↗
A role for the beta-subunit in the expression of functional Na+-K+-ATPase in Xenopus oocytes.
In all cellular systems studied so far, the catalytic alpha- and the glycosylated beta-subunit of Na+-K+-ATPase are coordinately synthesized and are assembled into stoichiometric alpha, beta-complexes. In contrast to these data, in this study we show that the fully grown oocyte of Xenopus laevis synthesizes much less beta-subunit than alpha-subunit. The alpha-subunit produced in excess over the beta-subunit is membrane associated but highly trypsin sensitive and can be compared with the immature alpha-subunit population identified in epithelial cells immediately after synthesis (K. Geering, J. P. Kraehenbuhl, and B.C. Rossier, J. Cell Biol. 105: 2613-2619, 1987). The Xenopus oocyte thus turns out to be a unique system to study the functional role of the beta-subunit. Injection of beta-subunit-specific mRNA transcribed in vitro from a beta-cDNA clone (derived from Xenopus kidney, A6 cells) into oocytes results in translation of a glycosylated beta-subunit. The synthesis of this exogenous beta-subunit increases significantly the proportion of trypsin-resistant oocyte alpha-subunits able to perform cation-dependent conformational changes. In addition, 25-65% more ouabian binding sites are expressed at the plasma membrane in beta-mRNA-injected oocytes. In contrast, newly synthesized alpha-subunit translated after injection of size-fractionated mRNA enriched in alpha-mRNA remains trypsin sensitive as the oocyte alpha-subunit. These data suggest that association of the beta-subunit to the alpha-subunit provokes a structural rearrangement of the alpha-subunit that might be a first step toward the functional maturation of the Na+-K+-ATPase and its expression at the plasma membrane.
Animals↗
Primary sequence of Xenopus laevis Na+-K+-ATPase and its localization in A6 kidney cells.
Polyclonal antibodies raised against the alpha- and beta-subunits of amphibian kidney Na+-K+-ATPase were used to screen an expression library from Xenopus laevis kidney epithelial cells (A6 cell line). cDNAs coding for each Na+-K+-ATPase subunit were identified and used to isolate near full-length cDNAs. The complete nucleotide sequence and the deduced amino acid sequence were determined. The alpha-subunit is an alpha (alpha I)-isoform. The alpha- and beta-subunits are more closely related to the mammalian and avian than the fish sequences. Antibodies raised against the fusion proteins produced by the two clones served to immunoprecipitate proteins from biosynthetically labeled or selectively surface-radioiodinated A6 cells grown on a porous substrate. The alpha- and the beta-subunits of Na+-K+-ATPase were found associated early in the course of biosynthesis and were restricted to the basolateral plasma membrane.
Amino Acid Sequence↗
Aldosterone-induced glycoproteins: electrophysiological-biochemical correlation.
Aldosterone induces the synthesis of a group of glycoproteins (GP65,70) in toad urinary bladders which are potential effectors of the natriferic action of this hormone. In the present study we have confirmed that aldosterone produces a two-phase electrophysiological response. During the early phase (less than 3 h) short-circuit current and transepithelial conductance increase in parallel, while during the late phase (greater than 3 h) short-circuit current continues to increase without any further change in conductance. By biosynthetically labeling aldosterone-treated toad bladders with [35S]methionine either during the early (h 0-2 or 1-3) or the late (h 4-6 or 7-9) phases of the natriferic response, we have demonstrated that GP65,70 is synthesized as a late effect of aldosterone. Since synthesis of GP65,70 occurs at a time when the electromotive force of the Na+ pump is increasing, and since GP65,70 biochemically resembles the beta subunit of Na+/K+-ATPase, studies were undertaken to examine whether GP65,70 is the beta subunit. Purified amphibian renal beta subunit was analyzed by two-dimensional polyacrylamide gel electrophoresis and was found to have an isoelectric point and Mr value similar to those of GP65,70. However, when nitrocellulose blots containing wheat germ agglutinin-purified proteins from aldosterone-treated bladders were stained with monospecific polyclonal antibodies developed against the beta subunit, GP65,70 was not recognized, whereas a group of slightly more acidic proteins of similar Mr were recognized. Thus, GP65,70 is not the beta subunit of Na+/Ka+-ATPase. Further studies are needed to determine the cellular function of GP65,70.
Aldosterone↗
Role of the Na,K-ATPase beta-subunit in the cellular accumulation and maturation of the enzyme as assessed by glycosylation inhibitors.
No functional role could yet be established for the glycosylated beta-subunit of the Na,K-ATPase. In this study, we describe the intracellular processing of the beta-subunit as a glycoprotein in toad bladder cells and the consequences of its structural perturbation with glycosylation inhibitors on the cellular expression of the alpha- and beta-subunits and on the structural and functional maturation of the enzyme. Controlled trypsinolysis of homogenates from pulse-labeled cells reveals that the beta-subunit is subjected to glycosylation-dependent structural rearrangements during its intracellular routing. Inhibition of correct terminal glycosylation of the beta-subunit with deoxynojirimycin or swainsonine has no effect on the trypsin sensitivity of the alpha-subunit, its ability to perform cation-dependent conformation changes or the cellular Na,K-ATPase activity. Acquisition of core-sugars is sufficient for the enzyme to assume its catalytic functions. On the other hand, complete inhibition of glycosylation with tunicamycin leads to a destabilization of both the beta- and the alpha-subunits as judged by their higher trypsin sensitivity. In addition, tunicamycin treatment results in a decrease of the amount of newly synthesized beta- and alpha-subunit indicating that a glycoprotein, possibly the beta-subunit itself, plays a role in the efficient accumulation of the alpha-subunit in the endoplasmic reticulum.
1-Deoxynojirimycin↗
Effects of neuropeptide Y on the blood pressure response to various vasoconstrictor agents.
Neuropeptide Y (NPY) is known to potentiate the pressor effect of norepinephrine. In the present work, we evaluated in unanesthetized normotensive rats the effect of NPY on blood pressure responsiveness not only to norepinephrine, but also to tyramine, a sympathomimetic agent acting indirectly to B-HT933, a selective alpha-2 adrenoceptor stimulant, to angiotensin II and vasopressin. Dose-response curves to the various pressor agents were established starting at the 45th min of an i.v. infusion with either NPY (0.025 and 0.1 microgram/min) or its vehicle. The two doses of NPY increased blood pressure by an average of approximately 6 mm Hg, which was not significantly different from the vehicle-induced blood pressure changes. NPY significantly enhanced the pressor effect of norepinephrine, tyramine and angiotensin II, but not that of B-HT933 and vasopressin. We also tested whether NPY inhibits the enzyme activity of Na, K-adenosine triphosphatase using a purified toad kidney preparation. Concentrations of NPY from 10(-14) M up to 10(-6) M had no effect on the enzyme activity. It appears therefore that the blood pressure potentiating effect of NPY is not restricted to alpha adrenoceptor stimulation with norepinephrine, but involves also the vasoconstrictor hormone angiotensin II. This NPY-induced potentiation does not seem to depend upon stimulation of alpha-2 adrenoceptors or inhibition of Na,K-adenosine triphosphatase.
Animals↗
Effects of tunicamycin on the cellular expression and structural arrangement of Na K-ATPase.
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Animals↗
Biosynthesis, membrane insertion and maturation of Na, K-ATPase.
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Animals↗
Structural organization of alpha-subunit from purified and microsomal toad kidney (Na+ + K+)-ATPase as assessed by controlled trypsinolysis.
The membrane organization of the alpha-subunit of purified (Na+ + K+)-ATPase ((Na+ + K+)-dependent adenosine triphosphate phosphorylase, EC 3.6.1.3) and of the microsomal enzyme of the kidney of the toad Bufo marinus was compared by using controlled trypsinolysis. With both enzyme preparations, digestions performed in the presence of Na+ yielded a 73 kDa fragment and in the presence of K+ a 56 kDa, a 40 kDa and small amounts of a 83 kDa fragment from the 96 kDa alpha-subunit. In contrast to mammalian preparations (Jørgensen, P.L. (1975) Biochim. Biophys. Acta 401, 399-415), trypsinolysis of the purified amphibian enzyme led to a biphasic loss of (Na+ + K+)-ATPase activity in the presence of both Na+ and K+. These data could be correlated with an early rapid cleavage of 3 kDa from the alpha-subunit in both ionic conditions and a slower degradation of the remaining 93 kDa polypeptide. On the other hand, in the microsomal enzyme, a 3 kDa shift of the alpha-subunit could only be produced in the presence of Na+. Our data indicate that (1) purification of the amphibian enzyme with detergent does not influence the overall topology of the alpha-subunit but produces a distinct structural alteration of its N-terminus and (2) the amphibian kidney enzyme responds to cations with similar conformational transitions as the mammalian kidney enzyme. In addition, anti alpha-serum used on digested enzyme samples revealed on immunoblots that the 40 kDa fragment was better recognized than the 56 kDa fragment. It is concluded that the NH2-terminal of the alpha-subunit contains more antigenic sites than the COOH-terminal domain in agreement with the results of Farley et al. (Farley, R.A., Ochoa, G.T. and Kudrow, A. (1986) Am. J. Physiol. 250, C896-C906).
Animals↗
Identification of two isoforms of the catalytic subunit of Na,K-ATPase in myocytes from adult rat heart.
The present study demonstrates that two forms of the alpha catalytic subunit of the Na,K-ATPase are present in rat heart and originate from cardiomyocytes. They were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction and alkylation of the sulfhydryl groups. The two forms were identified on immunoblots using two specific antisera against either the alpha subunit from Bufo marinus kidney and the alpha and beta subunits from lamb kidney. Comparison of the two forms to the alkylated Na,K-ATPase from rat kidney (containing one catalytic subunit) and from rat brain (containing alpha and alpha + subunits) suggested that, in rat cardiac myocytes, the form with a fast migration rate (alpha F) corresponds to the alpha subunit of low ouabain affinity and the one with a slow migration rate (alpha S), to a subunit of high ouabain affinity. Thus, the existence of two isoforms of the catalytic subunit in cardiac myocytes accounts well for the biphasic ouabain inhibition of the Na,K-ATPase activity and for the biphasic inotropic responsiveness to cardiac glycosides of the rat heart.
Alkylation↗
Regulation by aldosterone of Na+,K+-ATPase mRNAs, protein synthesis, and sodium transport in cultured kidney cells.
Transepithelial Na+ reabsorption across tight epithelia is regulated by aldosterone. Mineralocorticoids modulate the expression of a number of proteins. Na+,K+-ATPase has been identified as an aldosterone-induced protein (Geering, K., M. Girardet, C. Bron, J. P. Kraehenbuhl, and B. C. Rossier, 1982, J. Biol. Chem., 257:10338-10343). Using A6 cells (kidney of Xenopus laevis) grown on filters we demonstrated by Northern blot analysis that the induction of Na+,K+-ATPase was mainly mediated by a two- to fourfold accumulation of both alpha- and beta-subunit mRNAs. The specific competitor spironolactone decreased basal Na+ transport, Na+,K+-ATPase mRNA, and the relative rate of protein biosynthesis, and it blocked the response to aldosterone. Cycloheximide inhibited the aldosterone-dependent sodium transport but did not significantly affect the cytoplasmic accumulation of Na+,K+-ATPase mRNA induced by aldosterone.
Aldosterone↗
Maturation of the catalytic alpha-subunit of Na,K-ATPase during intracellular transport.
The protease sensitivity of the catalytic alpha-subunit of Na,K-ATPase during intracellular transport along the exocytic pathway has been investigated in two amphibian epithelial cell lines. Controlled trypsinolysis followed by immunoprecipitation of cell homogenates or microsomal fractions from [35S]methionine pulse-chased A6 kidney cells revealed distinct cleavage patterns by SDS-PAGE. Shortly after synthesis (7-min pulse), the 98-kD alpha-subunit is fully sensitive to trypsin digestion and is cleaved into a 35-kD membrane-bound and a 27.5-kD soluble peptide. With a 15-min pulse, 10% of the newly synthesized polypeptide becomes resistant to trypsin digestion. With longer chase time, the proportion of protease-resistant alpha-subunit further increases. Concomitantly, the alpha-subunit acquires the ability to undergo cation-dependent conformational transitions, as reflected by distinct tryptic digest patterns in the presence of Na+ or K+. Similar results were obtained in TBM cells, a toad bladder cell line. Our data indicate that the catalytic subunit of Na,K-ATPase is structurally rearranged during intracellular transport from its site of synthesis to its site of action at the cell surface, a modification which might mark the functional maturation of the enzyme.
Animals↗
Aldosterone regulation of Na+ transport and Na+-K+-ATPase in A6 cells: role of growth conditions.
The effects of aldosterone on transepithelial sodium transport (measured by the short-circuit current (SCC) and on Na+-K+-adenosine triphosphatase (ATPase) biogenesis have been studied in A6 kidney cells grown on collagen-coated filters in two different media. In medium A, base-line SCCA was close to zero but transmural electrical resistance (RA) was high. Aldosterone (100 nM, t24h) drastically increased SCCA and RA, but only after a 4-h latent period. In medium B, base-line SCCB and RB were significantly higher than in medium A. Aldosterone significantly enhanced SCCB and to a lesser extent RB after a much shorter latent period (approximately 45 min) than in medium A. In medium A, aldosterone elicited a fourfold increase in the relative rate of synthesis of alpha- and beta-subunits of Na+-K+-ATPase. A twofold increase was already observed within the observed latent period. This time course suggests that de novo synthesis of sodium pumps might be one of the critical factors underlying the increase in sodium transport in this growth medium. In medium B, aldosterone elicited a two- to fourfold increase in the relative rate of synthesis of the alpha- and beta-subunits of Na+-K+-ATPase that paralleled SCCB. Thus de novo synthesis of Na+-K+-ATPase is clearly not a prerequisite for the early mineralocorticoid response (t90 min - t180 min), but still could be part of the late mineralocorticoid response (t3 h - t24 h). In both media, the immunochemical cellular pool of Na+-K+-ATPase was apparently not modulated by aldosterone for up to 48 h of incubation.(ABSTRACT TRUNCATED AT 250 WORDS)
Aldosterone↗
Three ATPase activities have an abnormal developmental time course in trembler sciatic nerves.
Trembler mutant mice are affected by a peripheral neuropathy characterized by hypomyelination, demyelination, and Schwann cell proliferation. In adult mutants, supernumerary Schwann cells form membranous structures known as 'onion-bulb' formations. The activities of the Na+, K+-ATPase and of two ouabain-insensitive Mg2+-ATPases were investigated in sciatic nerves of young and adult mutants. The Na+, K+-ATPase activities were 92 and 76% of the control values in young and adult mutants, respectively. By immunoblot analysis, the alpha-subunit of the Na+, K+-ATPase had an identical apparent molecular weight in controls at both ages and in young mutants. In adult mutants, on the contrary, the alpha-subunit appeared smaller by about 2 kd, similar to that in kidney, indicating that the Na+, K+-ATPase was localized mainly on supernumerary Schwann cells. In addition, in the mutants, the developmental increase of both the mitochondrial and the nonmitochondrial Mg2+-ATPase was abnormally high. We suggest that the abnormal increase of the nonmitochondrial Mg2+-ATPase activity during development reflects an enrichment of that enzyme in 'onion-bulb' formations.
Animals↗
Control of transepithelial Na+ transport and Na-K-ATPase by oxytocin and aldosterone.
Short- and long-term effect of oxytocin on Na+ transport and Na-K-ATPase biosynthesis in the toad bladder, and the potential interaction of this hormone with aldosterone have been studied, leading to the following observations. An early Na+ transport response (oxytocin, 50 mU/ml) peaked at 10-15 min of hormone addition. At maximal stimulation a three- to fourfold increase in Na+ transport was observed, a sustained Na+ transport response (about two-fold control base line) was observed as long as the hormone was present in the medium and for up to 20 h of incubation. Pretreatment for 30 min with actinomycin D (2 micrograms/ml) did not inhibit the early response, but significantly impaired the sustained response, suggesting that de novo protein synthesis was required. The simultaneous addition of the two hormones led within 60 min to a marked potentiation of the action on Na+ transport. This synergism could be mimicked by exogenous cyclic adenosine monophosphate (cAMP). Oxytocin alone (18 h exposure, 50 mU/ml) increased the relative rate of synthesis of both alpha and beta subunits of Na-K-ATPase (1.9- and 1.6-fold, respectively; P less than 0.05), whereas aldosterone (80 nM) increased the relative rate of synthesis of the same subunits (2.6- and 2.2-fold, respectively; P less than 0.02). Finally, in contrast to what was observed at the physiological level, the interaction of oxytocin and aldosterone did not lead to a similar potentiation at the biochemical level, i.e., induction of Na-K-ATPase biosynthesis (2.7- and 2.9-fold, for alpha and beta subunits, respectively; P less than 0.025).
Aldosterone↗