Functional Role and Immunocytochemical Localization of the γa and γb Forms of the Na,K-ATPase γ Subunit

The γ subunit of the Na,K-ATPase is a member of the FXYD family of type 2 transmembrane proteins that probably function as regulators of ion transport. Rat γ is present primarily in the kidney as two main splice variants, γa and γb, which differ only at their extracellular N termini (TELSANH and MDRWYL, respectively; Kuster, B., Shainskaya, A., Pu, H. X., Goldshleger, R., Blostein, R., Mann, M., and Karlish, S. J. D. (2000) J. Biol. Chem. 275, 18441–18446). Expression in cultured cells indicates that both variants affect catalytic properties, without a detectable difference between γa and γb. At least two singular effects are seen, irrespective of whether the variants are expressed in HeLa or rat α1-transfected HeLa cells, i.e. (i) an increase in apparent affinity for ATP, probably secondary to a left shift in E1 ↔E2 conformational equilibrium and (ii) an increase in K+ antagonism of cytoplasmic Na+activation. Antibodies against the C terminus common to both variants (anti-γ) abrogate the first effect but not the second. In contrast, γa and γb show differences in their localization along the kidney tubule. Using anti-γ (C-terminal) and antibodies to the rat α subunit as well as antibodies to identify cell types, double immunofluorescence showed γ in the basolateral membrane of several tubular segments. Highest expression is in the medullary portion of the thick ascending limb (TAL), which contains both γa and γb. In fact, TAL is the only positive tubular segment in the medulla. In the cortex, most tubules express γ but at lower levels. Antibodies specific for γa and γb showed differences in their cortical location; γa is specific for cells in the macula densa and principal cells of the cortical collecting duct but not cortical TAL. In contrast, γb but not γa is present in the cortical TAL only. Thus, the importance of γa and γb may be related to their partially overlapping but distinct expression patterns and tissue-specific functions of the pump that these serve. The γ subunit of the Na,K-ATPase is a member of the FXYD family of type 2 transmembrane proteins that probably function as regulators of ion transport. Rat γ is present primarily in the kidney as two main splice variants, γa and γb, which differ only at their extracellular N termini (TELSANH and MDRWYL, respectively; Kuster, B., Shainskaya, A., Pu, H. X., Goldshleger, R., Blostein, R., Mann, M., and Karlish, S. J. D. (2000) J. Biol. Chem. 275, 18441–18446). Expression in cultured cells indicates that both variants affect catalytic properties, without a detectable difference between γa and γb. At least two singular effects are seen, irrespective of whether the variants are expressed in HeLa or rat α1-transfected HeLa cells, i.e. (i) an increase in apparent affinity for ATP, probably secondary to a left shift in E1 ↔E2 conformational equilibrium and (ii) an increase in K+ antagonism of cytoplasmic Na+activation. Antibodies against the C terminus common to both variants (anti-γ) abrogate the first effect but not the second. In contrast, γa and γb show differences in their localization along the kidney tubule. Using anti-γ (C-terminal) and antibodies to the rat α subunit as well as antibodies to identify cell types, double immunofluorescence showed γ in the basolateral membrane of several tubular segments. Highest expression is in the medullary portion of the thick ascending limb (TAL), which contains both γa and γb. In fact, TAL is the only positive tubular segment in the medulla. In the cortex, most tubules express γ but at lower levels. Antibodies specific for γa and γb showed differences in their cortical location; γa is specific for cells in the macula densa and principal cells of the cortical collecting duct but not cortical TAL. In contrast, γb but not γa is present in the cortical TAL only. Thus, the importance of γa and γb may be related to their partially overlapping but distinct expression patterns and tissue-specific functions of the pump that these serve. human embryonic kidney thick ascending limb of Henle's loop cortical and medullary portions, respectively, of the thick ascending limb of Henle's loop macula densa aquaporin 2 A small membrane protein, γ, first described over 20 years ago in purified kidney Na,K-ATPase preparations (1Forbush III, B. Kaplan J.H. Hoffman J.F. Biochemistry. 1978; 17: 3667-3676Crossref PubMed Scopus (224) Google Scholar, 2Reeves A.S. Collins J.H. Schwartz A. Biochem. Biophys. Res. Commun. 1980; 95: 1591-1598Crossref PubMed Scopus (56) Google Scholar) associates, in approximately equimolar amounts, with the α and β subunits (3Collins J.H. Forbush III, B. Lane L.K. Ling E. Schwartz A. Zot A. Biochim. Biophys. Acta. 1982; 686: 7-12Crossref PubMed Scopus (32) Google Scholar, 4Hardwicke P.M. Freytag J.W.A. Biochem. Biophys. Res. Commun. 1981; 102: 250-257Crossref PubMed Scopus (31) Google Scholar). Molecular cloning of the γ subunits of rat, mouse, cow, and sheep indicated a molecular weight of ∼6500 (5Mercer R.W. Biemesderfer D. Bliss Jr., D.P. Collins J.H. Forbush III, B. J. Cell Biol. 1993; 121: 579-586Crossref PubMed Scopus (184) Google Scholar). Cloning and sequencing of the human (6Kim J.W. Lee Y. Lee H.I.A. Kang B. Choe Y.K. Choe I.S.,. Biochim. Biophys. Acta. 1997; 1350: 133-135Crossref PubMed Scopus (31) Google Scholar) and Xenopus laevis (7Béguin P. Wang X. Firsov D. Puoti A. Claeys D. Horisberger J.D. Geering K. EMBO J. 1997; 16: 4250-4260Crossref PubMed Scopus (214) Google Scholar) γ subunits of γ subunits of the but is for only that γ a transmembrane and an N C (7Béguin P. Wang X. Firsov D. Puoti A. Claeys D. Horisberger J.D. Geering K. EMBO J. 1997; 16: 4250-4260Crossref PubMed Scopus (214) Google Scholar, J. Biol. Chem. 1997; PubMed Scopus Google Scholar). In two at the molecular as described the γ subunit as a molecular of and (5Mercer R.W. Biemesderfer D. Bliss Jr., D.P. Collins J.H. Forbush III, B. J. Cell Biol. 1993; 121: 579-586Crossref PubMed Scopus (184) Google Scholar, J. Biol. Chem. 1997; PubMed Scopus Google and a is expression in cells J. Biol. Chem. 1997; PubMed Scopus Google Scholar, E. J. Biol. Chem. PubMed Scopus Google Scholar) and in in expression in the (5Mercer R.W. Biemesderfer D. Bliss Jr., D.P. Collins J.H. Forbush III, B. J. Cell Biol. 1993; 121: 579-586Crossref PubMed Scopus (184) Google Scholar) but not of (5Mercer R.W. Biemesderfer D. Bliss Jr., D.P. Collins J.H. Forbush III, B. J. Cell Biol. 1993; 121: 579-586Crossref PubMed Scopus (184) Google Scholar, P. Wang X. Firsov D. Puoti A. Claeys D. Horisberger J.D. Geering K. EMBO J. 1997; 16: 4250-4260Crossref PubMed Scopus (214) Google Scholar). of the γ of rat kidney Na,K-ATPase showed that γa a of B. A. J. Biol. Chem. PubMed Scopus Google to that for the without the J. Biol. Chem. PubMed Scopus Google γb a of and sequencing that the of are in γb, but the two are are to the expressed E. PubMed Scopus Google Scholar). Expression of γa or γb in human embryonic kidney as well as HeLa cells against a the of the γ The showed that the expressed to γa or γb of the in and in HeLa cells that these are B. A. J. Biol. Chem. PubMed Scopus Google Scholar). showed that the γ subunit is with α and β subunits in kidney and not at the in the of α and β subunits (7Béguin P. Wang X. Firsov D. Puoti A. Claeys D. Horisberger J.D. Geering K. EMBO J. 1997; 16: 4250-4260Crossref PubMed Scopus (214) Google J. Cell Biol. 1997; PubMed Scopus Google Scholar) expression of γ in the of the pump the of In with the localization of α and β the γ subunit is expressed in a of J. Biol. Chem. 1997; PubMed Scopus Google Scholar). showed expression patterns of α and γ in tubules and collecting as well as of the γ subunit with both the α and β subunits in kidney (5Mercer R.W. Biemesderfer D. Bliss Jr., D.P. Collins J.H. Forbush III, B. J. Cell Biol. 1993; 121: 579-586Crossref PubMed Scopus (184) Google Scholar). The of the γ subunit only to be that the γ is not for function P.M. Freytag J.W.A. Biochem. Biophys. Res. Commun. 1981; 102: 250-257Crossref PubMed Scopus (31) Google Scholar, Biochim. Biophys. Acta. PubMed Scopus Google Scholar, R.W. J. Biol. Chem. 1993; PubMed Google Scholar) and γ subunit not be in in both (5Mercer R.W. Biemesderfer D. Bliss Jr., D.P. Collins J.H. Forbush III, B. J. Cell Biol. 1993; 121: 579-586Crossref PubMed Scopus (184) Google Scholar, J.W. Lee Y. Lee H.I.A. Kang B. Choe Y.K. Choe I.S.,. Biochim. Biophys. Acta. 1997; 1350: 133-135Crossref PubMed Scopus (31) Google Scholar) and (7Béguin P. Wang X. Firsov D. Puoti A. Claeys D. Horisberger J.D. Geering K. EMBO J. 1997; 16: 4250-4260Crossref PubMed Scopus (214) Google that γ an in Thus, of with γ subunit the of expressed γ subunit and a in as well as J. Cell Biol. 1997; PubMed Scopus Google Scholar). A a in γ in a family with a of γ in H. PubMed Scopus Google Scholar). in J. Biol. Chem. 1997; PubMed Scopus Google Scholar, J. Biol. Chem. PubMed Scopus Google Scholar) that γ is a tissue-specific of the Na,K-ATPase and that an increase in the apparent affinity of the for in a that is anti-γ The specific effect of anti-γ affinity a specific the γ subunit in in kidney cells in which is A of the γ subunit in of the Na,K-ATPase with K+ the of that the γ subunit is a of the in the of of K+ or the to E. Biochemistry. PubMed Scopus Google and K+ against of the γ subunit in J. Biol. Chem. 1997; PubMed Scopus Google Scholar). In Xenopus that the γ subunit an the apparent affinity of the Na,K-ATPase for K+ in a and (7Béguin P. Wang X. Firsov D. Puoti A. Claeys D. Horisberger J.D. Geering K. EMBO J. 1997; 16: 4250-4260Crossref PubMed Scopus (214) Google the of these A of and K+ to the γa subunit expressed in kidney cells E. J. Biol. Chem. PubMed Scopus Google Scholar). The γ subunit to ion in and in cells R.W. S. A. 95: PubMed Scopus Google Scholar). the of B. A. J. Biol. Chem. PubMed Scopus Google Scholar) two main γ in rat kidney and is that (7Béguin P. Wang X. Firsov D. Puoti A. Claeys D. Horisberger J.D. Geering K. EMBO J. 1997; 16: 4250-4260Crossref PubMed Scopus (214) Google Scholar, J. Biol. Chem. 1997; PubMed Scopus Google Scholar, E. J. Biol. Chem. PubMed Scopus Google Scholar, J. Biol. Chem. PubMed Scopus Google Scholar, Biochim. Biophys. Acta. PubMed Scopus Google Scholar, R.W. J. Biol. Chem. 1993; PubMed Google Scholar) effects of only the γa the of whether the two variants distinct The described in two of the of γ in of of the and the of and to the two variants distinct effects Na,K-ATPase The of the localization of the two γ variants along the Rat kidney of or partially purified Na,K-ATPase of as in PubMed Scopus Google preparations of anti-γ (C-terminal) in is against both of the kidney γ subunit a The purified an affinity of the to the of the γ is to as anti-γ The is described in J. Biol. Chem. 1997; PubMed Scopus Google against a of the and for without and antibodies against and respectively, to purified an affinity of to affinity to affinity or and the antibodies purified a The the in a of with and with and for with anti-γ (C-terminal) and and In the antibodies described in H. J. PubMed Google Scholar) for (i) 2 (ii) against the subunit of Na,K-ATPase Rat in as described H. J. 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The γa is in the basolateral membrane of cells and of cortical collecting duct principal cells as well as in the medullary portion of the thick ascending limb of Henle's The γb is expressed along the thick ascending limb of Henle's loop of the γa and γb of the γ subunit along the and and of the collecting and and of the collecting in a In showed that anti-γ against the C terminus of γ Na,K-ATPase of the but not of that not express γ J. Biol. Chem. 1997; PubMed Scopus Google Scholar). showed that anti-γ the apparent affinity for probably the of the Thus, the of the of is with a of anti-γ in the equilibrium of the K+ that anti-γ effects between the Na,K-ATPase and the γ that the of the γ subunit is to shift the equilibrium of the two γ variants cells, that is the at least for γa J. Biol. Chem. PubMed Scopus Google Scholar). 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