FXYD6 Is a Novel Regulator of Na,K-ATPase Expressed in the Inner Ear

The exquisite sensitivity of the cochlea, which mediates the transduction of sound waves into nerve impulses, depends on the endolymph ionic composition and the endocochlear potential. A key protein in the maintenance of the electrochemical composition of the endolymph is the Na,K-ATPase. In this study, we have looked for the presence in the rat inner ear of members of the FXYD protein family, recently identified as tissue-specific modulators of Na,K-ATPase. Only FXYD6 is detected at the protein level. FXYD6 is expressed in various epithelial cells bordering the endolymph space and in the auditory neurons. FXYD6 co-localizes with Na,K-ATPase in the stria vascularis and can be co-immunoprecipitated with Na,K-ATPase. After expression in Xenopus oocytes, FXYD6 associates with Na,K-ATPase α1-β1 and α1-β2 isozymes, which are preferentially expressed in different regions of the inner ear and also with gastric and non-gastric H,K-ATPases. The apparent K+ and Na+ affinities of α1-β1 and α1-β2 isozymes are different. Association of FXYD6 with Na,K-ATPase α1-β1 isozymes slightly decreases their apparent K+ affinity and significantly decreases their apparent Na+ affinity. On the other hand, association with α1-β2 isozymes increases their apparent K+ and Na+ affinity. The effects of FXYD6 on the apparent Na+ affinity of Na,K-ATPase and the voltage dependence of its K+ effect are distinct from other FXYD proteins. In conclusion, this study defines the last FXYD protein of unknown function as a modulator of Na,K-ATPase. Among FXYD protein, FXYD6 is unique in its expression in the inner ear, suggesting a role in endolymph composition. The exquisite sensitivity of the cochlea, which mediates the transduction of sound waves into nerve impulses, depends on the endolymph ionic composition and the endocochlear potential. A key protein in the maintenance of the electrochemical composition of the endolymph is the Na,K-ATPase. In this study, we have looked for the presence in the rat inner ear of members of the FXYD protein family, recently identified as tissue-specific modulators of Na,K-ATPase. Only FXYD6 is detected at the protein level. FXYD6 is expressed in various epithelial cells bordering the endolymph space and in the auditory neurons. FXYD6 co-localizes with Na,K-ATPase in the stria vascularis and can be co-immunoprecipitated with Na,K-ATPase. After expression in Xenopus oocytes, FXYD6 associates with Na,K-ATPase α1-β1 and α1-β2 isozymes, which are preferentially expressed in different regions of the inner ear and also with gastric and non-gastric H,K-ATPases. The apparent K+ and Na+ affinities of α1-β1 and α1-β2 isozymes are different. Association of FXYD6 with Na,K-ATPase α1-β1 isozymes slightly decreases their apparent K+ affinity and significantly decreases their apparent Na+ affinity. On the other hand, association with α1-β2 isozymes increases their apparent K+ and Na+ affinity. The effects of FXYD6 on the apparent Na+ affinity of Na,K-ATPase and the voltage dependence of its K+ effect are distinct from other FXYD proteins. In conclusion, this study defines the last FXYD protein of unknown function as a modulator of Na,K-ATPase. Among FXYD protein, FXYD6 is unique in its expression in the inner ear, suggesting a role in endolymph composition. The mammalian inner ear is composed of two distinct sensory organs, the cochlea (the organ of hearing) and the vestibule (the organ of equilibrium). Proper functioning of the sensory hair cells depends on the ionic composition of endolymph and perilymph. Whereas perilymph resembles most other interstitial fluids in containing low K+ and high Na+, endolymph has an ionic composition characterized by high K+ and low Na+, similar to the intracellular fluid (1Bosher S.K. Warren R.L. Proc. R. Soc. Lond. B Biol. Sci. 1968; 171: 227-247Crossref PubMed Google Scholar, 2Morgenstern C. Amano H. Orsulakova A. Am. J. Otolaryngol. 1982; 3: 323-327Crossref PubMed Scopus (38) Google Scholar, 3Smith C.A. Lowry O.H. Wu M.L. Laryngoscope. 1954; 64: 141-153PubMed Google Scholar). K+ is central to the inner ear physiology, as it is the charge-carrying ion for the sensory transduction. In the cochlea, K+ is secreted by the marginal cells of the stria vascularis to maintain a very high concentration (150 mm) in the endolymph, the extracellular fluid bathing the stereocilia of the sensory hair cells. This results in a positive potential (+90 mV), which provides a large driving force for K+ entry into the sensory hair cell. The unique ionic composition of endolymph, along with a highly positive endocochlear potential, are thus essential to mechanoelectric transduction (by hair cells) and to normal hearing (4Dallos P. J. Neurosci. 1992; 12: 4575-4585Crossref PubMed Google Scholar, 5Kuijpers W. Bonting S.L. Pfluegers Arch. 1970; 320: 348-358Crossref PubMed Scopus (108) Google Scholar, 6Kuijpers W. Bonting S.L. Pfluegers Arch. 1970; 320: 359-372Crossref PubMed Scopus (121) Google Scholar). Maintenance of both the ionic composition of endolymph and the endocochlear potential depends on the activity of Na,K-ATPase (5Kuijpers W. Bonting S.L. Pfluegers Arch. 1970; 320: 348-358Crossref PubMed Scopus (108) Google Scholar, 6Kuijpers W. Bonting S.L. Pfluegers Arch. 1970; 320: 359-372Crossref PubMed Scopus (121) Google Scholar). The Na,K-ATPase is an ubiquitous enzyme consisting of an α- and a β-subunit, which is responsible for the creation and maintenance of the Na+ and K+ gradients across the cell membrane by transporting three Na+ out of and two K+ into the cell. This function is crucial for cell survival and body homeostasis, because the Na+ gradient is used as an energy source to transport ions or solutes and is at the origin of the vectorial Na+ reabsorption in the kidney and of action potentials in excitable tissues. Regulation of the activity and expression of Na,K-ATPase is tight and governed by a variety of mechanisms. Short term regulation involves protein kinases and results in modulation of the cell surface expression of the Na,K-ATPase, whereas long term regulation, mediated by mineralocorticoid or thyroid hormones, leads to a change in the total number of Na,K-ATPase units (for review, see Refs. 7Therien A.G. Blostein R. Am. J. Physiol. 2000; 279: C541-C566Crossref PubMed Google Scholar and 8Feraille E. Doucet A. Physiol. Rev. 2001; 81: 345-418Crossref PubMed Scopus (437) Google Scholar). Moreover, the existence of multiple α and β isoforms permits the production of isozymes with different transport properties (9Crambert G. Hasler U. Beggah A.T. Yu C. Modyanov N.N. Horisberger J.D. Lelievre L. Geering K. J. Biol. Chem. 2000; 275: 1976-1986Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). Finally, recent experimental evidence shows that members of the FXYD protein family specifically associate with and modulate the transport properties of Na,K-ATPase (for review, see Refs. 10Crambert G. Geering K. Science's STKE. 2003; : RE1PubMed Google Scholar and 11Geering K. Am. J. Physiol. 2006; 290: F241-F250Crossref PubMed Scopus (293) Google Scholar). The mammalian FXYD family contains seven members that share a common signature sequence encompassing the transmembrane and adjacent regions (12Sweadner K.J. Rael E. Genomics. 2000; 68: 41-56Crossref PubMed Scopus (358) Google Scholar). Most characterized FXYD proteins exhibit a similar structure with a single transmembrane domain and a type I orientation that is achieved, in some but not all cases, by the cleavage of an N-terminal signal peptide (for references, see Ref. 10Crambert G. Geering K. Science's STKE. 2003; : RE1PubMed Google Scholar). So far, six of the seven members of the FXYD protein family have been shown to regulate Na,K-ATPase in a tissue- and isozyme-specific way. Four FXYD proteins (FXYD1, FXYD3, FXYD4, and FXYD7) affect, in a distinct way, the apparent affinity for extracellular K+ of the Na,K-ATPase, which in the case of the brain-specific FXYD7 may be physiologically relevant in neuronal excitability (13Beguin P. Crambert G. Monnet-Tschudi F. Uldry M. Horisberger J.D. Garty H. Geering K. EMBO J. 2002; 21: 3264-3273Crossref PubMed Scopus (119) Google Scholar). In addition, FXYD1 (phospholemman) (14Crambert G. Fuzesi M. Garty H. Karlish S. Geering K. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11476-11481Crossref PubMed Scopus (226) Google Scholar), FXYD2 (γ-subunit) (15Arystarkhova E. Wetzel R.K. Asinovski N.K. Sweadner K.J. J. Biol. Chem. 1999; 274: 33183-33185Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 16Beguin P. Wang X. Firsov D. Puoti A. Claeys D. Horisberger J.D. Geering K. EMBO J. 1997; 16: 4250-4260Crossref PubMed Scopus (215) Google Scholar, 17Pu H.X. Cluzeaud F. Goldshleger R. Karlish S.J. Farman N. Blostein R. J. Biol. Chem. 2001; 276: 20370-20378Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), FXYD3 (18Crambert G. Li C. Claeys D. Geering K. Mol. Biol. Cell. 2005; 16: 2363-2371Crossref PubMed Scopus (58) Google Scholar), and FXYD4 (19Beguin P. Crambert G. Guennoun S. Garty H. Horisberger J.D. Geering K. EMBO J. 2001; 20: 3993-4002Crossref PubMed Scopus (134) Google Scholar, 20Garty H. Lindzen M. Scanzano R. Aizman R. Fuzesi M. Goldshleger R. Farman N. Blostein R. Karlish S.J. Am. J. Physiol. 2002; 283: F607-F615Crossref PubMed Scopus (80) Google Scholar) affect the apparent affinity for internal Na+ of Na,K-ATPase in a way that is consistent with the physiological demands of the tissues in which they are expressed. These results suggest that at least one common function of all FXYD proteins could be the regulation of Na,K-ATPase transport properties. Given the key role of the Na,K-ATPase in the maintenance of the electrochemical composition of the endolymph and the modulatory role of FXYD proteins on its transport properties, we have looked in this study for the expression of several FXYD proteins in the inner ear. The only FXYD protein that we were able to detect by Western blot was FXYD6. We, therefore, characterized FXYD6, the last member of the FXYD family of unknown function. FXYD6, also known as phosphohippolin, was previously identified as a phospholemman-like protein expressed at high levels in brain and at a lesser level in lung, testis, and colon (21Kadowaki K. Sugimoto K. Yamaguchi F. Song T. Watanabe Y. Singh K. Tokuda M. Brain Res. Mol. Brain Res. 2004; 125: 105-112Crossref PubMed Scopus (33) Google Scholar). In this study, we investigated the association of FXYD6 with Na,K- and H,K-ATPases, the modulation of Na,K-ATPase function, and its cellular distribution in the inner ear and in PC12 cells. Our results show that FXYD6 has functional characteristics that are distinct from other FXYD proteins and that the protein may have a crucial role in endolymph homeostasis. cDNAs—Mouse FXYD6 cDNA, subcloned between the 5′ and 3′ domains of Xenopus β-globin, was kindly provided by H. Garty (Weizmann Institute of Science). Cloning of rat Na,K-ATPase α1, α2, α3, β1, and β2 cDNAs has been described previously (9Crambert G. Hasler U. Beggah A.T. Yu C. Modyanov N.N. Horisberger J.D. Lelievre L. Geering K. J. Biol. Chem. 2000; 275: 1976-1986Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). cDNAs for the rabbit gastric H,K-ATPase α- and β-subunits were kindly provided by G. Sachs (UCLA) and cDNA for rat colonic H,K-ATPase α-subunits by Fréderic Jaisser (INSERM, U478, Paris, France). All cDNAs were introduced into the pSD5 vector, and cRNAs were prepared by in vitro translation (22Melton D.A. Krieg P.A. Rebagliati M.R. Maniatis T. Zinn K. Green M.R. Nucleic Acids Res. 1984; 12: 7035-7056Crossref PubMed Scopus (4402) Google Scholar). Protein Expression in Xenopus laevis Oocytes—Stages V and VI oocytes were obtained from X. laevis as described previously (23Geering K. Beggah A. Good P. Girardet S. Roy S. Schaer D. Jaunin P. J. Cell Biol. 1996; 133: 1193-1204Crossref PubMed Scopus (125) Google Scholar). cRNAs coding for mouse FXYD6, rat Na,K-ATPase α1-, α2-, α3-, β1-, and β2-subunits, or rabbit gastric or rat colonic H,K-ATPase α and gastric H,K-ATPase β-subunits were injected into oocytes in different combinations, as described in the figure legends. To study protein expression and association, oocytes were incubated in modified Barth's solution in the presence of 0.7–1 mCi/ml [35S]methionine (Easy Tag were to a and to in modified Barth's solution containing After the and were prepared as described previously (23Geering K. Beggah A. Good P. Girardet S. Roy S. Schaer D. Jaunin P. J. Cell Biol. 1996; 133: 1193-1204Crossref PubMed Scopus (125) Google Scholar) and to with an FXYD6 of of were and in a The was in a containing Science). The was with a containing for at and at for The were used for and Western and Western FXYD6 a peptide of mouse FXYD6 was by The cDNA was into the After and used the protein was used for the of This was used for of Na,K- and H,K-ATPase expressed in Xenopus oocytes G. P. Modyanov N.N. Geering K. 2002; PubMed Scopus Google Scholar). were on or and by FXYD1 (14Crambert G. Fuzesi M. Garty H. Karlish S. Geering K. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11476-11481Crossref PubMed Scopus (226) Google Scholar), FXYD2 P. Wang X. Firsov D. Puoti A. Claeys D. Horisberger J.D. Geering K. EMBO J. 1997; 16: 4250-4260Crossref PubMed Scopus (215) Google Scholar), FXYD3 (18Crambert G. Li C. Claeys D. Geering K. Mol. Biol. Cell. 2005; 16: 2363-2371Crossref PubMed Scopus (58) Google Scholar), FXYD4 (19Beguin P. Crambert G. Guennoun S. Garty H. Horisberger J.D. Geering K. EMBO J. 2001; 20: 3993-4002Crossref PubMed Scopus (134) Google Scholar), FXYD6, and FXYD7 (13Beguin P. Crambert G. Monnet-Tschudi F. Uldry M. Horisberger J.D. Garty H. Geering K. EMBO J. 2002; 21: 3264-3273Crossref PubMed Scopus (119) Google Scholar) were used in Western to the expression of FXYD proteins in the inner ear. To the association of FXYD6 with Na,K-ATPase in rat cochlea, of the protein of were incubated with a Na,K-ATPase at were on a and FXYD6 was with the FXYD6 and were with rat Na,K-ATPase α1-β1 or α1-β2 cRNAs or with FXYD6 by the voltage of the apparent K+ affinity were out as described previously P. Wang X. Firsov D. Puoti A. Claeys D. Horisberger J.D. Geering K. EMBO J. 1997; 16: 4250-4260Crossref PubMed Scopus (215) Google Scholar) in the presence of which the but not the expressed rat Na,K-ATPase The and the apparent K+ affinity in the presence of Na+ were obtained by the to the with a of F. Jaunin P. Geering K. Horisberger J.D. J. Physiol. PubMed Scopus Google Scholar). of the apparent Na+ affinity of Na,K-ATPase in cells were as described previously U. Wang X. Crambert G. P. Jaisser F. Horisberger J.D. Geering K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) by rat with rat or β2 cRNAs with rat epithelial Na+ and cRNAs in the presence or of FXYD6 The and the apparent Na+ affinity were by the and a of U. Wang X. Crambert G. P. Jaisser F. Horisberger J.D. Geering K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). was by of were and with in were the and were and a was at the an at were to a containing for and were with a and at The were similar to described previously N. N. G. M. J. Neurosci. 2003; PubMed Scopus Google Scholar). The were in in normal with and incubated at with FXYD6 Na,K-ATPase and in with normal were in and incubated for with to in and The were in and in of FXYD6 and in the stria a T. H. K. T. Y. Y. Am. J. Physiol. 2006; PubMed Scopus Google Scholar) provided by was used at a of The were with a was the was in the or were PC12 Cell cells provided by a cell used to study were in in modified with and at To was with nerve for cells were at on All were in in for and with were with and with in for were with an FXYD6 and Na,K-ATPase in for at with and incubated with and for were with and with was a of PC12 cells were prepared in a containing and used for of FXYD6 in the and with Na,K-ATPase in expression of several FXYD has been in the inner ear of E. C. Y. P. J. Res. Otolaryngol. 2005; PubMed Scopus Google Scholar, J. Res. Otolaryngol. 2002; 3: PubMed Scopus Google Scholar). Western blot which of FXYD proteins only FXYD6 could be detected in of rat inner ear FXYD6 expression was low at but at and FXYD6 was as a with a at to the of FXYD6 expressed in brain (21Kadowaki K. Sugimoto K. Yamaguchi F. Song T. Watanabe Y. Singh K. Tokuda M. Brain Res. Mol. Brain Res. 2004; 125: 105-112Crossref PubMed Scopus (33) Google Scholar) and a at of unknown The cellular of the FXYD6 protein in the cochlea was investigated by In the rat cochlea, FXYD6 was expressed in various bordering the endolymph (the the cells and their and the stria Moreover, the protein was expressed in and of the neurons. FXYD6 with Na,K-ATPase and in several regions the stria A Na,K-ATPase was able to FXYD6 in the inner ear at and that FXYD6 is with Na,K-ATPase. of FXYD6 in the is expressed in the stria vascularis it co-localizes with Na,K-ATPase A signal was detected in the of the stria vascularis of marginal cells and of cells are The of was not to the of FXYD6, because the membrane of marginal cells and the membrane of cells are in of and R. Am. J. PubMed Scopus Google Scholar, Res. 2005; PubMed Scopus Google Scholar). is a of specifically expressed in the membrane of marginal cells R. T. R. E. F. 2001; PubMed Scopus Google Scholar) of FXYD6 and of the two proteins suggesting the presence of FXYD6 in the membrane of marginal cells but not expression in of the stria K+ into endolymph, and distribution of Na,K-ATPase and FXYD6. a see of FXYD6 in PC12 to (21Kadowaki K. Sugimoto K. Yamaguchi F. Song T. Watanabe Y. Singh K. Tokuda M. Brain Res. Mol. Brain Res. 2004; 125: 105-112Crossref PubMed Scopus (33) Google Scholar) have shown that FXYD6 is expressed in cells. To FXYD6 co-localizes with Na,K-ATPase in we with FXYD6 and Na,K-ATPase which of FXYD6 and Na,K-ATPase at the membrane of PC12 cells Moreover, in PC12 cells of neuronal FXYD6 with Na,K-ATPase in and the cell body in and PC12 FXYD6 was with Na,K-ATPase, because it could be co-immunoprecipitated with a Na,K-ATPase Association of FXYD6 with Na,K-ATPase and H,K-ATPase in Xenopus association of FXYD6 with Na,K-ATPase was investigated of FXYD6 in Xenopus oocytes with Na,K-ATPase or α1-β2 After and various were prepared and to with an FXYD6 The FXYD6 the of FXYD6 The of the FXYD6 was because FXYD6 contains only which can be Western blot the expression of FXYD6 and also the of FXYD6 in the inner ear not Na,K-ATPase α- and β-subunits of all isozymes could be co-immunoprecipitated with FXYD6 the and Moreover, FXYD6 co-immunoprecipitated Na,K-ATPase α1-β2 isozymes which is the Na,K-ATPase in the stria vascularis W. Arch. 2001; PubMed Scopus Google Scholar). were with gastric and non-gastric and they that both with FXYD6 could be co-immunoprecipitated with the FXYD6 of FXYD6 on Na,K-ATPase functional effects of FXYD6 on Na,K-ATPase transport properties were investigated by in Xenopus oocytes rat Na,K-ATPase α1-β1 or α1-β2 isozymes with or FXYD6. shown in Na,K-ATPase α1-β2 isozymes expressed FXYD6 a significantly apparent K+ affinity α1-β1 isozymes, and the voltage dependence of the K+ was at membrane FXYD6 a but in the apparent K+ affinity of the Na,K-ATPase α1-β1 at slightly and positive membrane In FXYD6 a in the apparent K+ affinity of Na,K-ATPase α1-β2 isozymes at membrane potentials FXYD6 also and distinct effects on the apparent affinity for internal Na+ of the two Na,K-ATPase isozymes and a in the apparent Na+ affinity of the α1-β1 significantly and a in the apparent Na+ affinity of the α1-β2 In this we evidence that FXYD6, the last FXYD protein, may also be a tissue-specific of Na,K-ATPase, similar to other members of the FXYD protein results show that FXYD6 is with Na,K-ATPase in the inner ear and that it the transport properties of different Na,K-ATPase isozymes in Xenopus Expression of FXYD6 in the expression of several members of the FXYD family in the inner ear has been previously E. C. Y. P. J. Res. Otolaryngol. 2005; PubMed Scopus Google Scholar, J. Res. Otolaryngol. 2002; 3: PubMed Scopus Google Scholar). In this study, we show of FXYD only FXYD6 was detected in this organ at the protein level. the other FXYD proteins are not expressed or at levels FXYD6. of FXYD6 with common of all FXYD proteins characterized is their with Na,K-ATPase, both in expression and in (for review, see Refs. 10Crambert G. Geering K. Science's STKE. 2003; : RE1PubMed Google Scholar and 11Geering K. Am. J. Physiol. 2006; 290: F241-F250Crossref PubMed Scopus (293) Google Scholar). FXYD6 this but shows a other FXYD proteins. to mouse FXYD3 (18Crambert G. Li C. Claeys D. Geering K. Mol. Biol. Cell. 2005; 16: 2363-2371Crossref PubMed Scopus (58) Google Scholar), FXYD6 also with both gastric and non-gastric expression in Xenopus In the cochlea, FXYD6 is in in the stria which Na,K-ATPase and gastric H,K-ATPase at the of marginal cells T. H. K. T. Y. Y. Am. J. Physiol. 2006; PubMed Scopus Google Scholar) are to the potential association of FXYD6 and H,K-ATPase in this of the Regulation of Na,K-ATPase by FXYD6 in the Maintenance of the with other FXYD proteins association of FXYD6 the transport properties of Na,K-ATPase in a distinct way. In of the cellular of FXYD6 in the cochlea, we could at least two of which preferentially Na,K-ATPase α1-β1 or α1-β2 study shows that two rat Na,K-ATPase isozymes have different functional properties These results that β isoforms can modulate the transport properties of Na,K-ATPase (9Crambert G. Hasler U. Beggah A.T. Yu C. Modyanov N.N. Horisberger J.D. Lelievre L. Geering K. J. Biol. Chem. 2000; 275: 1976-1986Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar, F. Jaunin P. Geering K. Horisberger J.D. J. Physiol. PubMed Scopus Google Scholar). The of action of FXYD6 be the auditory Na,K-ATPase α1-β1 Na+ in significantly neuronal from to 2002; PubMed Scopus Google Scholar). The in the apparent Na+ by the association of FXYD6 with Na,K-ATPase α1-β1 isozymes, may be for the of Na+ neuronal a in intracellular Na+ to Na,K-ATPase α1-β1 isozymes with a Na+ of transport at On the other hand, Na,K-ATPase with a Na+ of to association with FXYD6, have the to its transport at intracellular Na+ A similar in the apparent Na+ affinity of α1-β1 isozymes is by FXYD1 (phospholemman) expressed in and and has been to of intracellular Na+ action potentials in tissues (14Crambert G. Fuzesi M. Garty H. Karlish S. Geering K. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11476-11481Crossref PubMed Scopus (226) Google Scholar). at we or in to the Na+ effect on α1-β1 Na,K-ATPase FXYD6 may be in other of the expression or function of Na,K-ATPase isozymes in neuronal cells. The of action of FXYD6 be the stria The stria vascularis is of three of epithelial marginal and epithelial cells cells with cells and are by the K+ from the cells and cells a of it is secreted into the space by and by marginal cells Na,K-ATPase and other K+ Finally, K+ is secreted into the to the FXYD6 is expressed in the marginal cells and in the which α1-β2 Na,K-ATPase isozymes W. Arch. 2001; PubMed Scopus Google Scholar). α1-β2 Na,K-ATPase with FXYD6 with a high apparent affinity for Na+ be to maintain the low intracellular Na+ in marginal cells that have been by K. T. Res. PubMed Scopus Google Scholar). The space is for the of the endocochlear potential. the endocochlear potential is a K+ potential that is by the of two K+ and in the cells of the stria vascularis in with the very low K+ concentration in the fluid and a high K+ concentration in the of cells S. M. A. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, Wu T. P. P. Am. J. Physiol. 2002; PubMed Scopus Google Scholar, L. K.J. W. J. Neurosci. 2005; PubMed Scopus Google Scholar). cells as cells in the extracellular K+ H. PubMed Scopus Google Scholar). Na,K-ATPase as by L. K.J. W. J. Neurosci. 2005; PubMed Scopus Google Scholar), may be in this which the of K+ by the K+ in the space S. M. A. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), the α1-β2 with a K+ of not transport K+ at a membrane potential of of the cells S. M. A. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). an α1-β2 with a apparent affinity for K+ be able to transport at a and a low K+ concentration in the In conclusion, in this study, we have characterized the last FXYD protein of unknown function. Our results show that FXYD6 associates with Na,K-ATPase as other FXYD proteins and has a distinct functional effect on Na,K-ATPase, that all mammalian FXYD proteins associate with and modulate the transport properties of Na,K-ATPase in a tissue-specific way. The of FXYD6 in the inner ear an role in endolymph and Horisberger for in the of the also the of the of which to was kindly provided by and