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W2010243693 abstract "The two variants of the γ subunit of the rat renal sodium pump, γa and γb, have similar effects on the Na,K-ATPase. Both increase the affinity for ATP due to a shift in the enzyme's E1 ↔ E2 conformational equilibrium toward E1. In addition, both increase K+ antagonism of cytoplasmic Na+ activation. To gain insight into the structural basis for these distinct effects, extramembranous N-terminal and C-terminal mutants of γ were expressed in rat α1-transfected HeLa cells. At the N terminus, the variant-distinct region was deleted (γNΔ7) or replaced by alanine residues (γN7A). At the C terminus, four (γaCΔ4) or ten (γaCΔ10) residues were deleted. None of these mutations abrogates the K+/Na+ antagonism as evidenced in a similar increase in K′Na seen at high (100 mm) K+ concentration. In contrast, the C-terminal as well as N-terminal deletions (γNΔ7, γaCΔ4, and γaCΔ10) abolished the decrease in K′ATP seen with wild-type γa or γb. It is concluded that different regions of the γ chain mediate the distinct functional effects of γ, and the effects can be long-range. In the transmembrane region, the impact of G41R replacement was analyzed since this mutation is associated with autosomal dominant renal Mg2+-wasting in man (Meij, I. C., Koenderink, J. B., van Bokhoven, H., Assink, K. F. H., Groenestege, W. T., de Pont, J. J. H. H. M., Bindels, R. J. M., Monnens, L. A. H., Van den Heuvel, L. P. W. J., and Knoers, N. V. A. M. (2000) Nat. Genet. 26, 265–266). The results show that Gly-41 → Arg prevents trafficking of γ but not αβ pumps to the cell surface and abrogates functional effects of γ on αβ pumps. These findings underscore a potentially important role of γ in affecting solute transport, in this instance Mg2+ reabsorption, consequent to its primary effect on the sodium pump. The two variants of the γ subunit of the rat renal sodium pump, γa and γb, have similar effects on the Na,K-ATPase. Both increase the affinity for ATP due to a shift in the enzyme's E1 ↔ E2 conformational equilibrium toward E1. In addition, both increase K+ antagonism of cytoplasmic Na+ activation. To gain insight into the structural basis for these distinct effects, extramembranous N-terminal and C-terminal mutants of γ were expressed in rat α1-transfected HeLa cells. At the N terminus, the variant-distinct region was deleted (γNΔ7) or replaced by alanine residues (γN7A). At the C terminus, four (γaCΔ4) or ten (γaCΔ10) residues were deleted. None of these mutations abrogates the K+/Na+ antagonism as evidenced in a similar increase in K′Na seen at high (100 mm) K+ concentration. In contrast, the C-terminal as well as N-terminal deletions (γNΔ7, γaCΔ4, and γaCΔ10) abolished the decrease in K′ATP seen with wild-type γa or γb. It is concluded that different regions of the γ chain mediate the distinct functional effects of γ, and the effects can be long-range. In the transmembrane region, the impact of G41R replacement was analyzed since this mutation is associated with autosomal dominant renal Mg2+-wasting in man (Meij, I. C., Koenderink, J. B., van Bokhoven, H., Assink, K. F. H., Groenestege, W. T., de Pont, J. J. H. H. M., Bindels, R. J. M., Monnens, L. A. H., Van den Heuvel, L. P. W. J., and Knoers, N. V. A. M. (2000) Nat. Genet. 26, 265–266). The results show that Gly-41 → Arg prevents trafficking of γ but not αβ pumps to the cell surface and abrogates functional effects of γ on αβ pumps. These findings underscore a potentially important role of γ in affecting solute transport, in this instance Mg2+ reabsorption, consequent to its primary effect on the sodium pump. The Na,K-ATPase, or sodium pump, maintains the high Na+ and K+ gradients across the plasma membrane of animal cells. Accordingly, this pump plays a major role in determining the cytoplasmic Na+ concentration and hence the cytoplasmic concentration of protons and Ca2+, as well as other solutes whose accumulation is driven by secondary countertransport systems. The kinetic properties of the sodium pump are, in turn, subject to complex mechanisms of short- and long-term regulation. While the nature of the catalytic α subunit isoform may be a primary determinant of tissue-specific behavior of the pump, there are also diverse mechanisms underlying pump regulation. (For review, see Refs. 1Feraille E. Doucet A. Physiol. Rev. 2001; 81: 345-418Crossref PubMed Scopus (424) Google Scholar and 2Therien A.G. Blostein R. Amer. J. Physiol. Cell Physiol. 2000; 279: C541-C566Crossref PubMed Google Scholar).There is an increasing body of evidence that a family of small, single transmembrane proteins characterized by the motif FXYD are expressed in a tissue-specific manner. To date, at least two members have been identified in kidney, FXYD2 or γ (4Mercer R.W. Biemesderfer D. Bliss D.P., Jr. Collins J.H. Forbush III B. J. Cell Biol. 1993; 121: 579-586Crossref PubMed Scopus (184) Google Scholar, 5Therien A.G. Goldshleger R. Karlish S.J.D. R. B. J. Biol. Chem. 1997; 272: 32628-32634Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) and FXYD4 or the corticosteroid hormone-induced factor, CHIF 1The abbreviations used are: CHIFcorticosteroid hormone-induced factorPFOperfluorooctanoatePRMplasma-rich membranesHDMhigh density microsomal membranesLDMlow density membranesMES4-morpholineethanesulfonic acidWTwild-typeNHSN-hydroxysuccinimide1The abbreviations used are: CHIFcorticosteroid hormone-induced factorPFOperfluorooctanoatePRMplasma-rich membranesHDMhigh density microsomal membranesLDMlow density membranesMES4-morpholineethanesulfonic acidWTwild-typeNHSN-hydroxysuccinimide (6Attali B. Latter H. Rachamim N. Garty H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6092-6096Crossref PubMed Scopus (176) Google Scholar, 7Capurro C. Coutry N. Bonvalet J.P. Escoubet B. Garty H. Farman N. Amer. J. Physiol. Cell. Physiol. 1996; 271: C753-C762Crossref PubMed Google Scholar, 8Shi H. Levy-Holzman R. Cluzeaud F. Farman N. Garty H. Amer. J. Physiol. Renal. Physiol. 2001; 280: F505-F515Crossref PubMed Google Scholar). Both modulate the kinetic behavior of the Na,K-ATPase (see Refs. 5Therien A.G. Goldshleger R. Karlish S.J.D. R. B. J. Biol. Chem. 1997; 272: 32628-32634Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 9Béguin P. Wang X. Firsov D. Puoti A. Claeys D. Horisberger J.D. Geering K.E.J. EMBO J. 1997; 16: 4250-4260Crossref PubMed Scopus (213) Google Scholar, 10Therien A.G. Karlish S.J.D. Blostein R. J. Biol. Chem. 1999; 274: 12252-12256Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 11Arystarkhova E. Wetzel R.K. Asinovski N.K. Sweadner K.J. J. Biol. Chem. 1999; 274: 33183-33185Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 12Pu H.X. Cluzeaud F. Goldshleger R. Karlish S.J.D. Farman N. Blostein R. J. Biol. Chem. 2001; 276: 20370-20378Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 13Beguin P. Crambert G. Guennoun S. Garty H. Horisberger J-D. Geering K. EMBO J. 2001; 20: 3993-4002Crossref PubMed Scopus (132) Google Scholar for γ and 13, 14 for CHIF). Another related protein, phospholemman-like protein of shark (PLMS) (15Mahmooud Y.A. Vorum H. Cornelius F. J. Biol. Chem. 2000; 275: 35969-35977Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), related to FXYD1 (phospholemman) in mammalian heart (16Palmer C.J. Scott B.T. Jones L.R. J. Biol. Chem. 1991; 266: 11126-11130Abstract Full Text PDF PubMed Google Scholar), also modulates function in a phosphokinase C-dependent manner. To date, at least seven members of this family have been identified (3Sweadner K. Rael E. Genomics. 2001; 68: 41-56Crossref Scopus (352) Google Scholar).The γ subunit of the Na,K-ATPase was discovered over 20 years ago (17Forbush III, B. Kaplan J.H. Hoffman J.F. Biochemistry. 1978; 17: 3667-3676Crossref PubMed Scopus (223) Google Scholar, 18Collins J.H. Leszyk J. Biochemistry. 1987; 26: 8665-8668Crossref PubMed Scopus (47) Google Scholar) and was shown recently to exist as two major variants in the kidney, γa and γb (19Küster B. Shainskaya A. Mann M. Pu H.X. Blostein R. Goldshleger R. Karlish S.J.D. J. Biol. Chem. 2000; 275: 18441-18446Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), consistent with predictions based on the Expressed Sequence Tag (EST) data base (20Sweadner K.J. Wetzel R.K. Arystarkhova E. Biochem. Biophys. Res. Commun. 2000; 279: 196-201Crossref PubMed Scopus (40) Google Scholar). These are splice variants and differ only in their N-terminal residues. In the rat, the seven N-terminal amino acids TELSANH of γa are replaced by Ac-MDRWYL in γb (19Küster B. Shainskaya A. Mann M. Pu H.X. Blostein R. Goldshleger R. Karlish S.J.D. J. Biol. Chem. 2000; 275: 18441-18446Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Expression studies in fetal tissues suggest that a third form may be present (21Jones D.H. Golding M.C. Barr K.J. Fong G.-H. Kidder G.M. Physiol. Genom. 2001; 6: 129-135Crossref PubMed Scopus (23) Google Scholar).We have previously cloned and expressed the γa and γb variants in mammalian cells and characterized their two main regulatory roles (Refs. 10Therien A.G. Karlish S.J.D. Blostein R. J. Biol. Chem. 1999; 274: 12252-12256Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar and 12Pu H.X. Cluzeaud F. Goldshleger R. Karlish S.J.D. Farman N. Blostein R. J. Biol. Chem. 2001; 276: 20370-20378Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar and reviewed in Ref. 22Therien A.G. Pu H.X. Karlish S.J.D. Blostein R. J. Bioenerg. Biomemb. 2001; 33: 407-414Crossref PubMed Scopus (49) Google Scholar). One function of γ is to increase cytoplasmic K+antagonism of Na+ activation, which is apparent as an increase in K′Na, particularly at elevated K+ concentrations. The other function is a γ-mediated increase in the apparent affinity for ATP, concordant with our earlier finding that antibodies raised against the C terminus of γ decreased the affinity for ATP (5Therien A.G. Goldshleger R. Karlish S.J.D. R. B. J. Biol. Chem. 1997; 272: 32628-32634Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). We ascribed the latter decrease in K′ATP to a γ-mediated shift in the poise of the steady-state E1 ↔ E2 equilibrium toward E1. Consistent with this finding is the behavior of both γ subunits expressed in Xenopus oocytes (13Beguin P. Crambert G. Guennoun S. Garty H. Horisberger J-D. Geering K. EMBO J. 2001; 20: 3993-4002Crossref PubMed Scopus (132) Google Scholar). Thus, in the presence but not absence of Na+, both subunits alter the apparent affinity for extracellular K+ in a membrane potential-dependent manner, indicative of a γ-mediated shift in conformational equilibrium toward E1. Although no notable difference between γa and γbfunction could be detected (12Pu H.X. Cluzeaud F. Goldshleger R. Karlish S.J.D. Farman N. Blostein R. J. Biol. Chem. 2001; 276: 20370-20378Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), the significance of the presence of two major variants of γ may be related to their partially overlapping but distinct patterns of expression (12Pu H.X. Cluzeaud F. Goldshleger R. Karlish S.J.D. Farman N. Blostein R. J. Biol. Chem. 2001; 276: 20370-20378Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 23Wetzel R.K. Sweadner K.J. Amer. J. Physiol. Renal. Physiol. 2001; 281: F531-F545Crossref PubMed Google Scholar), which, in turn, may be relevant to specific functions along the nephron.One goal of this study was to gain insight into the structural basis for the two distinct kinetics effects of γ. To this end, we examined the consequences of altering both N- and C-terminal extramembranous regions of γ by deletion and alanine replacement of the variant-specific N terminus and deletion of up to ten residues from the C terminus. The other aim was to analyze the functional basis for the transmembrane Gly-41 → Arg mutation associated with familial magnesium-wasting in man (24Meij I.C. Koenderink J.B. van Bokhoven H. Assink K.F.H. Groenestege W.T. de Pont J.J.H.H. M. Bindels R.J.M. Monnens L.A.H. Van den Heuvel L.P.W.J. Knoers N.V.A.M. Nat. Genet. 2000; 26: 265-266Crossref PubMed Scopus (214) Google Scholar). This analysis underscores an important role of γ in affecting secondary transport as a result of primary effects on Na,K-ATPase function.DISCUSSIONThe γ subunit of the sodium pump is a member of the FXYD family of small single-span transmembrane proteins. There are at least seven members of this gene family in mammals (3Sweadner K. Rael E. Genomics. 2001; 68: 41-56Crossref Scopus (352) Google Scholar). In the kidney, two members, CHIF and γ, are regulators of the pump with opposite effects on apparent affinity for Na+ (13Beguin P. Crambert G. Guennoun S. Garty H. Horisberger J-D. Geering K. EMBO J. 2001; 20: 3993-4002Crossref PubMed Scopus (132) Google Scholar, 30Garty, H., Lindzen, M., Scanzano, R., Aizen, R., Füzesi, M., Carradus, M., Goldshleger, R., Farman, N., Blostein, R., and Karlish, S. J. D. (2002) Amer. J. Physiol. Renal Physiol., in pressGoogle Scholar). Immunolocalization studies indicate that their expression along the nephron is mutually exclusive. For example, CHIF is present exclusively in cortical and medullary collecting ducts and γ primarily in the medullary thick ascending limb (14Garty H. Cluzeaud F. Farman N. Goldshleger R. Karlish S.J.D. Biophys. J. 2001; 80: 501AGoogle Scholar). The two major γ variants have similar, if not identical, functional effects on the sodium pump complex. On the other hand, there are differences in their localization along the nephron. Although both co-localize to certain segments of the nephron and are abundant in the medullary thick ascending limb where rates of Na+ reabsorption are particularly high, they also exhibit distinct segment localizations. Thus, Pu et al. (12Pu H.X. Cluzeaud F. Goldshleger R. Karlish S.J.D. Farman N. Blostein R. J. Biol. Chem. 2001; 276: 20370-20378Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) found that γb but not γa was visualized in cortical thick ascending limb, whereas γa is present in the region of the macular densa in which γb is absent. More recent studies by Wetzel and Sweadner (23Wetzel R.K. Sweadner K.J. Amer. J. Physiol. Renal. Physiol. 2001; 281: F531-F545Crossref PubMed Google Scholar) have shown that γb is present in the distal convoluted tubule and connecting tubule; γa, if present, is less abundant.The functional effects of γ described earlier include the following: (i) an increase in apparent affinity for ATP reflecting a shift in the steady-state E1/E2 distribution toward the E1 conformation and (ii) an increase in K+/Na+ competition at cytoplasmic Na+ activation sites. Considering these effects in terms of the Albers-Post reaction mechanism, in particular E2(K+) ↔ E1 + K+ and E1 + ATP + Na+ ↔ Na·E1P + ADP, it is intriguing that the two effects of γ are paradoxically opposing. A higher affinity for K+ at cytoplasmic Na+ activation sites should shift the E1/E2 poise away from E1 and, conversely, a higher affinity for ATP should shift the poise away from E2, toward E1. The implications of these dichotomies are considered below.From our earlier observation that anti-γC treatment of the renal enzyme abrogated the effect of γ on K′ATP but had no effect on K+/Na+ antagonism, we suggested that the two effects of γ are relevant to different regions of the γ chain. The present mutagenesis study provides definitive evidence in support of this theory. Thus, deletion of ten and as few as four residues from the C terminus, as well as deletion of the variant-specific N terminus, completely abrogates the γ-mediated decrease in K′ATP seen with both WT variants but not the increase in K+/Na+ antagonism. An intriguing possibility is that there is interplay between the two opposing modifying effects of γ whereby the γ-mediated increase in K′ATP affinity may counteract and hence minimize the true K+/Na+ antagonism and vice versa. This may come about if γ effects on Na,K-ATPase behavior are, in turn, modulated by cell-specific interactions of α/β/γ complexes with other cell elements such as those of the cytoskeleton.The observation that none of the extramembranous mutants abrogated both effects of γ indicates that all of these mutant γ subunits associate with Na,K-ATPase α/β dimers. This finding is consistent with the recent report of Beguin et al. (13Beguin P. Crambert G. Guennoun S. Garty H. Horisberger J-D. Geering K. EMBO J. 2001; 20: 3993-4002Crossref PubMed Scopus (132) Google Scholar), which showed that the FXYD motif that is present in these mutants is critical for stable association.The finding that deletion of the N terminus, like removal of the C terminus (or addition of anti-γC-terminal antibodies), abrogates the effect of γ on the E1 ↔ E2 conformational equilibrium points to long-range effects of γ/αβ interactions on K′ATP. Because the N-terminal deletion but not N7A replacement abrogates the K′ATP effect, the γ effect to stabilize E1 does not involve TELSANH or MDRWYL interactions with αβ but rather the remainder of the γ chain.A physiological basis for the dual effects of γ is that it provides a fine-tuned, self-regulatory mechanism for balancing energy utilization and maintaining appropriate salt gradients across renal epithelial cells. Both γ variants are particularly abundant in the medullary thick ascending limb. As reasoned elsewhere (22Therien A.G. Pu H.X. Karlish S.J.D. Blostein R. J. Bioenerg. Biomemb. 2001; 33: 407-414Crossref PubMed Scopus (49) Google Scholar), it is in the anoxic regions of the medulla that the increased affinity for ATP effected by γ would serve to maintain pump activity, and the moderate decrease in Na+ affinity would serve to balance ATP depletion and maintain an appropriately low intracellular Na+concentration. Accordingly, its dual effect enables γ to imbue the pump with the ability to handle ATP under energy-compromised conditions and yet be self-regulated by having an appropriately modest increase in the Na+ concentration set point. Recent studies by Garty et al. (14Garty H. Cluzeaud F. Farman N. Goldshleger R. Karlish S.J.D. Biophys. J. 2001; 80: 501AGoogle Scholar) have shown that in certain regions with little if any γ in which the apparent affinity for Na+ is higher (29Barlet-Bas C. Cheval L. Marsy S. Khadouri C. Doucet A. Am. J. Physiol. Renal. Physiol. 1990; 259: F246-F250Crossref PubMed Google Scholar), in particular cortical and medullary collecting ducts, the renal pump is associated with CHIF. CHIF has the opposite effect of γ on K′Na (13Beguin P. Crambert G. Guennoun S. Garty H. Horisberger J-D. Geering K. EMBO J. 2001; 20: 3993-4002Crossref PubMed Scopus (132) Google Scholar); it increases the apparent affinity for Na+ at least 2-fold, which these authors suggest may be critical for aldosterone-responsive tissues, which have an important role in maintaining Na+ and K+ homeostasis. It is not known yet whether, in mirror image to the γ effect, the increase in apparent Na+ affinity effected by CHIF reflects a decrease in the apparent affinity for K+ acting as a competitive inhibitor of cytoplasmic Na+ activation.An important role of γ in renal cation homeostasis secondary to its association with, and modulation of, Na,K-ATPase is demonstrated by our results showing the functional consequences of mutating Gly-41 to Arg. This study provides evidence that the G41R substitution alters γ interaction with the αβ pump, resulting in the failure of γ both to traffic to the cell surface and to modulate pump kinetics. The former finding confirms the γ routing defect reported by Meij et al. (24Meij I.C. Koenderink J.B. van Bokhoven H. Assink K.F.H. Groenestege W.T. de Pont J.J.H.H. M. Bindels R.J.M. Monnens L.A.H. Van den Heuvel L.P.W.J. Knoers N.V.A.M. Nat. Genet. 2000; 26: 265-266Crossref PubMed Scopus (214) Google Scholar). In addition, our experiments indicate that αβ pump trafficking per se is not notably affected.The significance of the association of renal Mg2+-wasting with abrogation of γ modulation of Na,K-ATPase is uncertain. It is evident that the consequences of changes in Na+, K+, and Cl− transport along the different regions of the nephron are varied and complex. Reduced apparent ATP affinity of αβ pumps by abrogation of their modulation by γ may decrease pump activity and lead to secondary changes (reduction) in Mg2+ reabsorption. Accordingly, renal Mg2+-wasting seen in the dominant hypomagnesemia described by Meij et al. (24Meij I.C. Koenderink J.B. van Bokhoven H. Assink K.F.H. Groenestege W.T. de Pont J.J.H.H. M. Bindels R.J.M. Monnens L.A.H. Van den Heuvel L.P.W.J. Knoers N.V.A.M. Nat. Genet. 2000; 26: 265-266Crossref PubMed Scopus (214) Google Scholar) appears to be secondary to the loss of γ modulation of Na,K-ATPase function. The primary cellular mechanism remains to be determined. Also unexplained is the increase in renal calcium absorption and hypocalciuria that is consistently observed in these patients (24Meij I.C. Koenderink J.B. van Bokhoven H. Assink K.F.H. Groenestege W.T. de Pont J.J.H.H. M. Bindels R.J.M. Monnens L.A.H. Van den Heuvel L.P.W.J. Knoers N.V.A.M. Nat. Genet. 2000; 26: 265-266Crossref PubMed Scopus (214) Google Scholar).The experiments described in this study were carried out with cultured apolar cells. The extent to which γ trafficking and abrogation of the γ effects by G41R replacement are different in polar cells remains to be addressed. Current efforts are underway to address this aspect of γ function in polarized renal epithelial cells. The Na,K-ATPase, or sodium pump, maintains the high Na+ and K+ gradients across the plasma membrane of animal cells. Accordingly, this pump plays a major role in determining the cytoplasmic Na+ concentration and hence the cytoplasmic concentration of protons and Ca2+, as well as other solutes whose accumulation is driven by secondary countertransport systems. The kinetic properties of the sodium pump are, in turn, subject to complex mechanisms of short- and long-term regulation. While the nature of the catalytic α subunit isoform may be a primary determinant of tissue-specific behavior of the pump, there are also diverse mechanisms underlying pump regulation. (For review, see Refs. 1Feraille E. Doucet A. Physiol. Rev. 2001; 81: 345-418Crossref PubMed Scopus (424) Google Scholar and 2Therien A.G. Blostein R. Amer. J. Physiol. Cell Physiol. 2000; 279: C541-C566Crossref PubMed Google Scholar). There is an increasing body of evidence that a family of small, single transmembrane proteins characterized by the motif FXYD are expressed in a tissue-specific manner. To date, at least two members have been identified in kidney, FXYD2 or γ (4Mercer R.W. Biemesderfer D. Bliss D.P., Jr. Collins J.H. Forbush III B. J. Cell Biol. 1993; 121: 579-586Crossref PubMed Scopus (184) Google Scholar, 5Therien A.G. Goldshleger R. Karlish S.J.D. R. B. J. Biol. Chem. 1997; 272: 32628-32634Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) and FXYD4 or the corticosteroid hormone-induced factor, CHIF 1The abbreviations used are: CHIFcorticosteroid hormone-induced factorPFOperfluorooctanoatePRMplasma-rich membranesHDMhigh density microsomal membranesLDMlow density membranesMES4-morpholineethanesulfonic acidWTwild-typeNHSN-hydroxysuccinimide1The abbreviations used are: CHIFcorticosteroid hormone-induced factorPFOperfluorooctanoatePRMplasma-rich membranesHDMhigh density microsomal membranesLDMlow density membranesMES4-morpholineethanesulfonic acidWTwild-typeNHSN-hydroxysuccinimide (6Attali B. Latter H. Rachamim N. Garty H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6092-6096Crossref PubMed Scopus (176) Google Scholar, 7Capurro C. Coutry N. Bonvalet J.P. Escoubet B. Garty H. Farman N. Amer. J. Physiol. Cell. Physiol. 1996; 271: C753-C762Crossref PubMed Google Scholar, 8Shi H. Levy-Holzman R. Cluzeaud F. Farman N. Garty H. Amer. J. Physiol. Renal. Physiol. 2001; 280: F505-F515Crossref PubMed Google Scholar). Both modulate the kinetic behavior of the Na,K-ATPase (see Refs. 5Therien A.G. Goldshleger R. Karlish S.J.D. R. B. J. Biol. Chem. 1997; 272: 32628-32634Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 9Béguin P. Wang X. Firsov D. Puoti A. Claeys D. Horisberger J.D. Geering K.E.J. EMBO J. 1997; 16: 4250-4260Crossref PubMed Scopus (213) Google Scholar, 10Therien A.G. Karlish S.J.D. Blostein R. J. Biol. Chem. 1999; 274: 12252-12256Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 11Arystarkhova E. Wetzel R.K. Asinovski N.K. Sweadner K.J. J. Biol. Chem. 1999; 274: 33183-33185Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 12Pu H.X. Cluzeaud F. Goldshleger R. Karlish S.J.D. Farman N. Blostein R. J. Biol. Chem. 2001; 276: 20370-20378Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 13Beguin P. Crambert G. Guennoun S. Garty H. Horisberger J-D. Geering K. EMBO J. 2001; 20: 3993-4002Crossref PubMed Scopus (132) Google Scholar for γ and 13, 14 for CHIF). Another related protein, phospholemman-like protein of shark (PLMS) (15Mahmooud Y.A. Vorum H. Cornelius F. J. Biol. Chem. 2000; 275: 35969-35977Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), related to FXYD1 (phospholemman) in mammalian heart (16Palmer C.J. Scott B.T. Jones L.R. J. Biol. Chem. 1991; 266: 11126-11130Abstract Full Text PDF PubMed Google Scholar), also modulates function in a phosphokinase C-dependent manner. To date, at least seven members of this family have been identified (3Sweadner K. Rael E. Genomics. 2001; 68: 41-56Crossref Scopus (352) Google Scholar). corticosteroid hormone-induced factor perfluorooctanoate plasma-rich membranes high density microsomal membranes low density membranes 4-morpholineethanesulfonic acid wild-type N-hydroxysuccinimide corticosteroid hormone-induced factor perfluorooctanoate plasma-rich membranes high density microsomal membranes low density membranes 4-morpholineethanesulfonic acid wild-type N-hydroxysuccinimide The γ subunit of the Na,K-ATPase was discovered over 20 years ago (17Forbush III, B. Kaplan J.H. Hoffman J.F. Biochemistry. 1978; 17: 3667-3676Crossref PubMed Scopus (223) Google Scholar, 18Collins J.H. Leszyk J. Biochemistry. 1987; 26: 8665-8668Crossref PubMed Scopus (47) Google Scholar) and was shown recently to exist as two major variants in the kidney, γa and γb (19Küster B. Shainskaya A. Mann M. Pu H.X. Blostein R. Goldshleger R. Karlish S.J.D. J. Biol. Chem. 2000; 275: 18441-18446Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), consistent with predictions based on the Expressed Sequence Tag (EST) data base (20Sweadner K.J. Wetzel R.K. Arystarkhova E. Biochem. Biophys. Res. Commun. 2000; 279: 196-201Crossref PubMed Scopus (40) Google Scholar). These are splice variants and differ only in their N-terminal residues. In the rat, the seven N-terminal amino acids TELSANH of γa are replaced by Ac-MDRWYL in γb (19Küster B. Shainskaya A. Mann M. Pu H.X. Blostein R. Goldshleger R. Karlish S.J.D. J. Biol. Chem. 2000; 275: 18441-18446Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Expression studies in fetal tissues suggest that a third form may be present (21Jones D.H. Golding M.C. Barr K.J. Fong G.-H. Kidder G.M. Physiol. Genom. 2001; 6: 129-135Crossref PubMed Scopus (23) Google Scholar). We have previously cloned and expressed the γa and γb variants in mammalian cells and characterized their two main regulatory roles (Refs. 10Therien A.G. Karlish S.J.D. Blostein R. J. Biol. Chem. 1999; 274: 12252-12256Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar and 12Pu H.X. Cluzeaud F. Goldshleger R. Karlish S.J.D. Farman N. Blostein R. J. Biol. Chem. 2001; 276: 20370-20378Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar and reviewed in Ref. 22Therien A.G. Pu H.X. Karlish S.J.D. Blostein R. J. Bioenerg. Biomemb. 2001; 33: 407-414Crossref PubMed Scopus (49) Google Scholar). One function of γ is to increase cytoplasmic K+antagonism of Na+ activation, which is apparent as an increase in K′Na, particularly at elevated K+ concentrations. The other function is a γ-mediated increase in the apparent affinity for ATP, concordant with our earlier finding that antibodies raised against the C terminus of γ decreased the affinity for ATP (5Therien A.G. Goldshleger R. Karlish S.J.D. R. B. J. Biol. Chem. 1997; 272: 32628-32634Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). We ascribed the latter decrease in K′ATP to a γ-mediated shift in the poise of the steady-state E1 ↔ E2 equilibrium toward E1. Consistent with this finding is the behavior of both γ subunits expressed in Xenopus oocytes (13Beguin P. Crambert G. Guennoun S. Garty H. Horisberger J-D. Geering K. EMBO J. 2001; 20: 3993-4002Crossref PubMed Scopus (132) Google Scholar). Thus, in the presence but not absence of Na+, both subunits alter the apparent affinity for extracellular K+ in a membrane potential-dependent manner, indicative of a γ-mediated shift in conformational equilibrium toward E1. Although no notable difference between γa and γbfunction could be detected (12Pu H.X. Cluzeaud F. Goldshleger R. Karlish S.J.D. Farman N. Blostein R. J. Biol. Chem. 2001; 276: 20370-20378Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), the significance of the presence of two major variants of γ may be related to their partially overlapping but distinct patterns of expression (12Pu H.X. Cluzeaud F. Goldshleger R. Karlish S.J.D. Farman N. Blostein R. J. Biol. Chem. 2001; 276: 20370-20378Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 23Wetzel R.K. Sweadner K.J. Amer. J. Physiol. Renal. Physiol. 2001; 281: F531-F545Crossref PubMed Google Scholar), which, in turn, may be relevant to specific functions along the nephron. One goal of this study was to gain insight into the structural basis for the two distinct kinetics effects of γ. To this end, we examined the consequences of altering both N- and C-terminal extramembranous regions of γ by deletion and alanine replacement of the variant-specific N terminus and deletion of up to ten residues from the C terminus. The other aim was to analyze the functional basis for the transmembrane Gly-41 → Arg mutation associated with familial magnesium-wasting in man (24Meij I.C. Koenderink J.B. van Bokhoven H. Assink K.F.H. Groenestege W.T. de Pont J.J.H.H. M. Bindels R.J.M. Monnens L.A.H. Van den Heuvel L.P.W.J. Knoers N.V.A.M. Nat. Genet. 2000; 26: 265-266Crossref PubMed Scopus (214) Google Scholar). This analysis underscores an important role of γ in affecting secondary transport as a result of primary effects on Na,K-ATPase function. DISCUSSIONThe γ subunit of the sodium pump is a member of the FXYD family of small single-span transmembrane proteins. There are at least seven members of this gene family in mammals (3Sweadner K. Rael E. Genomics. 2001; 68: 41-56Crossref Scopus (352) Google Scholar). In the kidney, two members, CHIF and γ, are regulators of the pump with opposite effects on apparent affinity for Na+ (13Beguin P. Crambert G. Guennoun S. Garty H. Horisberger J-D. Geering K. EMBO J. 2001; 20: 3993-4002Crossref PubMed Scopus (132) Google Scholar, 30Garty, H., Lindzen, M., Scanzano, R., Aizen, R., Füzesi, M., Carradus, M., Goldshleger, R., Farman, N., Blostein, R., and Karlish, S. J. D. (2002) Amer. J. Physiol. Renal Physiol., in pressGoogle Scholar). Immunolocalization studies indicate that their expression along the nephron is mutually exclusive. For example, CHIF is present exclusively in cortical and medullary collecting ducts and γ primarily in the medullary thick ascending limb (14Garty H. Cluzeaud F. Farman N. Goldshleger R. Karlish S.J.D. Biophys. J. 2001; 80: 501AGoogle Scholar). The two major γ variants have similar, if not identical, functional effects on the sodium pump complex. On the other hand, there are differences in their localization along the nephron. Although both co-localize to certain segments of the nephron and are abundant in the medullary thick ascending limb where rates of Na+ reabsorption are particularly high, they also exhibit distinct segment localizations. Thus, Pu et al. (12Pu H.X. Cluzeaud F. Goldshleger R. Karlish S.J.D. Farman N. Blostein R. J. Biol. Chem. 2001; 276: 20370-20378Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) found that γb but not γa was visualized in cortical thick ascending limb, whereas γa is present in the region of the macular densa in which γb is absent. More recent studies by Wetzel and Sweadner (23Wetzel R.K. Sweadner K.J. Amer. J. Physiol. Renal. Physiol. 2001; 281: F531-F545Crossref PubMed Google Scholar) have shown that γb is present in the distal convoluted tubule and connecting tubule; γa, if present, is less abundant.The functional effects of γ described earlier include the following: (i) an increase in apparent affinity for ATP reflecting a shift in the steady-state E1/E2 distribution toward the E1 conformation and (ii) an increase in K+/Na+ competition at cytoplasmic Na+ activation sites. Considering these effects in terms of the Albers-Post reaction mechanism, in particular E2(K+) ↔ E1 + K+ and E1 + ATP + Na+ ↔ Na·E1P + ADP, it is intriguing that the two effects of γ are paradoxically opposing. A higher affinity for K+ at cytoplasmic Na+ activation sites should shift the E1/E2 poise away from E1 and, conversely, a higher affinity for ATP should shift the poise away from E2, toward E1. The implications of these dichotomies are considered below.From our earlier observation that anti-γC treatment of the renal enzyme abrogated the effect of γ on K′ATP but had no effect on K+/Na+ antagonism, we suggested that the two effects of γ are relevant to different regions of the γ chain. The present mutagenesis study provides definitive evidence in support of this theory. Thus, deletion of ten and as few as four residues from the C terminus, as well as deletion of the variant-specific N terminus, completely abrogates the γ-mediated decrease in K′ATP seen with both WT variants but not the increase in K+/Na+ antagonism. An intriguing possibility is that there is interplay between the two opposing modifying effects of γ whereby the γ-mediated increase in K′ATP affinity may counteract and hence minimize the true K+/Na+ antagonism and vice versa. This may come about if γ effects on Na,K-ATPase behavior are, in turn, modulated by cell-specific interactions of α/β/γ complexes with other cell elements such as those of the cytoskeleton.The observation that none of the extramembranous mutants abrogated both effects of γ indicates that all of these mutant γ subunits associate with Na,K-ATPase α/β dimers. This finding is consistent with the recent report of Beguin et al. (13Beguin P. Crambert G. Guennoun S. Garty H. Horisberger J-D. Geering K. EMBO J. 2001; 20: 3993-4002Crossref PubMed Scopus (132) Google Scholar), which showed that the FXYD motif that is present in these mutants is critical for stable association.The finding that deletion of the N terminus, like removal of the C terminus (or addition of anti-γC-terminal antibodies), abrogates the effect of γ on the E1 ↔ E2 conformational equilibrium points to long-range effects of γ/αβ interactions on K′ATP. Because the N-terminal deletion but not N7A replacement abrogates the K′ATP effect, the γ effect to stabilize E1 does not involve TELSANH or MDRWYL interactions with αβ but rather the remainder of the γ chain.A physiological basis for the dual effects of γ is that it provides a fine-tuned, self-regulatory mechanism for balancing energy utilization and maintaining appropriate salt gradients across renal epithelial cells. Both γ variants are particularly abundant in the medullary thick ascending limb. As reasoned elsewhere (22Therien A.G. Pu H.X. Karlish S.J.D. Blostein R. J. Bioenerg. Biomemb. 2001; 33: 407-414Crossref PubMed Scopus (49) Google Scholar), it is in the anoxic regions of the medulla that the increased affinity for ATP effected by γ would serve to maintain pump activity, and the moderate decrease in Na+ affinity would serve to balance ATP depletion and maintain an appropriately low intracellular Na+concentration. Accordingly, its dual effect enables γ to imbue the pump with the ability to handle ATP under energy-compromised conditions and yet be self-regulated by having an appropriately modest increase in the Na+ concentration set point. Recent studies by Garty et al. (14Garty H. Cluzeaud F. Farman N. Goldshleger R. Karlish S.J.D. Biophys. J. 2001; 80: 501AGoogle Scholar) have shown that in certain regions with little if any γ in which the apparent affinity for Na+ is higher (29Barlet-Bas C. Cheval L. Marsy S. Khadouri C. Doucet A. Am. J. Physiol. Renal. Physiol. 1990; 259: F246-F250Crossref PubMed Google Scholar), in particular cortical and medullary collecting ducts, the renal pump is associated with CHIF. CHIF has the opposite effect of γ on K′Na (13Beguin P. Crambert G. Guennoun S. Garty H. Horisberger J-D. Geering K. EMBO J. 2001; 20: 3993-4002Crossref PubMed Scopus (132) Google Scholar); it increases the apparent affinity for Na+ at least 2-fold, which these authors suggest may be critical for aldosterone-responsive tissues, which have an important role in maintaining Na+ and K+ homeostasis. It is not known yet whether, in mirror image to the γ effect, the increase in apparent Na+ affinity effected by CHIF reflects a decrease in the apparent affinity for K+ acting as a competitive inhibitor of cytoplasmic Na+ activation.An important role of γ in renal cation homeostasis secondary to its association with, and modulation of, Na,K-ATPase is demonstrated by our results showing the functional consequences of mutating Gly-41 to Arg. This study provides evidence that the G41R substitution alters γ interaction with the αβ pump, resulting in the failure of γ both to traffic to the cell surface and to modulate pump kinetics. The former finding confirms the γ routing defect reported by Meij et al. (24Meij I.C. Koenderink J.B. van Bokhoven H. Assink K.F.H. Groenestege W.T. de Pont J.J.H.H. M. Bindels R.J.M. Monnens L.A.H. Van den Heuvel L.P.W.J. Knoers N.V.A.M. Nat. Genet. 2000; 26: 265-266Crossref PubMed Scopus (214) Google Scholar). In addition, our experiments indicate that αβ pump trafficking per se is not notably affected.The significance of the association of renal Mg2+-wasting with abrogation of γ modulation of Na,K-ATPase is uncertain. It is evident that the consequences of changes in Na+, K+, and Cl− transport along the different regions of the nephron are varied and complex. Reduced apparent ATP affinity of αβ pumps by abrogation of their modulation by γ may decrease pump activity and lead to secondary changes (reduction) in Mg2+ reabsorption. Accordingly, renal Mg2+-wasting seen in the dominant hypomagnesemia described by Meij et al. (24Meij I.C. Koenderink J.B. van Bokhoven H. Assink K.F.H. Groenestege W.T. de Pont J.J.H.H. M. Bindels R.J.M. Monnens L.A.H. Van den Heuvel L.P.W.J. Knoers N.V.A.M. Nat. Genet. 2000; 26: 265-266Crossref PubMed Scopus (214) Google Scholar) appears to be secondary to the loss of γ modulation of Na,K-ATPase function. The primary cellular mechanism remains to be determined. Also unexplained is the increase in renal calcium absorption and hypocalciuria that is consistently observed in these patients (24Meij I.C. Koenderink J.B. van Bokhoven H. Assink K.F.H. Groenestege W.T. de Pont J.J.H.H. M. Bindels R.J.M. Monnens L.A.H. Van den Heuvel L.P.W.J. Knoers N.V.A.M. Nat. Genet. 2000; 26: 265-266Crossref PubMed Scopus (214) Google Scholar).The experiments described in this study were carried out with cultured apolar cells. The extent to which γ trafficking and abrogation of the γ effects by G41R replacement are different in polar cells remains to be addressed. Current efforts are underway to address this aspect of γ function in polarized renal epithelial cells. The γ subunit of the sodium pump is a member of the FXYD family of small single-span transmembrane proteins. There are at least seven members of this gene family in mammals (3Sweadner K. Rael E. Genomics. 2001; 68: 41-56Crossref Scopus (352) Google Scholar). In the kidney, two members, CHIF and γ, are regulators of the pump with opposite effects on apparent affinity for Na+ (13Beguin P. Crambert G. Guennoun S. Garty H. Horisberger J-D. Geering K. EMBO J. 2001; 20: 3993-4002Crossref PubMed Scopus (132) Google Scholar, 30Garty, H., Lindzen, M., Scanzano, R., Aizen, R., Füzesi, M., Carradus, M., Goldshleger, R., Farman, N., Blostein, R., and Karlish, S. J. D. (2002) Amer. J. Physiol. Renal Physiol., in pressGoogle Scholar). Immunolocalization studies indicate that their expression along the nephron is mutually exclusive. For example, CHIF is present exclusively in cortical and medullary collecting ducts and γ primarily in the medullary thick ascending limb (14Garty H. Cluzeaud F. Farman N. Goldshleger R. Karlish S.J.D. Biophys. J. 2001; 80: 501AGoogle Scholar). The two major γ variants have similar, if not identical, functional effects on the sodium pump complex. On the other hand, there are differences in their localization along the nephron. Although both co-localize to certain segments of the nephron and are abundant in the medullary thick ascending limb where rates of Na+ reabsorption are particularly high, they also exhibit distinct segment localizations. Thus, Pu et al. (12Pu H.X. Cluzeaud F. Goldshleger R. Karlish S.J.D. Farman N. Blostein R. J. Biol. Chem. 2001; 276: 20370-20378Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) found that γb but not γa was visualized in cortical thick ascending limb, whereas γa is present in the region of the macular densa in which γb is absent. More recent studies by Wetzel and Sweadner (23Wetzel R.K. Sweadner K.J. Amer. J. Physiol. Renal. Physiol. 2001; 281: F531-F545Crossref PubMed Google Scholar) have shown that γb is present in the distal convoluted tubule and connecting tubule; γa, if present, is less abundant. The functional effects of γ described earlier include the following: (i) an increase in apparent affinity for ATP reflecting a shift in the steady-state E1/E2 distribution toward the E1 conformation and (ii) an increase in K+/Na+ competition at cytoplasmic Na+ activation sites. Considering these effects in terms of the Albers-Post reaction mechanism, in particular E2(K+) ↔ E1 + K+ and E1 + ATP + Na+ ↔ Na·E1P + ADP, it is intriguing that the two effects of γ are paradoxically opposing. A higher affinity for K+ at cytoplasmic Na+ activation sites should shift the E1/E2 poise away from E1 and, conversely, a higher affinity for ATP should shift the poise away from E2, toward E1. The implications of these dichotomies are considered below. From our earlier observation that anti-γC treatment of the renal enzyme abrogated the effect of γ on K′ATP but had no effect on K+/Na+ antagonism, we suggested that the two effects of γ are relevant to different regions of the γ chain. The present mutagenesis study provides definitive evidence in support of this theory. Thus, deletion of ten and as few as four residues from the C terminus, as well as deletion of the variant-specific N terminus, completely abrogates the γ-mediated decrease in K′ATP seen with both WT variants but not the increase in K+/Na+ antagonism. An intriguing possibility is that there is interplay between the two opposing modifying effects of γ whereby the γ-mediated increase in K′ATP affinity may counteract and hence minimize the true K+/Na+ antagonism and vice versa. This may come about if γ effects on Na,K-ATPase behavior are, in turn, modulated by cell-specific interactions of α/β/γ complexes with other cell elements such as those of the cytoskeleton. The observation that none of the extramembranous mutants abrogated both effects of γ indicates that all of these mutant γ subunits associate with Na,K-ATPase α/β dimers. This finding is consistent with the recent report of Beguin et al. (13Beguin P. Crambert G. Guennoun S. Garty H. Horisberger J-D. Geering K. EMBO J. 2001; 20: 3993-4002Crossref PubMed Scopus (132) Google Scholar), which showed that the FXYD motif that is present in these mutants is critical for stable association. The finding that deletion of the N terminus, like removal of the C terminus (or addition of anti-γC-terminal antibodies), abrogates the effect of γ on the E1 ↔ E2 conformational equilibrium points to long-range effects of γ/αβ interactions on K′ATP. Because the N-terminal deletion but not N7A replacement abrogates the K′ATP effect, the γ effect to stabilize E1 does not involve TELSANH or MDRWYL interactions with αβ but rather the remainder of the γ chain. A physiological basis for the dual effects of γ is that it provides a fine-tuned, self-regulatory mechanism for balancing energy utilization and maintaining appropriate salt gradients across renal epithelial cells. Both γ variants are particularly abundant in the medullary thick ascending limb. As reasoned elsewhere (22Therien A.G. Pu H.X. Karlish S.J.D. Blostein R. J. Bioenerg. Biomemb. 2001; 33: 407-414Crossref PubMed Scopus (49) Google Scholar), it is in the anoxic regions of the medulla that the increased affinity for ATP effected by γ would serve to maintain pump activity, and the moderate decrease in Na+ affinity would serve to balance ATP depletion and maintain an appropriately low intracellular Na+concentration. Accordingly, its dual effect enables γ to imbue the pump with the ability to handle ATP under energy-compromised conditions and yet be self-regulated by having an appropriately modest increase in the Na+ concentration set point. Recent studies by Garty et al. (14Garty H. Cluzeaud F. Farman N. Goldshleger R. Karlish S.J.D. Biophys. J. 2001; 80: 501AGoogle Scholar) have shown that in certain regions with little if any γ in which the apparent affinity for Na+ is higher (29Barlet-Bas C. Cheval L. Marsy S. Khadouri C. Doucet A. Am. J. Physiol. Renal. Physiol. 1990; 259: F246-F250Crossref PubMed Google Scholar), in particular cortical and medullary collecting ducts, the renal pump is associated with CHIF. CHIF has the opposite effect of γ on K′Na (13Beguin P. Crambert G. Guennoun S. Garty H. Horisberger J-D. Geering K. EMBO J. 2001; 20: 3993-4002Crossref PubMed Scopus (132) Google Scholar); it increases the apparent affinity for Na+ at least 2-fold, which these authors suggest may be critical for aldosterone-responsive tissues, which have an important role in maintaining Na+ and K+ homeostasis. It is not known yet whether, in mirror image to the γ effect, the increase in apparent Na+ affinity effected by CHIF reflects a decrease in the apparent affinity for K+ acting as a competitive inhibitor of cytoplasmic Na+ activation. An important role of γ in renal cation homeostasis secondary to its association with, and modulation of, Na,K-ATPase is demonstrated by our results showing the functional consequences of mutating Gly-41 to Arg. This study provides evidence that the G41R substitution alters γ interaction with the αβ pump, resulting in the failure of γ both to traffic to the cell surface and to modulate pump kinetics. The former finding confirms the γ routing defect reported by Meij et al. (24Meij I.C. Koenderink J.B. van Bokhoven H. Assink K.F.H. Groenestege W.T. de Pont J.J.H.H. M. Bindels R.J.M. Monnens L.A.H. Van den Heuvel L.P.W.J. Knoers N.V.A.M. Nat. Genet. 2000; 26: 265-266Crossref PubMed Scopus (214) Google Scholar). In addition, our experiments indicate that αβ pump trafficking per se is not notably affected. The significance of the association of renal Mg2+-wasting with abrogation of γ modulation of Na,K-ATPase is uncertain. It is evident that the consequences of changes in Na+, K+, and Cl− transport along the different regions of the nephron are varied and complex. Reduced apparent ATP affinity of αβ pumps by abrogation of their modulation by γ may decrease pump activity and lead to secondary changes (reduction) in Mg2+ reabsorption. Accordingly, renal Mg2+-wasting seen in the dominant hypomagnesemia described by Meij et al. (24Meij I.C. Koenderink J.B. van Bokhoven H. Assink K.F.H. Groenestege W.T. de Pont J.J.H.H. M. Bindels R.J.M. Monnens L.A.H. Van den Heuvel L.P.W.J. Knoers N.V.A.M. Nat. Genet. 2000; 26: 265-266Crossref PubMed Scopus (214) Google Scholar) appears to be secondary to the loss of γ modulation of Na,K-ATPase function. The primary cellular mechanism remains to be determined. Also unexplained is the increase in renal calcium absorption and hypocalciuria that is consistently observed in these patients (24Meij I.C. Koenderink J.B. van Bokhoven H. Assink K.F.H. Groenestege W.T. de Pont J.J.H.H. M. Bindels R.J.M. Monnens L.A.H. Van den Heuvel L.P.W.J. Knoers N.V.A.M. Nat. Genet. 2000; 26: 265-266Crossref PubMed Scopus (214) Google Scholar). The experiments described in this study were carried out with cultured apolar cells. The extent to which γ trafficking and abrogation of the γ effects by G41R replacement are different in polar cells remains to be addressed. Current efforts are underway to address this aspect of γ function in polarized renal epithelial cells. We thank Drs. Alex Therien, Steven Karlish, and Gary Quamme for helpful comments on the article and Dr. Edward Chan for the gift of anti-giantin." @default.
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W2010243693 title "Distinct Regulatory Effects of the Na,K-ATPase γ Subunit" @default.
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