The Functional Unit of the Renal Type IIa Na+/Pi Cotransporter Is a Monomer
2000; Elsevier BV; Volume: 275; Issue: 34 Linguagem: Inglês
10.1074/jbc.m003564200
ISSN1083-351X
AutoresKatja Köhler, Ian C. Forster, Georg Lambert, Jürg Biber, Heini Murer,
Tópico(s)Amino Acid Enzymes and Metabolism
ResumoThe composition of the functional unit of the rat renal type IIa Na+/Pi cotransporter (NaPi-IIa) was investigated by using two approaches based on the differential sensitivities of the wild type (WT) and mutant S460C proteins to 2-aminoethylmethanethiosulfonate hydrobromide (MTSEA), a charged cysteine modifier. Transport activity of S460C is completely blocked after incubation in MTSEA, whereas that of the WT remains unaffected. First, Xenopus laevis oocytes were coinjected with cRNAs coding for the WT and S460C in different proportions, and the transport inhibition after MTSEA incubation was assayed by electrophysiology. The relationship between MTSEA inhibition and proportion of cRNA was consistent with that for a functional monomer. Second, concatameric proteins were constructed that either comprised two WT proteins (WT-WT), two S460C mutants (S460C-S460C), or one of each (WT-S460C). Western blots of oocytes injected with fusion protein cRNA showed bands at ∼200 kDa, whereas a main band at ∼90 kDa was obtained for the WT cRNA alone. The kinetic properties of concatamers were the same as for the single proteins. Transport activity of the WT-WT concatamer was unaffected by MTSEA incubation, fully inhibited for S460C-S460C, but 50% inhibited for WT-S460C. This behavior was also consistent with NaPi-IIa being a functional monomer. The composition of the functional unit of the rat renal type IIa Na+/Pi cotransporter (NaPi-IIa) was investigated by using two approaches based on the differential sensitivities of the wild type (WT) and mutant S460C proteins to 2-aminoethylmethanethiosulfonate hydrobromide (MTSEA), a charged cysteine modifier. Transport activity of S460C is completely blocked after incubation in MTSEA, whereas that of the WT remains unaffected. First, Xenopus laevis oocytes were coinjected with cRNAs coding for the WT and S460C in different proportions, and the transport inhibition after MTSEA incubation was assayed by electrophysiology. The relationship between MTSEA inhibition and proportion of cRNA was consistent with that for a functional monomer. Second, concatameric proteins were constructed that either comprised two WT proteins (WT-WT), two S460C mutants (S460C-S460C), or one of each (WT-S460C). Western blots of oocytes injected with fusion protein cRNA showed bands at ∼200 kDa, whereas a main band at ∼90 kDa was obtained for the WT cRNA alone. The kinetic properties of concatamers were the same as for the single proteins. Transport activity of the WT-WT concatamer was unaffected by MTSEA incubation, fully inhibited for S460C-S460C, but 50% inhibited for WT-S460C. This behavior was also consistent with NaPi-IIa being a functional monomer. type IIa Na+/Pi cotransporter brush border membrane vesicle wild type cotransporter Ser-460-Cys mutant cotransporter 2-aminoethylmethanethiosulfonate hydrobromide phosphonoformic acid dithiothreitol radiation inactivation size The reabsorption of inorganic phosphate (Pi) at the luminal brush border membrane of the renal proximal tubule is mediated chiefly by a Na+-coupled cotransport process. Three unique types of Na+/Pi cotransporters have been identified in proximal tubular epithelia as follows: type I, type IIa, and type III (1Murer H. Forster I. Hernando N. Lambert G. Traebert M. Biber J. Am. J. Physiol. 1999; 277: F676-F684PubMed Google Scholar, 2Murer, H., Hernando, N., Forster, I., and Biber, J. (2000)Physiol. Rev., in pressGoogle Scholar). The type IIa Na+/Picotransporter (NaPi-IIa)1contributes the major component of luminal Pi transport (1Murer H. Forster I. Hernando N. Lambert G. Traebert M. Biber J. Am. J. Physiol. 1999; 277: F676-F684PubMed Google Scholar, 2Murer, H., Hernando, N., Forster, I., and Biber, J. (2000)Physiol. Rev., in pressGoogle Scholar, 3Beck L. Karaplis A.C. Amizuka N. Hewson A.S. Ozawa H. Tenenhouse H.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5372-5377Crossref PubMed Scopus (511) Google Scholar). Type IIa isoforms, cloned from various species, display similar kinetic properties when expressed in Xenopus oocytes (4Busch A. Waldegger S. Herzer T. Biber J. Markovich D. Hayes G. Murer H. Lang F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8205-8208Crossref PubMed Scopus (74) Google Scholar, 5Busch A.E. Wagner C.A. Schuster A. Waldegger S. Biber J. Murer H. Lang F. J. Am. Soc. Nephrol. 1995; 6: 1547-1551PubMed Google Scholar, 6Forster I. Hernando N. Biber J. Murer H. J. Gen. Physiol. 1998; 112: 1-18Crossref PubMed Scopus (110) Google Scholar, 7Hartmann C.M. Wagner C.A. Busch A.E. Markovich D. Biber J. Lang F. Murer H. Pfluegers Arch. 1995; 430: 830-836Crossref PubMed Scopus (73) Google Scholar,10Forster I. Loo D.D. Eskandari S. Am. J. Physiol. 1999; 276: F644-F649PubMed Google Scholar).On Western blots made from brush border membrane vesicles (BBMVs) the rat type IIa isoform (NaPi-IIa) protein is detected as an 80–90-kDa glycoprotein (8Custer M. Lotscher M. Biber J. Murer H. Kaissling B. Am. J. Physiol. 1994; 266: F767-F774PubMed Google Scholar, 9Biber J. Custer M. Magagnin S. Hayes G. Werner A. Lotscher M. Kaissling B. Murer H. Kidney Int. 1996; 49: 981-985Abstract Full Text PDF PubMed Scopus (98) Google Scholar). When expressed in Xenopus laevisoocytes, a similar band, specific to NaPi-IIa, at 70–110 kDa is observed (11Hayes G. Busch A. Lotscher M. Waldegger S. Lang F. Verrey F. Biber J. Murer H. J. Biol. Chem. 1994; 269: 24143-24149Abstract Full Text PDF PubMed Google Scholar, 12Lambert G. Forster I.C. Stange G. Biber J. Murer H. J. Gen. Physiol. 1999; 114: 637-652Crossref PubMed Scopus (38) Google Scholar). This molecular weight corresponds to that predicted from the amino acid sequence (68.7 kDa) after taking account of protein glycosylation (11Hayes G. Busch A. Lotscher M. Waldegger S. Lang F. Verrey F. Biber J. Murer H. J. Biol. Chem. 1994; 269: 24143-24149Abstract Full Text PDF PubMed Google Scholar).From a number of studies using the radiation inactivation technique applied to Pi uptake in BBMVs, estimates of the functional size of the Na+/Pi system have ranged from 124 to 242 kDa depending on the species and assay conditions (13Beliveau R. Demeule M. Ibnoul-Khatib H. Bergeron M. Beauregard G. Potier M. Biochem. J. 1988; 252: 807-813Crossref PubMed Scopus (42) Google Scholar, 14Delisle M.C. Vachon V. Giroux S. Potier M. Laprade R. Beliveau R. Biochim. Biophys. Acta. 1992; 1104: 132-136Crossref PubMed Scopus (14) Google Scholar, 15Delisle M.C. Giroux S. Vachon V. Boyer C. Potier M. Beliveau R. Biochemistry. 1994; 33: 9105-9109Crossref PubMed Scopus (12) Google Scholar, 16Jette M. Vachon V. Potier M. Beliveau R. Biochemistry. 1996; 35: 15209-15214Crossref PubMed Scopus (7) Google Scholar, 17Tenenhouse H.S. Lee J. Harvey N. Potier M. Jette M. Beliveau R. Biochem. Biophys. Res. Commun. 1990; 170: 1288-1293Crossref PubMed Scopus (13) Google Scholar, 18Xiao Y. Boyer C.J. Vincent E. Dugre A. Vachon V. Potier M. Beliveau R. Biochem. J. 1997; 323: 401-408Crossref PubMed Scopus (22) Google Scholar). The significant difference between the molecular weight of the type IIa protein (see above) and radiation inactivation size (RIS) was taken as evidence for an oligomeric functional unit of Na+/Pi cotransport. However, since Pi uptake in BBMVs most likely includes contributions from other Pi transport systems, e.g. type I, or other as yet unidentified proteins, estimation of the number of cotransporter subunits of a particular type that contribute to the functional transport unit is not possible. Moreover, the membrane target of radiation may include other proteins and/or membrane domains associated with the cotransporter itself, which could result in an overestimation of the functional mass contributed by type IIa proteins alone.Alternative approaches have been recently used to determine the functional composition of membrane transport proteins. These take advantage of the X. laevis oocyte expression system to overexpress only the protein of interest and thereby avoid contamination of the functional assay. A means of functional identification is then used to assay the contribution made by the constitutive subunits such as non-conductive mutations for the potassium channel (MinK) (19Wang K.W. Goldstein S.A. Neuron. 1995; 14: 1303-1309Abstract Full Text PDF PubMed Scopus (112) Google Scholar), differential sensitivity of subunits to methane sulfonate reagents that react specifically with cysteine residues for the epithelial Na+ channel (20Kosari F. Sheng S. Li J. Mak D.O. Foskett J.K. Kleyman T.R. J. Biol. Chem. 1998; 273: 13469-13474Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 21Eskandari S. Snyder P.M. Kreman M. Zampighi G.A. Welsh M.J. Wright E.M. J. Biol. Chem. 1999; 274: 27281-27286Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) and the voltage-gated chloride channel (ClC-1) (22Fahlke C. Rhodes T.H. Desai R.R. George Jr., A.L. Nature. 1998; 394: 687-690Crossref PubMed Scopus (59) Google Scholar), sensitivity of subunits to channel blockers for the shaker K+channel (23MacKinnon R. Nature. 1991; 350: 232-235Crossref PubMed Scopus (761) Google Scholar), and mammalian delayed rectifier K+ channel (KV1.1) (24Liman E.R. Tytgat J. Hess P. Neuron. 1992; 9: 861-871Abstract Full Text PDF PubMed Scopus (971) Google Scholar) and epithelial Na+ channel (21Eskandari S. Snyder P.M. Kreman M. Zampighi G.A. Welsh M.J. Wright E.M. J. Biol. Chem. 1999; 274: 27281-27286Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 25Firsov D. Gautschi I. Merillat A.M. Rossier B.C. Schild L. EMBO J. 1998; 17: 344-352Crossref PubMed Scopus (368) Google Scholar). To express functional proteins with a predefined subunit composition, an extension of this approach involves the construction of concatameric cDNAs that encode for linked subunits as used, for example to determine the stoichiometry of KV1.1(24) and epithelial Na+channel (25Firsov D. Gautschi I. Merillat A.M. Rossier B.C. Schild L. EMBO J. 1998; 17: 344-352Crossref PubMed Scopus (368) Google Scholar).Here, we determined the functional unit of the type IIa Na+/Pi cotransporter (NaP-IIa) by overexpression of the wild type (WT) NaPi-IIa protein and a functionally distinguishable mutant protein (S460C) in X. laevis oocytes. The electrogenic response of oocytes was quantified by means of the two-electrode voltage clamp. We took advantage of the differential sensitivity of the WT NaPi-IIa and mutant cotransporter (S460C) to nominally membrane-impermeant methanethiosulfonate (MTS) reagents; the WT is insensitive to externally applied MTSEA, whereas Pi uptake and the electrogenic cotransport mode are fully inhibited for S460C (12Lambert G. Forster I.C. Stange G. Biber J. Murer H. J. Gen. Physiol. 1999; 114: 637-652Crossref PubMed Scopus (38) Google Scholar). We injected oocytes either with different proportions of cRNAs coding for the WT and S460C protein or for novel fusion proteins (concatamers) that comprised two fused WT or S460C proteins alone, or a mixed protein that comprised the WT and S460C. In both cases we assayed the Pi-induced cotransport activity before and after MTS incubation. The results of each approach were consistent with the NaPi-IIa protein being a functional monomer.DISCUSSIONThis study documents the identification of the functional molecular unit of the type IIa NaPi cotransporter (rat NaPIIa) expressed in Xenopus oocytes. We took advantage of the marked difference in sensitivity of the WT transporter and the S460C mutant to the cysteine-modifying reagent, MTSEA. Whereas the cotransport function of the WT is unchanged after external application of this reagent, that of the S460C mutant is fully inhibited (12Lambert G. Forster I.C. Stange G. Biber J. Murer H. J. Gen. Physiol. 1999; 114: 637-652Crossref PubMed Scopus (38) Google Scholar).The underlying assumption in our use of externally applied MTSEA was that this reagent modified only the substituted cysteine residue at site 460. It has been previously reported that MTSEA can permeate membranes and modify exogenously expressed membrane proteins from the cytosolic side of the membrane (30Holmgren M. Liu Y. Xu Y. Yellen G. Neuropharmacology. 1996; 35: 797-804Crossref PubMed Scopus (191) Google Scholar, 31Kaplan R.S. Mayor J.A. Brauer D. Kotaria R. Walters D.E. Dean A.M. J. Biol. Chem. 2000; 275: 12009-12016Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Therefore, part of the observed inhibition of transport function might be attributable to modification of other functionally important cysteine residues present in both the WT and S460C proteins. For example, the membrane-permeant MTS reagent methyl methanethiosulfonate can inhibit the WT transport activity (12Lambert G. Forster I.C. Stange G. Biber J. Murer H. J. Gen. Physiol. 1999; 114: 637-652Crossref PubMed Scopus (38) Google Scholar). However, we have shown that for externally applied MTSEA, even at 10-fold higher concentrations than those required to fully suppress S460C function, no alteration in WT transport activity occurs. Moreover, external application of the impermeant reagent 2-(trimethylammonium)ethyl methanethiosulfonate bromide (30Holmgren M. Liu Y. Xu Y. Yellen G. Neuropharmacology. 1996; 35: 797-804Crossref PubMed Scopus (191) Google Scholar) leads to the same full inhibition of transport function for mutant S460C as MTSEA under the same conditions. These findings established the following: 1) external application of either reagent is able to access the Cys residue at site 460 from the extracellular side, and 2) if MTSEA does permeate the membrane, it is unable to access functionally important cysteine residues associated with NaPi-II protein via this pathway. Therefore, under our experimental conditions MTSEA can be considered impermeant, and any loss of transport function can be attributed only to the chemical modification of Cys-460.To interpret our data, we tacitly assumed that if the transport function of one subunit of an oligomeric functional unit is inhibited through chemical modification of Cys-460, the transport of the entire functional complex is blocked. Given the validity of this assumption, we could make predictions concerning the functional composition of NaPi-II protein and interpret our results accordingly. The results of both approaches matched well with the prediction that NaPi-II protein exists as a functional monomer. For example, for the coinjection experiments if 50% WT and 50% S460C cRNA is injected, we would expect, on average, for random protein association as dimers 25% S460C + S460C, 25% WT + WT, and 50% WT + S460C transporters. Thus, the transport rate should be inhibited by 75%, whereas for a functional monomer, an inhibition of 50% is predicted (Equation 3) that is close to the observed value. Similarly, for the mixed concatamer (WT-S460C), the observed 50% loss of function is consistent with there being two functionally independent transport pathways per concatamer and a total inhibition of the transport pathway established by the S460C protein component. Our interpretation of the data therefore also implies the existence of one transport pathway or, rather unlikely, multiple and identical transport pathways for each functional unit. Although we cannot fully exclude this latter possibility, the simplest interpretation, consistent with our findings, is that there is only one transport pathway per unit of protein.The Coinjection MethodFrom the coinjection experiments, in which the proportion of S460C cRNA varied from 15 to 88%, the best agreement between the measured % inhibition and that predicted from Equation 3 was for N = 1 (i.e. a monomeric functional unit). For all intermediate proportions of cRNA, the measured mean % inhibition always remained slightly below that predicted for N = 1, and the deviation increased at the upper limit (88% S460C). This suggested the influence of a systematic error, which may arise from intrinsic uncertainties in the coinjection method. In the first place, coinjection of equal quantities of two different cRNAs into oocytes does not guarantee that a given percentage of cRNA leads to the corresponding amount of protein either expressed or actually inserted into the membrane as functional cotransporters. For example, membrane expression of one protein could be favored over another, depending on the state of the oocyte expression machinery. Second, expression levels can vary significantly between different isoforms of the same cotransporter. We have observed that the human (5Busch A.E. Wagner C.A. Schuster A. Waldegger S. Biber J. Murer H. Lang F. J. Am. Soc. Nephrol. 1995; 6: 1547-1551PubMed Google Scholar) and mouse (7Hartmann C.M. Wagner C.A. Busch A.E. Markovich D. Biber J. Lang F. Murer H. Pfluegers Arch. 1995; 430: 830-836Crossref PubMed Scopus (73) Google Scholar) NaPi-IIa isoforms, which show very high molecular identity with the rat isoform, give significantly lower functional expression (up to 5-fold) for the same quantity of cRNA injected and same batch of oocytes. 2K. Köhler, I. C. Forster, G. Lambert, J. Biber, and H. Murer, unpublished observations. In the present study, although we found that the amount of cRNA injected correlated well with the electrogenic activity of both WT and the mutant S460C alone, nevertheless coexpression of both cRNAs might still lead to disproportionate functional expression. This could therefore account for the deviation from monomeric prediction at higher proportions of S460C cRNA. Third, time-dependent expression rates of the two proteins could differ depending on the individual oocyte or between donor frogs. In practice, we observed no qualitative differences between WT and S460C with respect to their functional expression in oocytes measured from day 4 to day 6 post-injection, and therefore we think that this would most likely not lead to significant errors in the final membrane expression levels.For the particular case that inhibition of transport would be directly proportional to the number of S460C subunits present in a putative functional oligomer of stoichiometry N, we would be unable to distinguish between a functional unit comprising Nindependent transport pathways and a single composite transport pathway formed by the N subunits, i.e. a 1:1 relation between % inhibition and fraction of mutant cRNA would also be predicted, as for the functional monomer. These two possibilities could be best resolved by morphological means (21Eskandari S. Snyder P.M. Kreman M. Zampighi G.A. Welsh M.J. Wright E.M. J. Biol. Chem. 1999; 274: 27281-27286Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar).Concatameric ConstructsThe kinetic behavior of both the mixed (WT-S460C) and control concatamers (WT-WT, S460C-S460C) confirmed that the transport characteristics of the protein were unaltered compared with the original WT and S460C alone. Moreover, for the WT-WT concatamer, MTSEA application neither altered the transport rate nor the steady-state voltage dependence, which provided further validation of our assumption regarding its use in the present study.The Western blots for oocytes that displayed transport function confirmed the integrity of the fusion proteins by the presence of a single band that had twice the molecular weight of the individual proteins. Based on the Western blot data, the protein levels of the concatamers appeared comparable to those of the WT; however, these data alone do not provide any information concerning the amount of functional protein in the membrane, since the Western blots were obtained from whole cell lysate. Taking together the Western blot and electrophysiological data would therefore suggest that the concatameric protein may not be as efficiently inserted in the membrane as the WT.The concatamer that comprised one WT and one mutant (S460C) transporter as a fusion protein (WT-S460C) provided further evidence of a monomeric functional unit for the NaPi-IIa protein. An alternative proposition is that the dimers themselves associate to form functional tetramers. If this were the case, given random association of dimers and full block of transport for any tetramer that comprised at least one S460C as a subunit, we would then predict a 25% inhibition of function after MTSEA treatment. Our data clearly exclude this possibility. As for the coinjection approach, with the exception of the case in which the S460C half of the concatamer contributes 50% to a common transport pathway that is only 50% inhibited after Cys modification, the behavior of the mixed concatamer is fully consistent with that expected for a functional monomer.Comparison with Other StudiesThe radiation-inactivation method has been used extensively to estimate the molecular size of the functional unit of membrane enzymes (32Kempner E.S. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 107-147PubMed Google Scholar). By using this approach, the Béliveau group (13Beliveau R. Demeule M. Ibnoul-Khatib H. Bergeron M. Beauregard G. Potier M. Biochem. J. 1988; 252: 807-813Crossref PubMed Scopus (42) Google Scholar, 14Delisle M.C. Vachon V. Giroux S. Potier M. Laprade R. Beliveau R. Biochim. Biophys. Acta. 1992; 1104: 132-136Crossref PubMed Scopus (14) Google Scholar, 15Delisle M.C. Giroux S. Vachon V. Boyer C. Potier M. Beliveau R. Biochemistry. 1994; 33: 9105-9109Crossref PubMed Scopus (12) Google Scholar, 16Jette M. Vachon V. Potier M. Beliveau R. Biochemistry. 1996; 35: 15209-15214Crossref PubMed Scopus (7) Google Scholar, 17Tenenhouse H.S. Lee J. Harvey N. Potier M. Jette M. Beliveau R. Biochem. Biophys. Res. Commun. 1990; 170: 1288-1293Crossref PubMed Scopus (13) Google Scholar, 18Xiao Y. Boyer C.J. Vincent E. Dugre A. Vachon V. Potier M. Beliveau R. Biochem. J. 1997; 323: 401-408Crossref PubMed Scopus (22) Google Scholar) has proposed that the functional unit responsible for Na+-dependent Picotransport in BBMVs is a multimer. Although uncertainties concerning the contribution of Pi uptake by proteins other than NaPi-IIa have been recently addressed by combining Western blot analysis with radiation inactivation (18Xiao Y. Boyer C.J. Vincent E. Dugre A. Vachon V. Potier M. Beliveau R. Biochem. J. 1997; 323: 401-408Crossref PubMed Scopus (22) Google Scholar), a study by Tatsumi et al. (33Tatsumi S. Miyamoto K. Kouda T. Motonaga K. Katai K. Ohkido I. Morita K. Segawa H. Tani Y. Yamamoto H. Taketani Y. Takeda E. J. Biol. Chem. 1998; 273: 28568-28575Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) has reported the existence of other NaPi-II-related proteins in BBMVs. Since these share the same NH2 or COOH-terminal regions, they would also be detected by Western blot together with the full NaPi-IIa protein. This lack of discrimination of the Western blot analysis could therefore oversimplify the identification of the protein constituents of a functional unit.A discrepancy in estimating the functional size of a transport protein has also been reported for the sodium glucose cotransport system. Freeze fracture analysis of SGLT-1 expressed in Xenopusoocytes reveals membrane-bound particles that correlate with expression of SGLT-1 protein (34Zampighi G.A. Kreman M. Boorer K.J. Loo D.D. Bezanilla F. Chandy G. Hall J.E. Wright E.M. J. Membr. Biol. 1995; 148: 65-78Crossref PubMed Scopus (206) Google Scholar), and the particle size strongly suggests a monomeric assembly for SGLT-1 (35Eskandari S. Wright E.M. Kreman M. Starace D.M. Zampighi G.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11235-11240Crossref PubMed Scopus (160) Google Scholar). However, from radiation inactivation studies (36Stevens B.R. Fernandez A. Hirayama B. Wright E.M. Kempner E.S. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1456-1460Crossref PubMed Scopus (71) Google Scholar), the reported RIS of ≈290 kDa, taken together with the molecular masses from Western blots of 70–75 kDa, suggests a tetrameric functional unit in situ. Since the conditions of the transport activity assay during irradiation also alter the predicted RIS as in the case of Pi uptake in BBMVs (16Jette M. Vachon V. Potier M. Beliveau R. Biochemistry. 1996; 35: 15209-15214Crossref PubMed Scopus (7) Google Scholar), this could indicate that the target size changes according to which transporter species are active. The assay conditions used in the present study are similar to those encountered in vivo(high inwardly directed Na+ gradient and membrane potentials in the physiological range), and therefore our findings are relevant to the function of NaPi-IIa protein in the intact renal proximal tubule.ConclusionsThis study presents evidence that NaPi-IIa protein is a functional monomer when expressed in Xenopusoocytes. Although we cannot exclude that NaPi-IIa molecules may aggregate either with themselves, to give rise to multiple, independent transport pathways within the same complex, or with other associated proteins in situ, oligomeric assembly of NaPi-IIa proteins is not a necessary condition for type II Na+-dependent Pi transport. The reabsorption of inorganic phosphate (Pi) at the luminal brush border membrane of the renal proximal tubule is mediated chiefly by a Na+-coupled cotransport process. Three unique types of Na+/Pi cotransporters have been identified in proximal tubular epithelia as follows: type I, type IIa, and type III (1Murer H. Forster I. Hernando N. Lambert G. Traebert M. Biber J. Am. J. Physiol. 1999; 277: F676-F684PubMed Google Scholar, 2Murer, H., Hernando, N., Forster, I., and Biber, J. (2000)Physiol. Rev., in pressGoogle Scholar). The type IIa Na+/Picotransporter (NaPi-IIa)1contributes the major component of luminal Pi transport (1Murer H. Forster I. Hernando N. Lambert G. Traebert M. Biber J. Am. J. Physiol. 1999; 277: F676-F684PubMed Google Scholar, 2Murer, H., Hernando, N., Forster, I., and Biber, J. (2000)Physiol. Rev., in pressGoogle Scholar, 3Beck L. Karaplis A.C. Amizuka N. Hewson A.S. Ozawa H. Tenenhouse H.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5372-5377Crossref PubMed Scopus (511) Google Scholar). Type IIa isoforms, cloned from various species, display similar kinetic properties when expressed in Xenopus oocytes (4Busch A. Waldegger S. Herzer T. Biber J. Markovich D. Hayes G. Murer H. Lang F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8205-8208Crossref PubMed Scopus (74) Google Scholar, 5Busch A.E. Wagner C.A. Schuster A. Waldegger S. Biber J. Murer H. Lang F. J. Am. Soc. Nephrol. 1995; 6: 1547-1551PubMed Google Scholar, 6Forster I. Hernando N. Biber J. Murer H. J. Gen. Physiol. 1998; 112: 1-18Crossref PubMed Scopus (110) Google Scholar, 7Hartmann C.M. Wagner C.A. Busch A.E. Markovich D. Biber J. Lang F. Murer H. Pfluegers Arch. 1995; 430: 830-836Crossref PubMed Scopus (73) Google Scholar,10Forster I. Loo D.D. Eskandari S. Am. J. Physiol. 1999; 276: F644-F649PubMed Google Scholar). On Western blots made from brush border membrane vesicles (BBMVs) the rat type IIa isoform (NaPi-IIa) protein is detected as an 80–90-kDa glycoprotein (8Custer M. Lotscher M. Biber J. Murer H. Kaissling B. Am. J. Physiol. 1994; 266: F767-F774PubMed Google Scholar, 9Biber J. Custer M. Magagnin S. Hayes G. Werner A. Lotscher M. Kaissling B. Murer H. Kidney Int. 1996; 49: 981-985Abstract Full Text PDF PubMed Scopus (98) Google Scholar). When expressed in Xenopus laevisoocytes, a similar band, specific to NaPi-IIa, at 70–110 kDa is observed (11Hayes G. Busch A. Lotscher M. Waldegger S. Lang F. Verrey F. Biber J. Murer H. J. Biol. Chem. 1994; 269: 24143-24149Abstract Full Text PDF PubMed Google Scholar, 12Lambert G. Forster I.C. Stange G. Biber J. Murer H. J. Gen. Physiol. 1999; 114: 637-652Crossref PubMed Scopus (38) Google Scholar). This molecular weight corresponds to that predicted from the amino acid sequence (68.7 kDa) after taking account of protein glycosylation (11Hayes G. Busch A. Lotscher M. Waldegger S. Lang F. Verrey F. Biber J. Murer H. J. Biol. Chem. 1994; 269: 24143-24149Abstract Full Text PDF PubMed Google Scholar). From a number of studies using the radiation inactivation technique applied to Pi uptake in BBMVs, estimates of the functional size of the Na+/Pi system have ranged from 124 to 242 kDa depending on the species and assay conditions (13Beliveau R. Demeule M. Ibnoul-Khatib H. Bergeron M. Beauregard G. Potier M. Biochem. J. 1988; 252: 807-813Crossref PubMed Scopus (42) Google Scholar, 14Delisle M.C. Vachon V. Giroux S. Potier M. Laprade R. Beliveau R. Biochim. Biophys. Acta. 1992; 1104: 132-136Crossref PubMed Scopus (14) Google Scholar, 15Delisle M.C. Giroux S. Vachon V. Boyer C. Potier M. Beliveau R. Biochemistry. 1994; 33: 9105-9109Crossref PubMed Scopus (12) Google Scholar, 16Jette M. Vachon V. Potier M. Beliveau R. Biochemistry. 1996; 35: 15209-15214Crossref PubMed Scopus (7) Google Scholar, 17Tenenhouse H.S. Lee J. Harvey N. Potier M. Jette M. Beliveau R. Biochem. Biophys. Res. Commun. 1990; 170: 1288-1293Crossref PubMed Scopus (13) Google Scholar, 18Xiao Y. Boyer C.J. Vincent E. Dugre A. Vachon V. Potier M. Beliveau R. Biochem. J. 1997; 323: 401-408Crossref PubMed Scopus (22) Google Scholar). The significant difference between the molecular weight of the type IIa protein (see above) and radiation inactivation size (RIS) was taken as evidence for an oligomeric functional unit of Na+/Pi cotransport. However, since Pi uptake in BBMVs most likely includes contributions from other Pi transport systems, e.g. type I, or other as yet unidentified proteins, estimation of the number of cotransporter subunits of a particular type that contribute to the functional transport unit is not possible. Moreover, the membrane target of radiation may include other proteins and/or membrane domains associated with the cotransporter itself, which could result in an overestimation of the functional mass contributed by type IIa proteins alone. Alternative approaches have been recently used to determine the functional composition of membrane transport pro
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