Mutational Analysis of the α-1 Repeat of the Cardiac Na+-Ca2+ Exchanger

The Na+-Ca2+ exchanger contains internal regions of sequence homology known as the α repeats. The first region (α-1 repeat) includes parts of transmembrane segments (TMSs) 2 and 3 and a linker modeled to be a reentrant loop. To determine the involvement of the reentrant loop and TMS 3 portions of the α-1 repeat in exchanger function, we generated a series of mutants and examined ion binding and transport and regulatory properties. Mutations in the reentrant loop did not substantially modify transport properties of the exchanger though the Hill coefficient for Na+ and the rate of Na+-dependent inactivation were decreased. Mutations in TMS 3 had more striking effects on exchanger activity. Of mutations at 10 positions, 3 behaved like the wild-type exchanger (V137C, A141C, M144C). Mutants at two other positions expressed no activity (Ser139) or very low activity (Gly138). Six different mutations were made at position 143; only N143D was active, and it displayed wild-type characteristics. The highly specific requirement for an asparagine or aspartate residue at this position may indicate a key role for Asn143 in the transport mechanism. Mutations at residues Ala140 and Ile147 decreased affinity for intracellular Na+, whereas mutations at Phe145 increased Na+ affinity. The cooperativity of Na+ binding was also altered. In no case was Ca2+ affinity changed. TMS 3 may form part of a site that binds Na+ but not Ca2+. We conclude that TMS 3 is involved in Na+ binding and transport, but previously proposed roles for the reentrant loop need to be reevaluated. The Na+-Ca2+ exchanger contains internal regions of sequence homology known as the α repeats. The first region (α-1 repeat) includes parts of transmembrane segments (TMSs) 2 and 3 and a linker modeled to be a reentrant loop. To determine the involvement of the reentrant loop and TMS 3 portions of the α-1 repeat in exchanger function, we generated a series of mutants and examined ion binding and transport and regulatory properties. Mutations in the reentrant loop did not substantially modify transport properties of the exchanger though the Hill coefficient for Na+ and the rate of Na+-dependent inactivation were decreased. Mutations in TMS 3 had more striking effects on exchanger activity. Of mutations at 10 positions, 3 behaved like the wild-type exchanger (V137C, A141C, M144C). Mutants at two other positions expressed no activity (Ser139) or very low activity (Gly138). Six different mutations were made at position 143; only N143D was active, and it displayed wild-type characteristics. The highly specific requirement for an asparagine or aspartate residue at this position may indicate a key role for Asn143 in the transport mechanism. Mutations at residues Ala140 and Ile147 decreased affinity for intracellular Na+, whereas mutations at Phe145 increased Na+ affinity. The cooperativity of Na+ binding was also altered. In no case was Ca2+ affinity changed. TMS 3 may form part of a site that binds Na+ but not Ca2+. We conclude that TMS 3 is involved in Na+ binding and transport, but previously proposed roles for the reentrant loop need to be reevaluated. Na+-Ca2+ exchangers are found in a wide variety of tissues. These plasma membrane proteins are electrogenic transporters that utilize the electrochemical gradient of Na+ to exchange three extracellular Na+ ions for one intracellular Ca2+. As a Ca2+ efflux mechanism, the exchanger helps maintain intracellular Ca2+ homeostasis. Cloning of the Na+-Ca2+ exchanger (1Nicoll D.A. Longoni S. Philipson K.D. Science. 1990; 250: 562-565Crossref PubMed Scopus (628) Google Scholar) revealed a protein composed of 938 amino acids organized in nine membrane-spanning regions with the first five transmembrane segments (TMSs) 1The abbreviations used are: TMSs, transmembrane segments; MES, 4-morpholineethanesulfonic acid; PIP2, phosphatidylinositol phosphate; TEAOH, tetraethylammonium hydroxide. separated from the four C-terminal TMSs by a large cytoplasmic loop (2Nicoll D.A. Ottolia M. Lu L. Lu Y. Philipson K.D. J. Biol. Chem. 1999; 274: 910-917Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 3Nicoll D.A. Ottolia M. Philipson K.D. Ann. N. Y. Acad. Sci. 2002; 976: 11-18Crossref PubMed Scopus (24) Google Scholar, 4Iwamoto T. Uehara A. Imanaga I. Shigekawa M. J. Biol. Chem. 2000; 275: 38571-38580Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 5Iwamoto T. Nakamura T.Y. Pan Y. Uehara A. Imanaga I. Shigekawa M. FEBS Lett. 1999; 446: 264-268Crossref PubMed Scopus (90) Google Scholar). Much progress has been made in understanding exchanger topology, function, and regulation. Modulation by intracellular factors such as Na+ and Ca2+ ions, ATP, and PIP2, have been extensively studied, and the domains involved in regulation have been localized to the large cytoplasmic loop (6Philipson K.D. Nicoll D.A. Ann. Rev. Physiol. 2000; 62: 111-133Crossref PubMed Scopus (446) Google Scholar). Less is known about the regions of the Na+-Ca2+ exchanger involved in the translocation of Na+ and Ca2+. Two regions with similar sequences, called the α-1 and α-2 repeats, likely have key roles. The α-1 repeat consists of residues spanning TMSs 2 and 3 and an extracellular loop connecting the two TMSs. The homologous α-2 repeat encompasses TMS 7 and part of the following intracellular loop (Fig. 1). Several observations emphasize the functional importance of the α repeats. First, they are highly conserved among all Na+-Ca2+ exchangers and are the only regions of sequence conservation between members of the NCX and NCKX families (7Schwarz E.M. Benzer S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10249-10254Crossref PubMed Scopus (181) Google Scholar). Second, data suggest that the portions of the α repeats connecting TMS 2–3 and TMS 7–8 form reentrant membrane loops (Fig. 1 and Ref. 2Nicoll D.A. Ottolia M. Lu L. Lu Y. Philipson K.D. J. Biol. Chem. 1999; 274: 910-917Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Reentrant loops have been identified in ion channels (“P-Loops”), and in the aquaporin family of proteins and form a portion of the hydrophilic pathway for ions or other molecules to cross the membrane (8Hille B. Armstrong C.M. MacKinnon R. Nat. Med. 1999; 5: 1105-1109Crossref PubMed Scopus (123) Google Scholar, 9Fujiyoshi Y. Mitsuoka K. de Groot B.L. Philippsen A. Grubmuller H. Agre P. Engel A. Curr. Opin. Struct. Biol. 2002; 12: 509-515Crossref PubMed Scopus (236) Google Scholar). By analogy, the reentrant loops within the α repeats may be involved in the translocation pathway for Na+ and Ca2+. This hypothesis is supported by helix packing studies, which demonstrate that the two α repeats are in proximity (10Qiu Z. Nicoll D.A. Philipson K.D. J. Biol. Chem. 2001; 276: 194-199Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). In addition, site-directed mutagenesis reveals that mutations in the α repeats drastically alter the transport of Na+ and Ca2+ (4Iwamoto T. Uehara A. Imanaga I. Shigekawa M. J. Biol. Chem. 2000; 275: 38571-38580Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 11Nicoll D.A. Hryshko L.V. Matsuoka S. Frank J.S. Philipson K.D. J. Biol. Chem. 1996; 271: 13385-13391Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 12Shigekawa M. Iwamoto T. Uehara A. Kita S. Ann. N. Y. Acad. Sci. 2002; 976: 19-30Crossref PubMed Scopus (19) Google Scholar), supporting a role in ion translocation.Fig. 1Membrane topology of the Na+-Ca2+ exchanger. The top panel shows a schematic representation of Na+-Ca2+ exchanger topology. Numbers indicate transmembrane segments while dark areas mark the α repeats. The alignment in the bottom panel shows the sequence similarity of the α-1 repeat within diverse members of the Na+-Ca2+ exchanger family. Residues marked with an asterisk were individually mutated and analyzed. The symbol † indicates that mutation of Ser139 produced an inactive exchanger.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The aim of this study was to further define the role of the α-1 repeat in ion translocation by combining mutational and biophysical analysis. Mutations were made in the first reentrant loop and in TMS 3. We show that mutations within the reentrant loop do not cause major perturbations in ion translocation; all mutations lead to active exchangers with little alteration in ion affinities or regulatory properties. These data seem to exclude the reentrant loop of the α-1 repeat as a key component of ion translocation. In contrast, residues within TMS 3 are important in determining Na+ affinity. Mutations of residues Ala140, Phe145, and Ile147 drastically alter the apparent affinity for Na+, without affecting the binding of transported Ca2+. Thus, TMS 3 is important in determining the ion dependence of the Na+-Ca2+ exchanger. Molecular Biology and 45Ca2+Uptake Measurements—Mutations were generated in 200–400-bp cassettes from the full-length exchanger using QuikChange Mutagenesis (Stratagene). Cassettes were then sequenced and subcloned back into the full-length exchanger. RNA was synthesized using T3 mMessage mMachine (Ambion) after linearization with HindIII. RNA was injected into Xenopus oocytes and 45Ca2+ uptake measured as described previously (13Doering A.E. Nicoll D.A. Lu Y. Lu L. Weiss J.N. Philipson K.D. J. Biol. Chem. 1998; 273: 778-783Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). To measure extracellular Ca2+ dependence, mutants were transiently expressed in BHK or HEK cells, and 45Ca2+ uptake was measured at different [Ca2+]. Data were fit to the Michaelis-Menten equation using GraphPad Prism version 4.0 for Macintosh. The dog/squid NCX was generated by a of mutagenesis and The exchanger has a site at the position and an site at the The exchanger also has an site was at the site in the by The exchanger was then with and and the from the exchanger was into the exchanger. This in a exchanger the with the reentrant loop between TMSs 2 and 3. Na+-Ca2+ exchange were using the Lu 1998; PubMed Scopus Google Scholar). were and to a of about and with a of and to the membrane of the was in an to the for 10 2 to 7 using membrane were using a of the exchanger were using the following 10 7 or TEAOH, 10 10 or 1 7 were measured using the following 10 TEAOH, 10 2 7 TEAOH, 10 10 7 were to the Na+ and Ca2+ were by with different ion were to a Hill and to are were different at a of was used for and analysis. Data were at and at using an were at and at a of of Reentrant generated mutants and of the first reentrant loop of the Na+-Ca2+ exchanger and biophysical properties. Mutants were expressed in Xenopus oocytes and by 45Ca2+ uptake and by using the In a Ca2+ is within the at the extracellular Na+-Ca2+ exchange is by the of Na+ to the intracellular In this the of two different regulatory properties 1990; PubMed Scopus Google Scholar). First, Na+ to transport, Na+ at the intracellular also a inactivation known as and Ref. Matsuoka S. A. J. Physiol. PubMed Scopus Google Scholar). Second, intracellular Ca2+ binds to a regulatory site on the large intracellular loop to transport A. Matsuoka S. J. Physiol. PubMed Scopus Google Scholar, S. Nicoll D.A. Hryshko L.V. Weiss J.N. Philipson K.D. J. Physiol. PubMed Scopus Google Scholar). To measure apparent ion affinities at transport without effects of regulatory the exchanger be first by with at the intracellular We first apparent affinities for Na+, measured at the cytoplasmic of data and show that the of Na+ to of the mutated exchangers decreased in and and increased for of did not apparent and also a in the Hill coefficient with the effects for and (Fig. The of the Na+ dependence for and mutants to transport more at Na+ the 2 shows that at intracellular Na+, and wild-type exchangers had about and of To determine the affinity of the mutated exchangers for transported exchange were generated by of different cytoplasmic Ca2+ with extracellular Na+ within the We found no in the apparent Ca2+ affinity for reentrant loop a of the Ca2+ dependence 2 and and are of a study that of of residues with a decreased the apparent affinity of the binding site for transported Ca2+ (4Iwamoto T. Uehara A. Imanaga I. Shigekawa M. J. Biol. Chem. 2000; 275: 38571-38580Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). In the the exchangers had apparent affinities for Ca2+. The of and wild-type exchangers were measured at different extracellular Ca2+ as Na+ 45Ca2+ uptake into and behaved to (Fig. This is with the on the apparent affinities for intracellular Ca2+. We also examined the Na+-dependent of the mutated exchangers (Fig. Mutations and substantially the of Na+-dependent were to a to of for for and for the wild-type exchanger. also indicate that the exchanger had inactivation while mutations at position and no in the inactivation We have previously on the inactivation of D.A. Ottolia M. Philipson K.D. Ann. N. Y. Acad. Sci. 2002; 976: 11-18Crossref PubMed Scopus (24) Google Scholar). indicate that residues the reentrant loop connecting TMSs 2 and 3 do not cytoplasmic affinities for transported ions but alter the cooperativity of Na+ binding and inactivation of the in the portion of the α-1 repeat that the membrane is highly conserved in the three Na+-Ca2+ exchangers and but in such as the exchanger has the following and has biophysical properties similar to the Z. Philipson K.D. J. Physiol. 1998; PubMed Scopus Google Scholar). the reentrant loop is involved in ion transport, then other parts of the protein for the amino within the reentrant loop. To this we the α-1 reentrant loop of the Na+-Ca2+ exchanger with that from we have that the from the exchanger and inactivation D.A. Ottolia M. Philipson K.D. Ann. N. Y. Acad. Sci. 2002; 976: 11-18Crossref PubMed Scopus (24) Google Scholar). We of the regulatory properties of the a cytoplasmic Ca2+ of 1 the for inactivation of exchangers was increased as with the wild-type Na+-Ca2+ exchanger 3 The affinities of the exchanger for transported ions at the cytoplasmic were after the of the and inactivation and using As in and the exchanger displayed a but in Na+ affinity and a in cooperativity bottom panel of The fit of the Na+ dependence to a Hill apparent affinities of for the and for the with Hill of and The Ca2+ dependence for the transport site on the cytoplasmic was also and no were found between the Na+-Ca2+ exchanger and the exchanger residues to of the of shows of and the Ca2+ for The indicate that of the extracellular portion of the α-1 repeat not cause major perturbations of Na+ or Ca2+ were effects on the apparent Na+ affinity and more effects on the cooperativity of Na+ of within TMS made mutations at residues and of mutations at three of the residues and have been previously D.A. Hryshko L.V. Matsuoka S. Frank J.S. Philipson K.D. J. Biol. Chem. 1996; 271: 13385-13391Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). mutations of to an or have in low activity and were not Mutants and are inactive D.A. Hryshko L.V. Matsuoka S. Frank J.S. Philipson K.D. J. Biol. Chem. 1996; 271: 13385-13391Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), and we examined the effects of mutations at position of Asn143 with a or also all produced inactive the N143D produced an exchanger with Na+ affinity and activity (Fig. The N143D exchanger also displayed regulatory properties. and are in Na+-dependent inactivation is by intracellular and activity the of regulatory Ca2+ as also with the wild-type exchanger. Of the residues three (V137C, A141C, and displayed Na+-Ca2+ exchange and had biophysical properties similar to and Mutations at positions Ala140, Phe145, and displayed characteristics. of residues Ala140, Phe145, and Ile147 in the α-1 region of TMS 3 in the apparent affinity for intracellular As in mutation of at position or at position decreased the Na+ affinity of the exchanger. In contrast, exchanger had an increased Na+ affinity. The apparent Na+ affinities for for and for The for the wild-type exchanger was The of the in the Na+ affinity by the of with the of the residue and The the affinity for Na+ and the the affinity of produced two an in the apparent Na+ affinity and a in the Hill coefficient for Na+ and Ca2+. The in Na+ affinity was in mutants and and did not with or of the residue and The in the of the Na+ dependence was in mutants and and may with or and are of and are or and are also or Thus, the of the Na+ dependence may be by the or the of the amino at position mutation at a also had a decreased Hill coefficient for Na+ in this apparent Na+ affinity was (Fig. We also the of exchangers and to cytoplasmic Ca2+ (Fig. We no in the apparent affinities for Ca2+. mutation of to the Hill coefficient for the binding of cytoplasmic Hill were and for wild-type and the of the Na+-Ca2+ exchanger in (1Nicoll D.A. Longoni S. Philipson K.D. Science. 1990; 250: 562-565Crossref PubMed Scopus (628) Google Scholar), has been progress in understanding exchanger and regulatory regions have been identified S. Nicoll D.A. Hryshko L.V. Weiss J.N. Philipson K.D. J. Physiol. PubMed Scopus Google Scholar, S. Nicoll D.A. Philipson K.D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), and a topology has been proposed (2Nicoll D.A. Ottolia M. Lu L. Lu Y. Philipson K.D. J. Biol. Chem. 1999; 274: 910-917Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 3Nicoll D.A. Ottolia M. Philipson K.D. Ann. N. Y. Acad. Sci. 2002; 976: 11-18Crossref PubMed Scopus (24) Google Scholar, 5Iwamoto T. Nakamura T.Y. Pan Y. Uehara A. Imanaga I. Shigekawa M. FEBS Lett. 1999; 446: 264-268Crossref PubMed Scopus (90) Google Scholar). The indicate a protein of nine transmembrane segments with the five TMSs separated from the four TMSs by a large cytoplasmic loop. The hydrophilic connecting TMSs 2 and 3 and TMSs 7 and have been proposed to form reentrant loops as residues are to to of the membrane (2Nicoll D.A. Ottolia M. Lu L. Lu Y. Philipson K.D. J. Biol. Chem. 1999; 274: 910-917Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 5Iwamoto T. Nakamura T.Y. Pan Y. Uehara A. Imanaga I. Shigekawa M. FEBS Lett. 1999; 446: 264-268Crossref PubMed Scopus (90) Google Scholar). These reentrant loops are on of the membrane (Fig. and are in proximity to one (10Qiu Z. Nicoll D.A. Philipson K.D. J. Biol. Chem. 2001; 276: 194-199Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). These two are parts of the α repeats, homologous within the exchanger of about amino acids parts of TMSs 2 and 3 (α-1 repeat) and a portion of TMS 7 Reentrant membrane loops were first described in ion channels they form a portion of the By analogy, the reentrant loops found in the Na+-Ca2+ exchanger family may be important for ion In of a described four mutations within the hydrophilic portion of the α-1 reentrant loop and that to a in the apparent affinity for Ca2+ T. Nakamura T.Y. Pan Y. Uehara A. Imanaga I. Shigekawa M. FEBS Lett. 1999; 446: 264-268Crossref PubMed Scopus (90) Google Scholar). In this we further the role of the first reentrant loop in ion translocation and to residues the first portion of TMS 3. data demonstrate that amino acids the extracellular linker between TMSs 2 and 3 are and that do not the transport of exchangers and and the exchanger the and in intracellular Na+ though intracellular Ca2+ affinities were These data to the first reentrant loop as a key component of the ion binding and translocation This with ion loops that form the ion within the with is the conservation of residues within the reentrant loop. a important reentrant loop to demonstrate the of conservation in the TMSs 2 and 3 (Fig. 1). The of of the in the and a key role for the reentrant loop. The of reentrant loop mutations was on the cooperativity of Na+ the of the all mutations within the reentrant loop decreased the Hill coefficient of the Na+ dependence, a in the Hill coefficient decreased from the wild-type of 2 to a to 1 for the and The data suggest that residues are involved in the between Na+ binding We found that reentrant loop mutations had little on the cytoplasmic or extracellular Ca2+ This is have that mutations at position and in the reentrant loop decreased the affinity for Ca2+ T. Nakamura T.Y. Pan Y. Uehara A. Imanaga I. Shigekawa M. FEBS Lett. 1999; 446: 264-268Crossref PubMed Scopus (90) Google Scholar). We do not have an for this apparent Na+ is transported by the Na+-Ca2+ exchanger but also exchange activity. of internal Na+ the exchanger into a Matsuoka S. A. J. Physiol. PubMed Scopus Google Scholar). The that lead to the Na+-dependent inactivation are not it that Na+ to the internal transport the Matsuoka S. A. J. Physiol. PubMed Scopus Google Scholar). two mutants within the first reentrant loop of the exchanger and show a of exchange in the of cytoplasmic The data suggest that residues within the first reentrant loop to regulation and involved in the Na+-dependent inactivation This is of inactivation found in ion channels T. Science. 1990; 250: PubMed Scopus Google Scholar, T. Full Text PDF PubMed Scopus Google Scholar). inactivation a of the to of the Mutations within the of the alter inactivation P. R. FEBS Lett. 1998; PubMed Scopus Google Scholar, Y. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Physiol. 1998; PubMed Scopus Google Scholar). By analogy, cytoplasmic Na+ may a of the region of the exchanger and of this region of transport The functional importance of TMS 2 and the linker between TMS 2 and 3 has been extensively mutagenesis (2Nicoll D.A. Ottolia M. Lu L. Lu Y. Philipson K.D. J. Biol. Chem. 1999; 274: 910-917Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 4Iwamoto T. Uehara A. Imanaga I. Shigekawa M. J. Biol. Chem. 2000; 275: 38571-38580Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 5Iwamoto T. Nakamura T.Y. Pan Y. Uehara A. Imanaga I. Shigekawa M. FEBS Lett. 1999; 446: 264-268Crossref PubMed Scopus (90) Google Scholar, 11Nicoll D.A. Hryshko L.V. Matsuoka S. Frank J.S. Philipson K.D. J. Biol. Chem. 1996; 271: 13385-13391Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, A.E. Nicoll D.A. Lu Y. Lu L. Weiss J.N. Philipson K.D. J. Biol. Chem. 1998; 273: 778-783Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) with the role of TMS 2 in ion translocation. In contrast, TMS 3 has little Nicoll D.A. Hryshko L.V. Matsuoka S. Frank J.S. Philipson K.D. J. Biol. Chem. 1996; 271: 13385-13391Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) that exchange activity is highly to mutation of or Asn143 within TMS 3. data this portion of the exchanger in with TMSs and to form a that may define a pathway for ion (10Qiu Z. Nicoll D.A. Philipson K.D. J. Biol. Chem. 2001; 276: 194-199Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). These data and the conservation of this region suggest an involvement in the of To TMS 3 a role in ion and transport, we the effects of mutations within TMS 3. We mutated of the residues to the α repeat portion of TMS 3 and measured the effects of mutations on Na+ and Ca2+ translocation. Several mutations produced First, as previously D.A. Hryshko L.V. Matsuoka S. Frank J.S. Philipson K.D. J. Biol. Chem. 1996; 271: 13385-13391Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), mutants and have low activity and has no activity. Of the active of and produced the in the biophysical properties of the Na+-Ca2+ exchanger. Mutations at the apparent affinity of the exchanger for Na+, with the at position The effects of mutations at position may be to the of the with a large amino produced the in the Na+ dependence, while of a residue did not modify is no to the effects of mutations at position Ca2+ and Na+ are to at the mutations that alter Na+ dependence also be to modify the apparent affinity for transported Ca2+. the apparent affinity for Ca2+ of exchangers and was not different from that measured for the exchanger. has previously been proposed J. Biol. Chem. Full Text PDF PubMed Google Scholar, PubMed Scopus Google Scholar) that the Na+-Ca2+ exchanger has two of binding of binds Ca2+ or two Na+ and the binds one Residues and may be involved in the of and mutations Na+, but not affinity. is little on the of Na+ binding within the exchanger. The only TMS mutation previously to alter Na+ affinity is the intracellular of (13Doering A.E. Nicoll D.A. Lu Y. Lu L. Weiss J.N. Philipson K.D. J. Biol. Chem. 1998; 273: 778-783Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). In this the cooperativity of Na+ transport was also be to the asparagine at position in TMS 3. of this residue with an or residue exchange activity. of Asn143 with an aspartate residue produced an exchanger from is striking that of an aspartate properties while of of five other residues activity. The of this position to mutations may indicate the involvement of this residue in ion transport and also indicates a requirement for specific amino acids or at this and are all of similar but only aspartate and asparagine are Thus, not only but also of at this position is in exchanger activity. may seem that of a aspartate residue into the of a transmembrane not transport in a this aspartate may in an and The three Na+-Ca2+ exchanger mutations in TMS 3 that do not properties and all be modeled to be on the of a transmembrane the mutations that alter exchanger are from this These may TMSs and that have been modeled to with TMS 3 and form an ion translocation pathway (10Qiu Z. Nicoll D.A. Philipson K.D. J. Biol. Chem. 2001; 276: 194-199Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). data indicate that residues the first reentrant membrane loop of the Na+-Ca2+ exchanger do not have a key role in ion transport but be involved in with Na+-dependent inactivation and in cooperativity between Na+ binding In contrast, at amino residues within the extracellular of TMS 3 are important of exchanger and may be involved in the binding of The Na+ binding site with TMS 3 may be a site that not in Ca2+ The data the importance of TMS 3 and the α-1 repeat in exchanger We Hryshko for on this