Topology of a Functionally Important Region of the Cardiac Na+/Ca2+ Exchanger

The cardiac Na+/Ca2+ exchanger, NCX1, has been modeled to consist of 11 transmembrane segments and a large cytoplasmic loop (loop f). Cysteine mutagenesis and sulfhydryl modification experiments demonstrate that the loop connecting transmembrane segments 1 and 2 (loop b) is located on the cytoplasmic side of the membrane, as previously modeled. A mutation in loop b, asparagine 101 to cysteine (N101C), renders the exchanger insensitive to regulation by cytoplasmic Na+ and Ca2+. Nearby mutations at residue threonine 103 (T103C or T103V) increase the apparent affinity of the exchanger for cytoplasmic Na+ and also produce a significant Li+ transport capacity. The evidence suggests that the region at the interface of cytoplasmic loop b and transmembrane segment 2 is important in Na+ transport and also in secondary regulation. Thus, this region may form part of the link between the ion translocation pathway formed by the transmembrane segments and regulatory sites that have previously been localized to loop f. The cardiac Na+/Ca2+ exchanger, NCX1, has been modeled to consist of 11 transmembrane segments and a large cytoplasmic loop (loop f). Cysteine mutagenesis and sulfhydryl modification experiments demonstrate that the loop connecting transmembrane segments 1 and 2 (loop b) is located on the cytoplasmic side of the membrane, as previously modeled. A mutation in loop b, asparagine 101 to cysteine (N101C), renders the exchanger insensitive to regulation by cytoplasmic Na+ and Ca2+. Nearby mutations at residue threonine 103 (T103C or T103V) increase the apparent affinity of the exchanger for cytoplasmic Na+ and also produce a significant Li+ transport capacity. The evidence suggests that the region at the interface of cytoplasmic loop b and transmembrane segment 2 is important in Na+ transport and also in secondary regulation. Thus, this region may form part of the link between the ion translocation pathway formed by the transmembrane segments and regulatory sites that have previously been localized to loop f. The Na+/Ca2+ exchanger transports one Ca2+ across the cell membrane in exchange for three Na+ and is important in maintaining Ca2+homeostasis in many cell types, including muscle, nerve, and epithelium. In cardiac muscle, Na+/Ca2+exchange has been well characterized as the primary mechanism for extrusion of the Ca2+ that enters the cell to initiate contraction. Na+/Ca2+ exchange is therefore essential to cardiac relaxation, and an understanding of the function and regulation of this protein is essential to understanding the basis of cardiac excitation-contraction coupling. Na+/Ca2+ exchange activity can be readily observed using inside-out giant excised patches in which “reverse” exchange is initiated by applying Na+ to the intracellular surface. This generates an outward current as intracellular Na+ is exchanged for extracellular Ca2+. This protocol, and other studies, have demonstrated that the exchanger is subject to modulation by several cytoplasmic factors, including Na+ and Ca2+. Secondary Na+ regulation is demonstrated by rapidly raising the intracellular Na+ concentration to activate outward Na+/Ca2+ exchange current. Current peaks and then declines over several seconds to a reduced steady-state level. The extent and rate of this partial inactivation are dependent on the concentration of intracellular Na+. This regulatory process has been referred to as Na+-dependent inactivation (1Matsuoka S. Hilgemann D.W. J. Physiol. 1994; 476: 443-458Crossref PubMed Scopus (67) Google Scholar, 2Hilgemann D.W. Nature. 1990; 344: 242-245Crossref PubMed Scopus (247) Google Scholar). Regulation of the Na+/Ca2+ exchanger by intracellular Ca2+ has been observed in several preparations. For example, squid axon and mammalian cardiac Na+/Ca2+ exchangers have an absolute requirement for intracellular Ca2+. The stimulatory effect of intracellular Ca2+ is most easily demonstrated by measuring reverse Na+/Ca2+ exchange. Under these conditions, the nontransported regulatory Ca2+ is on the opposite side of the membrane from the transported extracellular Ca2+. When NCX1 is expressed in oocytes, a reduction in the intracellular Ca2+ markedly decreases subsequent Ca2+ influx via reverse exchange (3Matsuoka S. Nicoll D.A. Hryshko L.V. Levitsky D.O. Weiss J.N. Philipson K.D. J. Gen. Physiol. 1995; 105: 403-420Crossref PubMed Scopus (203) Google Scholar). These and other regulatory effects can be abolished by partial proteolysis with chymotrypsin (2Hilgemann D.W. Nature. 1990; 344: 242-245Crossref PubMed Scopus (247) Google Scholar). Based on hydropathy analysis, the exchanger is modelled to consist of 11 transmembrane segments with a large intracellular loop (loop f) between transmembrane segments 5 and 6 (Fig. 1). The extracellular location of the amino terminus (4Hryshko L.V. Nicoll D.A. Weiss J.N. Philipson K.D. Biochim. Biophys. Acta. 1993; 1151: 35-42Crossref PubMed Scopus (62) Google Scholar) and the intracellular location of loop f (6Matsuoka S. Nicoll D.A. Reilly R.F. Hilgemann D.W. Philipson K.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3870-3874Crossref PubMed Scopus (199) Google Scholar, 7Porzig H. Li Z.P. Nicoll D.A. Philipson K.D. Am. J. Physiol. 1993; 265: C748-C756Crossref PubMed Google Scholar) have been experimentally verified. Also, loop e, between transmembrane segments 4 and 5, appears to be extracellular, based on the glycosylation of an exchanger truncated at that site (5Sahin-Toth M. Nicoll D.A. Frank J.S. Philipson K.D. Friedlander M. Biochem. Biomed. Res. Commun. 1995; 212: 968-974Crossref PubMed Scopus (25) Google Scholar). The transmembrane segments are essential for ion transport, whereas loop f is involved in regulation (6Matsuoka S. Nicoll D.A. Reilly R.F. Hilgemann D.W. Philipson K.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3870-3874Crossref PubMed Scopus (199) Google Scholar). Through mutagenesis studies, it has been determined that the endogenous XIP 1The abbreviations used are: XIP, exchanger inhibitory peptide; MTS, methanethiosulfonate; MTSET, (2-(trimethylammonium)ethyl) methanethiosulfonate; MTSES, (2-sulfonatoethyl) methanethiosulfonate; NEM,N-ethylmaleimide; TEAOH, tetraethylammonium hydroxide. 1The abbreviations used are: XIP, exchanger inhibitory peptide; MTS, methanethiosulfonate; MTSET, (2-(trimethylammonium)ethyl) methanethiosulfonate; MTSES, (2-sulfonatoethyl) methanethiosulfonate; NEM,N-ethylmaleimide; TEAOH, tetraethylammonium hydroxide. region in loop f (8Li Z. Nicoll D.A. Collins A. Hilgemann D.W. Filoteo A.G. Penniston J.T. Weiss J.N. Tomich J.M. Philipson K.D. J. Biol. Chem. 1991; 266: 1014-1020Abstract Full Text PDF PubMed Google Scholar) is involved in Na+-dependent inactivation (9Matsuoka S. Nicoll D.A. He Z. Philipson K.D. J. Gen. Physiol. 1997; 109: 273-286Crossref PubMed Scopus (142) Google Scholar) and that another portion of loop f is involved in the binding of regulatory Ca2+ (Fig. 1) (3Matsuoka S. Nicoll D.A. Hryshko L.V. Levitsky D.O. Weiss J.N. Philipson K.D. J. Gen. Physiol. 1995; 105: 403-420Crossref PubMed Scopus (203) Google Scholar, 10Levitsky D.O. Nicoll D.A. Philipson K.D. J. Biol. Chem. 1994; 269: 22847-22852Abstract Full Text PDF PubMed Google Scholar). The two regulatory processes, however, are not independent. Binding of regulatory Ca2+decreases Na+-dependent inactivation (11Hilgemann D.W. Matsuoka S. Nagel G.A. Collins A. J. Gen. Physiol. 1992; 100: 905-932Crossref PubMed Scopus (236) Google Scholar), and mutations in the endogenous XIP region alter the kinetics of Ca2+ regulation (9Matsuoka S. Nicoll D.A. He Z. Philipson K.D. J. Gen. Physiol. 1997; 109: 273-286Crossref PubMed Scopus (142) Google Scholar). Other mutagenesis studies have implicated the involvement of two highly conserved and internally homologous regions (the α-repeats) in ion transport (12Nicoll 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 (126) Google Scholar). However, the molecular details of ion transport are unknown, as are the mechanisms by which loop f interacts with the transmembrane segments to effect regulation. We used substituted cysteine mutagenesis and sulfhydryl modification reagents to determine the membrane topology and explore the function of residues modelled to be at the interface of loop b and transmembrane segment 2. Wild-type NCX1 mutants were expressed in Xenopusoocytes, and Na+/Ca2+ exchange activity was measured as 45Ca2+ uptake into intact oocytes or as Na+i-activated outward current in giant patches excised from oocyte membranes. We show that this region is located on the cytoplasmic side of the membrane and may form part of the link between transport and regulation. Mutations in the wild-type exchanger were generated using the Sculptor in vitro mutagenesis kit (Amersham Corp.). cRNA was synthesized with mMessage mMachine (Ambion) and injected intoXenopus laevis oocytes. Na+/Ca2+exchange activities were measured after incubation of the oocytes for 3–6 days at 17–19 °C. To directly measure 45Ca2+ fluxes, oocytes were first loaded with Na+ by incubation in ice-cold Barth's solution containing 20 μm nystatin, 0.2% dimethyl sulfoxide for 10 min. Oocytes were then washed four times in 4 ml of 90 mm NaCl, 5 mm HEPES, pH 7.5, and placed into45Ca2+ uptake medium containing either NaCl (90 mm) (to measure nonspecific Ca2+ uptake) or KCl (90 mm) (to measure Na+gradient-dependent Ca2+ uptake) and 10 μm CaCl2, 6.4 mm4-morpholinepropanesulfonic acid, pH 7.4, and 6 μCi/ml45CaCl2 for 10 min, at which time Ca2+ uptake was still in a linear phase. Unincorporated45Ca2+ was then removed by washing the oocytes four times in 10 ml of ice-cold 90 mm KCl, 1 mmEGTA. Oocytes were individually placed into scintillation vials and dissolved in 0.25 ml of 1 m NaOH at 100 °C for 20 min. Scintillation fluid (10 ml) was added, and the amount of45Ca2+ uptake was determined by liquid scintillation counting. The small nonspecific Ca2+ uptake was subtracted from the Na+ gradient-dependent Ca2+ uptake as a measure of Na+/Ca2+ exchange activity. We have previously used this technique extensively to quantitate Na+/Ca2+ exchange activity (6Matsuoka S. Nicoll D.A. Reilly R.F. Hilgemann D.W. Philipson K.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3870-3874Crossref PubMed Scopus (199) Google Scholar, 12Nicoll 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 (126) Google Scholar, 13Nicoll D.A. Longoni S. Philipson K.D. Science. 1990; 250: 562-565Crossref PubMed Scopus (624) Google Scholar). Where indicated, oocytes were preloaded with Li+, instead of Na+. To investigate the effects of extracellular sulfhydryl reagents NEM, MTSES, and MTSET, oocytes were preincubated with reagents (10 mm) for 10 min and then washed four times. To determine the effects of intracellular sulfhydryl reagents, 100 mm MTSES or MTSET was injected into oocytes 10 min before measuring45Ca2+ uptake to produce an estimated cytoplasmic concentration of 10 mm (assuming an intracellular oocyte volume of 0.5 μl). MTS reagents were purchased from Toronto Research Chemicals. Na+/Ca2+ exchange current was measured under voltage clamp in inside-out membrane patches using the giant excised patch technique (2Hilgemann D.W. Nature. 1990; 344: 242-245Crossref PubMed Scopus (247) Google Scholar, 3Matsuoka S. Nicoll D.A. Hryshko L.V. Levitsky D.O. Weiss J.N. Philipson K.D. J. Gen. Physiol. 1995; 105: 403-420Crossref PubMed Scopus (203) Google Scholar, 6Matsuoka S. Nicoll D.A. Reilly R.F. Hilgemann D.W. Philipson K.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3870-3874Crossref PubMed Scopus (199) Google Scholar, 11Hilgemann D.W. Matsuoka S. Nagel G.A. Collins A. J. Gen. Physiol. 1992; 100: 905-932Crossref PubMed Scopus (236) Google Scholar). Pipettes were filled with the following external solution: 120 mm N-methyl-d-glucamine·2-(N-morpholino) ethanesulfonic acid, 30 mm HEPES, 8 mmCaCO3, 2 mm Ba(OH)2, 2 mm Mg(OH)2, 0.025 mm ouabain, 0.05 mm niflumic acid, 30 mm TEAOH. The bath, or cytoplasmic, solution contained 100 mm2-(N-morpholino) ethanesulfonic acid, 20 mmCsOH, 20 mm HEPES, 10 mm EGTA, 1 mmMg(OH)2, 20 mm TEAOH, and 100 mmCsOH, NaOH, or LiOH. CaCO3 was added to give the indicated free Ca2+ concentration. In experiments involving deregulation of the exchanger by partial proteolysis, 1 mg/ml chymotrypsin was added to a cytoplasmic solution containing 0 Na+ and 1 μm free Ca2+. All solutions were pH 7.0, membrane holding potential was 0 mV, and experiments were conducted at 34–35 °C. Data summaries are presented as an average of nexperiments ± S.E. Results from 6–8 oocytes were averaged for each experiment measuring 45Ca2+ flux. Fig. 1 shows a model for the Na+/Ca2+ exchange protein, based primarily on hydropathy analysis. Loop b consists of amino acids 63–101 and is modeled to be on the cytoplasmic side of the membrane. To determine the orientation of loop b with respect to the membrane, we used substituted cysteine mutagenesis. Specifically, residues modeled to be near the surface of the membrane were mutated to cysteine, and the mutants were exposed to sulfhydryl modifying reagents that form covalent bonds with accessible cysteines. We then examined the effects on exchange activity of membrane-permeable reagents and of impermeable reagents applied either extracellularly or intracellularly. The wild-type and mutated canine NCX1 exchangers were expressed inXenopus oocytes. Amino acids Ser-100, Asn-101, Leu-102, Thr-103, and Ala-106 were each individually mutated to cysteine. The mutant T103V (12Nicoll 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 (126) Google Scholar) was also examined in this study. Fig.2 shows the activity of each of the mutants relative to the wild-type exchanger. Except for mutant N101C, which had a wild-type level of activity, all of the mutants displayed approximately half the activity of the wild-type exchanger. This may reflect either a reduction in the level of protein expression or a direct effect of the mutation on exchange activity. The membrane-permeable reagent NEM had no significant effect on the wild-type exchanger but inhibited each of the cysteine mutants except A106C (Fig. 3). T103C was inhibited least. T103C and A106C were modeled to be within the membrane, and may be to membrane-permeable sulfhydryl reagents, the other cysteine The effects of intracellular or extracellular of MTSET or MTSES are in Fig. MTS reagents have been used extensively in sulfhydryl modification experiments D.A. M. A. Science. 1992; PubMed Scopus Google Scholar, J. A. 1995; Full Text PDF PubMed Scopus Google Scholar, Biophys. J. 1995; Full Text PDF PubMed Scopus Google Scholar). These is and MTSES is are The MTS reagents were either applied by oocytes in medium containing 10 mm or applied by 100 mm MTS solution into the oocyte for an estimated cytoplasmic concentration of 10 The wild-type exchanger is insensitive to either MTSET or MTSES, intracellular MTSET a of MTSET or MTSES had no effects on of the cysteine the other intracellular of MTSET or MTSES inhibited mutants and T103C may also be inhibited by intracellular The that the loop segment 2 interface is located at the cytoplasmic surface of the membrane. The also that the region is significant the activity of mutants was inhibited by sulfhydryl that two mutations in this region had important effects on of exchange to intracellular Na+ and which suggests that loop b is involved in secondary regulation by these A the regulatory effects of intracellular Na+ and Ca2+ on wild-type Na+/Ca2+ exchange current. Current is by of intracellular the is to the secondary inhibitory effect of D.W. Nature. 1990; 344: 242-245Crossref PubMed Scopus (247) Google Scholar). When intracellular Ca2+ is the current is the Ca2+ regulatory These regulatory effects are also of cardiac Na+/Ca2+ exchange activity measured in (1Matsuoka S. Hilgemann D.W. J. Physiol. 1994; 476: 443-458Crossref PubMed Scopus (67) Google Scholar). of these effects are abolished following partial proteolysis of the exchanger by Fig. 5 shows that has regulatory was no in current after current was by raising and was to was a small reduction in current In a of the wild-type Na+/Ca2+exchange current to ± of after Na+-dependent inactivation but of current was of regulatory Ca2+ reduced the wild-type current by ± after Na+-dependent but it reduced the current by ± to function the wild-type exchanger after deregulation by In exchangers and displayed regulatory mutant the wild-type exchanger in of a of to regulatory chymotrypsin still the current of This was small in the experiment in Fig. 5 but in other the was ± chymotrypsin also abolished NEM of N101C, as in In the experiment in outward current by raising cytoplasmic Na+ was inhibited by NEM applied in the cytoplasmic and this inhibitory effect was The that NEM the cysteine at residue 101 in a as to with with the (Fig. 3). When the exchanger was by cytoplasmic chymotrypsin and then exposed to NEM, no inhibitory effect was (Fig. 6 The that mutation the of the Na+/Ca2+ exchanger to regulatory We also that mutations in transmembrane segment near loop b, ion translocation of threonine 103 the of the on cytoplasmic shows the of outward Na+/Ca2+ exchange current on cytoplasmic Na+ concentration. The excised patches were first exposed to chymotrypsin to regulatory effects in these The wild-type was with at The for mutant T103V was to the with at mm) and with a T103V has a apparent affinity for cytoplasmic Na+ and an apparent of The observed increase in apparent affinity for cytoplasmic Na+ the that mutant T103V also have an for We this using 45Ca2+ Oocytes were loaded with Li+ in of and 45Ca2+ uptake was The T103V mutant is of a uptake the wild-type exchanger (Fig. were after with or not T103V therefore appears to have a reduced for Na+ over other and may be of other Na+ in exchange for Ca2+. is that T103V a level of exchange the wild-type exchanger, that be in exchange for endogenous intracellular of the In this be no membrane exchange is not However, in voltage clamp experiments Li+ a outward current in oocytes The wild-type exchanger shows no in current is Li+ significant outward current in oocytes not The of activate was not not to (Fig. exchange may have been the of for T103V oocytes were loaded with these A of the of outward membrane current on or mutant T103V is in Fig. The estimated for Na+ is 8 and for Li+, it is 100 Thus, in to an apparent increase in affinity for (Fig. T103V a significant current. T103C has not In this we have a previously region of the Na+/Ca2+ exchange Mutations in loop b and the cytoplasmic of transmembrane segment 2 that this region is located at the cytoplasmic and that it is involved in ion translocation and regulation by cytoplasmic The transmembrane segments of the Na+/Ca2+exchange protein have been based on hydropathy of the amino D.A. Longoni S. Philipson K.D. Science. 1990; 250: 562-565Crossref PubMed Scopus (624) Google Scholar). the extracellular of the amino terminus (4Hryshko L.V. Nicoll D.A. Weiss J.N. Philipson K.D. Biochim. Biophys. Acta. 1993; 1151: 35-42Crossref PubMed Scopus (62) Google Scholar) and loop e, between transmembrane segments 4 and 5 (5Sahin-Toth M. Nicoll D.A. Frank J.S. Philipson K.D. Friedlander M. Biochem. Biomed. Res. Commun. 1995; 212: 968-974Crossref PubMed Scopus (25) Google Scholar), and the intracellular of loop between transmembrane segments 5 and 6 (6Matsuoka S. Nicoll D.A. Reilly R.F. Hilgemann D.W. Philipson K.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3870-3874Crossref PubMed Scopus (199) Google Scholar, 7Porzig H. Li Z.P. Nicoll D.A. Philipson K.D. Am. J. Physiol. 1993; 265: C748-C756Crossref PubMed Google Scholar). In this mutants and located in loop b, were to intracellular of reagents MTSET and MTSES, these residues to the intracellular surface. These understanding of the of the exchanger by loop b, between transmembrane segments 2 and to the of the These were dependent the that of the sulfhydryl modifying reagents we used inhibited the wild-type exchanger. appears that the endogenous are to modifying reagents or that the accessible are not involved in exchanger is that the mutations in loop b a that a cysteine to cytoplasmic MTS We are in the process of a mutant to investigate this are two in the effect of the membrane-permeable reagent NEM on the loop b the effect of NEM was reduced as the mutations were into transmembrane segment 2 (Fig. 3). This may be an that the mutated residues with reduced NEM are in the transmembrane segment as modeled. mutants and T103C are to by NEM but not by the MTS Thus, these two residues are but are to residues or may be or with the MTS Secondary regulation by cytoplasmic Na+ and Ca2+ has previously been localized to sites in loop but no other regions of the exchanger were implicated in the regulatory Mutations in the endogenous XIP region (Fig. 1) can Na+-dependent inactivation but not Ca2+ (9Matsuoka S. Nicoll D.A. He Z. Philipson K.D. J. Gen. Physiol. 1997; 109: 273-286Crossref PubMed Scopus (142) Google Scholar), whereas of amino acids has the opposite effect (6Matsuoka S. Nicoll D.A. Reilly R.F. Hilgemann D.W. Philipson K.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3870-3874Crossref PubMed Scopus (199) Google Scholar), but not Na+-dependent A important binding site for regulatory been at amino acids D.O. Nicoll D.A. Philipson K.D. J. Biol. Chem. 1994; 269: 22847-22852Abstract Full Text PDF PubMed Google Scholar). mutations in loop f alter Na+ and that the two (9Matsuoka S. Nicoll D.A. He Z. Philipson K.D. J. Gen. Physiol. 1997; 109: 273-286Crossref PubMed Scopus (142) Google Scholar). Loop f regulatory effects on transport by the transmembrane segments that ion We show that a mutation in loop b with secondary regulation by Na+ and Ca2+. are the residue at 101 may with loop f. residue to cysteine may then alter between b and f and regulation. the mutation may alter the protein that loop f is no to and the transmembrane We are to these in the interface of loop b and transmembrane segment 2 is as a site in the of regulation to We the that the cysteine in regulatory mechanisms by an with a cysteine. We that the not regulation to the mutant as be under these not Thus, it not that is the of of regulation in proteolysis by cytoplasmic which secondary regulatory effects of intracellular Na+ and Ca2+ in the wild-type also the inhibitory effect of the sulfhydryl reagent NEM on may secondary regulation in the wild-type exchanger by the between regulatory sites in loop f and the translocation is that the site for proteolysis of the exchanger is within loop f 1995; PubMed Scopus Google Scholar). The that proteolysis the NEM effect on mutant suggests a between b and f. the be either a direct or an The cytoplasmic of transmembrane segment 2 appears to a in the translocation of Na+ T103V shows an apparent affinity for cytoplasmic Na+ and also to exchange. T103C has but mutant not show current not The current of mutant T103V is 100 mm the exchange current is of exchange current. mutant T103V has a affinity for the wild-type exchanger, the that Na+ in the cytoplasmic Li+ solution activate significant current in the mutant but not the wild-type exchanger. This is we used and mm Na+ have to be in the 100 mm Li+ solution to produce a The 45Ca2+ show that mutant T103V transports Ca2+ across the membrane a Na+ (Fig. and the outward current that an process this Ca2+ transport This suggests that mutant T103V can directly exchange. Other as the exchanger A. and of and Scholar), the J. Physiol. Sci. 1992; Scholar), and the Na+/Ca2+ exchanger Am. J. Physiol. 1990; PubMed Google Scholar), can Li+ for Na+ to However, this is the first of a membrane Na+/Ca2+ exchanger of The relative level of exchange may also be for mutant T103V for the wild-type exchanger. uptake was measured in the of a Li+ (Fig. 45Ca2+ is to a of exchange and exchange for endogenous the other (Fig. are to exchange exchange is not For mutant exchanger the 45Ca2+ into oocytes was of that into oocytes, but the current was of the current. Thus, exchange activity of T103V to be that of the wild-type exchanger. In experiments oocytes were into45Ca2+ uptake medium containing or Li+ Li+ 45Ca2+ uptake for the wild-type exchanger were and of that in the of a Na+ For mutant these were and These are with the that the level of is in mutant T103V in the wild-type exchanger. The in Fig. the relative transport activity of Na+/Ca2+ exchange and exchange for were well with a a Na+/Ca2+exchange to the wild-type exchanger, we added Li+ binding and exchange. In this the is Li+ with a of 100 mm and the Li+ translocation rate is of the Na+ translocation is that residue is part of a Na+ binding site and that the mutation the affinity of the site for Na+ and also for is to the of the residue to 1 (Fig. which at The are highly conserved of the Na+/Ca2+ exchanger and an important in function (12Nicoll 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 (126) Google Scholar). The of the Na+/Ca2+ exchange protein is by the presented Loop b has been localized to the cytoplasmic side of the membrane and a potential in secondary regulation of the exchanger by cytoplasmic The cytoplasmic of transmembrane segment 2 may have an important in the transport of Na+. The loop segment 2 interface be an important link between ion translocation and regulation.