Helix Packing of the Cardiac Na+-Ca2+ Exchanger

The cardiac Na+-Ca2+ exchanger (NCX1) is a membrane protein that extrudes Ca2+ from cells using the energy of the Na+ gradient and is a key protein in regulating intracellular Ca2+ and contractility. Based on the current topological model, NCX1 consists of nine transmembrane segments (TMSs). The N-terminal five TMSs are separated from the C-terminal four TMSs by a large intracellular loop. Cysteine 768 is modeled to be in TMS 6 close to the intracellular surface. In this study, the proximity of TMS 6 to TMSs 1 and 2 was examined. Insect High Five cells were transfected with cDNAs encoding mutant NCX1 proteins. Each mutant contained cysteine 768 and an introduced cysteine in TMS 1 or 2. Cross-linking between cysteines was determined after reaction with thiol-specific cross-linkers containing spacer arms of 6.5-12 Å. The data indicate that residues in TMSs 1 and 2 are close to cysteine 768 in TMS 6. Cysteine 768 cross-linked with residues at both ends of TMSs 1 and 2 and is likely located toward the middle of TMS 6. Based on these results, we present an expanded helix-packing model for NCX1. The cardiac Na+-Ca2+ exchanger (NCX1) is a membrane protein that extrudes Ca2+ from cells using the energy of the Na+ gradient and is a key protein in regulating intracellular Ca2+ and contractility. Based on the current topological model, NCX1 consists of nine transmembrane segments (TMSs). The N-terminal five TMSs are separated from the C-terminal four TMSs by a large intracellular loop. Cysteine 768 is modeled to be in TMS 6 close to the intracellular surface. In this study, the proximity of TMS 6 to TMSs 1 and 2 was examined. Insect High Five cells were transfected with cDNAs encoding mutant NCX1 proteins. Each mutant contained cysteine 768 and an introduced cysteine in TMS 1 or 2. Cross-linking between cysteines was determined after reaction with thiol-specific cross-linkers containing spacer arms of 6.5-12 Å. The data indicate that residues in TMSs 1 and 2 are close to cysteine 768 in TMS 6. Cysteine 768 cross-linked with residues at both ends of TMSs 1 and 2 and is likely located toward the middle of TMS 6. Based on these results, we present an expanded helix-packing model for NCX1. Na+-Ca2+ exchangers (NCX) 2The abbreviations used are: NCX, Na+-Ca2+ exchanger; TMS, transmembrane segment;MTS,methanethiosulfonate;MTSET,[2-(trimethylammonium)ethyl]-methanethiosulfonatebromide; NEM, N-ethylmaleimide; Ch-MβCD, cholesterol-cyclodextrin complex; MOPS, 4-morpholinepropanesulfonic acid; CuPhe, CuSO4/phenanthroline; o-PDM, N′, N′-o-phenylenedimaleimide; p-PDM, N′, N′-p-phenylenedimaleimide; M3M, 1,3-propanediyl bismethanethiosulfonate; M6M, 1,6-hexanediyl bismethanethiosulfonate; WT, wild-type. are found in many tissues. The cardiac Na+-Ca2+ exchanger (NCX1) plays a key role in myocardial contraction and relaxation. NCX exchanges three Na+ ions for one Ca2+ and can move Ca2+ either into or out of cells, depending on the electrochemical driving force. During the depolarization phase of the action potential, Ca2+ enters the cardiomyocyte primarily through L-type Ca2+ channels to trigger sarcoplasmic reticular Ca2+ release and to initiate contraction. During the relaxation phase, Ca2+ is transported back into the sarcoplasmic reticulum by an ATP-dependent Ca2+ pump and is extruded from the cell by NCX1 (1Bers D.M. Excitation-Contraction Coupling and Cardiac Contractile Force. Second Edition. Kluwer Academic Publishers, Norwell, MA2001: 133-160Google Scholar). The current topological model of the NCX protein contains nine transmembrane segment (TMS) helices and two reentrant loops (Fig. 1). There is a large cytoplasmic loop between TMSs 5 and 6, and the N and C termini are located on the extracellular and intracellular sides, respectively. The native Na+-Ca2+ exchanger protein has a molecular mass of 110 kDa based on amino acid sequence (2Nicoll D.A. Longoni S. Philipson K.D. Science. 1990; 250: 562-565Crossref PubMed Scopus (628) Google Scholar). Electrophoretic analysis of purified NCX1 displays three protein bands with molecular masses of 160, 120, and 70 kDa. The 70-kDa band is likely a proteolytic fragment (3Philipson K.D. Longoni S. Ward R. Biochim. Biophys. Acta. 1988; 945: 298-306Crossref PubMed Scopus (159) Google Scholar, 4Durkin J.T. Ahrens D.C. Pan Y.C. Reeves J.P. Arch Biochem. Biophys. 1991; 290: 369-375Crossref PubMed Scopus (64) Google Scholar). Under reducing conditions, the 160-kDa band is weak, and the 120-kDa band is more intense. Under nonreducing conditions, the 120-kDa band disappears, and the 160-kDa band becomes prominent (3Philipson K.D. Longoni S. Ward R. Biochim. Biophys. Acta. 1988; 945: 298-306Crossref PubMed Scopus (159) Google Scholar). This mobility shift is due to an intramolecular disulfide bond between cysteine 792 and either cysteine 14 or cysteine 20 as shown by mutagenesis and biochemical analysis (5Santacruz-Toloza L. Ottolia M. Nicoll D.A. Philipson K.D. J. Biol. Chem. 2000; 275: 182-188Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Thus, cross-linking between the N- and C-terminal halves of the exchanger results in decreased mobility on SDS-PAGE (6Qiu Z. Nicoll D.A. Philipson K.D. J. Biol. Chem. 2001; 276: 194-199Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). To understand the molecular mechanism of the Na+-Ca2+ exchanger, it is necessary to learn about the three-dimensional arrangement of the TMSs within the plasma membrane. However, hydrophobic membrane proteins are difficult to crystallize, and a high resolution structure of NCX1 is not available. Qiu et al. (6Qiu Z. Nicoll D.A. Philipson K.D. J. Biol. Chem. 2001; 276: 194-199Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) developed an alternative method for obtaining structural information about NCX1 by taking advantage of the shift in electrophoretic mobility when there is a cross-link between the two halves of the protein. A helix-packing model of TMSs 2, 3, 7, and 8 of NCX1 was formulated by introducing pairs of cysteine mutants into a cysteine-less background and testing for disulfide cross-linker-induced mobility shifts. Here, we extend the packing model of NCX1 to include TMSs 1 and 6. Cysteine mutations were introduced into the N or C terminus of TMSs 1 or 2 and then paired with cysteine 768 in TMS 6. cDNAs of NCX1 mutants were expressed in insect High Five cells. Intramolecular cross-linking was detected by mobility shifts on SDS-PAGE gels following treatment of intact cells with cross-linking agents. The results indicate that TMSs 1 and 2 are in proximity with TMS 6. Strikingly, cysteine 768 in TMS 6 could form cross-links with residues modeled to be near both the intracellular and the extracellular surfaces of TMS 1 and TMS 2. Cysteine 768 is likely near the center of TMS 6. Construction of Exchanger Cysteine Mutants—The QuikChange site-directed mutagenesis method (Stratagene) was used to prepare mutants (7Nicoll 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). Mutations were generated in 300-500-bp cassettes and verified by sequencing. Full-length exchangers with single or double mutations were constructed by subcloning the mutated cassettes into the cysteine-less exchanger. Expression of the NCX1 Cysteine Mutants in Insect High Five Cells—A lepidopteran insect cell expression system, BTI-TN-5B1-4 (High Five, Invitrogen), was used for transient transfection of NCX1 cysteine mutants. High Five cells were cultured at 27 °C in Express Five SFM (Invitrogen) supplemented with 20 mm glutamine and 1% penicillin-streptomycin. Mutant NCX1 cDNA was subcloned into the pIB/V5-His vector (Invitrogen) and transfected into High Five insect cells using lipid-mediated transfection with Cellfectin reagent (Invitrogen). 4 h after transfection, 0.1 mm cholesterol-cyclodextrin complex (Ch-MβCD) (8Gimpl G. Klein U. Reilander H. Fahrenholz F. Biochemistry. 1995; 34: 13794-13801Crossref PubMed Scopus (145) Google Scholar) was added to the transfected cells to increase exchanger protein expression. 24-48 h after transfection, Na+ gradient-dependent 45Ca2+ uptake in transfected High Five insect cells was measured. Cells were harvested and washed twice with washing buffer (10 mm MOPS, pH 7.4, 140 mm NaCl) and then loaded with Na+ by incubation with 10 mm MOPS (pH 7.4), 140 mm NaCl, 1 mm MgCl2, 0.4 mm ouabain, and 25 μm nystatin for 10 min at room temperature. Nystatin was removed from the cells by two washes with washing buffer plus 0.4 mm ouabain. Uptake was initiated by resuspending the cell pellet in assay medium: 10 mm MOPS (pH 7.4), 140 mm KCl (or NaCl as control), 25 μm CaCl2, 0.4 mm ouabain, and 5 μCi/ml 45Ca2+. After 10 min, the reaction was stopped by adding 1 ml of ice-cold quenching solution (140 mm KCl, 1 mm EGTA) followed by two additional washes with quenching solution. Cell pellets were dissolved in 1 n NaOH at 60 °C for 30 min. Aliquots of samples were subjected to scintillation counting and protein assay (MicroBCA, Pierce). Cross-linking Procedures—Intact cells were washed with washing buffer, and cross-linking was carried out at room temperature by the addition of oxidative reagent (CuSO4/phenanthroline (CuPhe)), thiol-specific homobifunctional cross-linker, or methanethiosulfonate (MTS) cross-linkers (Toronto Research Chemicals) to the intact cell suspension. The final concentrations of reagents were 1 mm CuSO4, 3 mm phenanthroline, or 0.5 mm N′, N′-o-phenylenedimaleimide (o-PDM) or 0.5 mm p-PDM, 0.5 mm 1,3-propanediyl bismethanethiosulfonate (M3M), or 0.5 mm 1,6-hexanediyl bismethanethiosulfonate (M6M). Some samples were preincubated with 10 mm [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET) or 10 mm N-ethylmaleimide (NEM). Reactions were terminated after 20 min by the addition of NEM (10 mm). Cells were lysed with 1% Triton X-100 plus protease inhibitors (Complete, EDTA-free, Roche Applied Science). Aliquots were subjected to 7.5% SDS-PAGE in the absence of reducing reagents, and immunoblot analysis was carried out with NCX1 antibody R3F1 (SWant). Cysteine Residues Introduced into the Cysteine-less Na+-Ca2+ Exchanger—Fig. 1 shows residues mutated to cysteine in this study. The residues labeled with asterisks, 14, 20, 122, 768, and 792, are cysteines in the wild-type canine NCX1 but were mutated to alanine to produce the cysteine-less exchanger (7Nicoll 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). The cysteine-less exchanger was used as our background protein for introduction of cysteines. Thus, each mutant contains only one or two cysteines as indicated. In the single-cysteine mutant A122C, for example, the alanine at position 122 in the cysteine-less exchanger has been mutated back to a cysteine. In addition to the five reintroduced cysteines, we also used cysteine mutants at positions 101 and 102, as described previously (9Doering 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 and cysteine at positions and cysteines or pairs of cysteines were introduced into the cysteine-less NCX1 and the proximity of the paired cysteine residues was then by mobility shifts following treatment with disulfide or cross-linking reagents (6Qiu Z. Nicoll D.A. Philipson K.D. J. Biol. Chem. 2001; 276: 194-199Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Expression and Na+ Uptake of Cysteine Mutants in High Five cells by a expression system, High Five cells as cell and been used to the Na+-Ca2+ exchangers J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, M. L. Biochemistry. PubMed Scopus Google Scholar). found that transient transfection of High Five cells with the wild-type Na+-Ca2+ exchanger of NCX1 and protein not protein expression were determined by 45Ca2+ uptake into cells and immunoblot analysis and with expression of NCX1. The mutant proteins expressed a of from to about of NCX1 1). of mutants and detected by a with of NCX1 cysteine mutants The 45Ca2+ uptake into High Five cells each of the mutants was determined and to in a has been that the of in the insect cell membrane is in cell (8Gimpl G. Klein U. Reilander H. Fahrenholz F. Biochemistry. 1995; 34: 13794-13801Crossref PubMed Scopus (145) Google and for membrane expression is by membrane with (8Gimpl G. Klein U. Reilander H. Fahrenholz F. Biochemistry. 1995; 34: 13794-13801Crossref PubMed Scopus (145) Google Scholar). NCX1 is by R. Philipson K.D. Biochim. Biophys. Acta. 1988; PubMed Scopus Google Scholar, R. Philipson K.D. J. Biol. Chem. Full Text PDF PubMed Google we that the of NCX1 mutants be by membrane the on exchanger of adding to the after The of mutants was by the addition of 1). Thus, cross-linking were carried out using transfected cells with Ch-MβCD, and only mutants were examined. Cross-linking of TMSs 1 and 6 of the Na+-Ca2+ To the proximity of cysteine many cross-linkers are and is for of membrane proteins. is an oxidative can disulfide between and are cross-linkers with of and and been used with NCX1 in a (6Qiu Z. Nicoll D.A. Philipson K.D. J. Biol. Chem. 2001; 276: 194-199Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). cross-linkers with cysteines, in a disulfide of the spacer used the two of and cross-linkers in this study. The reagents we and cross-link as and the cross-linkers for this are and the cross-linking were carried out using intact cells. The NCX1 expressed in High Five insect cells as a 120-kDa band in SDS-PAGE reducing not Under nonreducing conditions, an additional band at 140 kDa is NCX1 expressed in cell or the additional band is at kDa. This be due to S. Y.C. J. PubMed Scopus Google Scholar). The (or band results from an intramolecular disulfide bond between cysteine 792 and either cysteine 14 or cysteine 20 (5Santacruz-Toloza L. Ottolia M. Nicoll D.A. Philipson K.D. J. Biol. Chem. 2000; 275: 182-188Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) and reducing in cells, the exchanger protein is not in molecular on SDS-PAGE when expressed in High Five cells, this a cell to for mobility shifts of the exchanger protein. The we to between TMSs was to a of single-cysteine mutants near the of a TMS example, in TMS we mutations at residues and we paired each single-cysteine mutant with a cysteine mutant in the of the exchanger and for shifts. more 20 mutants one that a There are we not cross-linking from of proximity between of the or an in in the cross-linked protein for on A of shift for double mutant information and mutants mutants were not The mutant we found for cross-linking was This that residues located in TMSs 1 and 6 were close in the protein to in cross-linking using o-PDM, p-PDM, M3M, or (Fig. The of that a band was μm M3M, the of NCX1 protein in the band was This of cross-linking was within 1 min of to were that residues and 768 cross-linked were modeled to be near of the membrane (Fig. 1). To the between TMSs 1 and 6, we paired with residues and modeled to be on the of TMS 1 as Residues and are modeled to be toward the center of TMS is modeled to be at the intracellular surface. shifts in SDS-PAGE nonreducing following of cross-linkers were for mutants (Fig. and (Fig. but not for mutant (Fig. that 768 can with residues the of TMS also found that mutants and of cross-linking (Fig. shifts could be by the cells with 10 mm NEM of cross-linkers (Fig. 2, a of TMS residues and are within a of each is that residues of the packing not we with and residues within one of However, of these mutants NCX we also paired with and NCX1 protein could be detected with mutant Mutant is but mobility shift of cross-linkers not However, mutant both is and cross-linker-induced shifts (Fig. we paired with and protein expressed in the mutants and Mutant and cross-linker-induced mobility shift in SDS-PAGE (Fig. data that TMSs 1 and 6 one of Cross-linking of TMSs 2 and 6 of the Na+-Ca2+ also for proximity of 768 to residues on either of TMS 2. In the wild-type there is a cysteine at position Cysteine mutagenesis indicate that this is located in TMS 2 or the reentrant loop close to the extracellular (7Nicoll 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, Pan Y. M. 1999; PubMed Scopus Google Scholar). reintroduced the two native cysteine residues 122 and 768 into the cysteine-less background and transfected the cDNA into High Five insect cells. The mutant as a 120-kDa band in SDS-PAGE nonreducing (Fig. 120-kDa and bands were of or the cross-linkers or The band was with (Fig. of a disulfide bond between cysteines. Thus, residues 122 and 768 are close to form a disulfide with either 10 mm NEM (Fig. or 10 mm not not cross-linking of residues 122 and Thus, residues 122 and 768 be in or protein and is not to Residues and were previously shown to be near the intracellular of TMS 2 the of mutants and were by intracellular of (9Doering 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). paired with either or on the cysteine-less background and the of cross-linking reagents or on SDS-PAGE mobility (Fig. 3, and both cross-linker-induced mobility could be by with NEM, were were to the of disulfide cross-linking on the of exchanger mutants expressed in High Five cells as the of the cysteine-less exchanger was in a by cross-linking This be to of by the cross-linking to the of 122, and data the topological model in 1 (7Nicoll 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, 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, Pan Y. M. 1999; PubMed Scopus Google Scholar). However, as the and for are Thus, we to the positions of cysteines at positions 122, and at positions and 122 were paired with native cysteine 792, has been shown to be at the extracellular of the membrane (5Santacruz-Toloza L. Ottolia M. Nicoll D.A. Philipson K.D. J. Biol. Chem. 2000; 275: 182-188Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Residues and 122, modeled to be at the extracellular surfaces of TMS 1 and 2, mobility shifts when paired with and with cross-linkers (Fig. A and the when modeled to be at the intracellular of TMS was paired with cross-linker-induced mobility shifts were (Fig. To the of it was with native cysteines and residues are both located at the extracellular of NCX1 (5Santacruz-Toloza L. Ottolia M. Nicoll D.A. Philipson K.D. J. Biol. Chem. 2000; 275: 182-188Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 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). shown in mobility shift was for this mutant following that is not to the extracellular surface. To more information on the of we the of reagents on the of the single-cysteine mutant expressed in used this previously to NCX1 (7Nicoll 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, 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). of NCX1 exchanger mutant was into and exchanger was following treatment with the reagents or is a and NEM is form with cysteines. or NEM were either by in containing 10 mm or by solution into the for an cytoplasmic of 10 mm (Fig. of on mutant the intracellular of or extracellular of NEM the of The results that 768 is more from the cytoplasmic extracellular of the membrane. The results also that 768 be in a was by intracellular of on the of NCX1 (9Doering 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 also contains a cysteine at position of native cysteines to or an disulfide bond with native cysteine. The likely is to the intracellular and is in a reducing of between cross-linking from intramolecular single-cysteine mutants A122C, and were transfected into High Five cells. mobility shift band was for single-cysteine mutant after the addition of cross-linking reagents not pairs of single-cysteine mutants were into High Five cells followed by cross-linking shown in 6, cross-linking was not when single-cysteine mutant or was with single mutant Thus, the cross-linking that was in the mutants was due to and not A helix-packing of NCX1 that TMSs 2, 3, 7, and reentrant loops are in proximity (6Qiu Z. Nicoll D.A. Philipson K.D. J. Biol. Chem. 2001; 276: 194-199Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The results of this current extend these to include TMS 1 and 6. data that 768 in TMS 6 is in proximity to residues in TMS 1 and 2. A of NCX1 that TMSs 1 and 6 with TMSs 2, 3, 7, and 8 is shown in This the results in the present and also the results from Qiu et al. (6Qiu Z. Nicoll D.A. Philipson K.D. J. Biol. Chem. 2001; 276: 194-199Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). this model, it was that each TMS the membrane by and that each of the residues is in a of the TMS and not in This model and for and model The only TMSs not for in our helix-packing model are TMSs and of the loops TMSs to be with the of the large loop TMSs 5 and 6. has also been that TMSs to be near one in of membrane proteins J. Biol. PubMed Scopus Google Scholar). it likely that TMSs 4 and 5 in the of TMS TMS is likely to be near TMS Qiu et al. (6Qiu Z. Nicoll D.A. Philipson K.D. J. Biol. Chem. 2001; 276: 194-199Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) cross-linking in the mutant also TMS into the by TMSs 2, and The that residues and are to residues modeled to be near both the extracellular and the intracellular of TMSs 1 and 2 2 and has previously been modeled to be in TMS 6 toward the intracellular of the membrane (Fig. K.D. Nicoll D.A. Ottolia M. H. S. Qiu Z. Y. PubMed Scopus Google Scholar). However, the position of can as be modeled to be more toward the center of TMS 6 reducing of the results indicate that this is the of to both of the membrane is TMS 1 and TMS 2 are both then the residues that with be separated by five or a of about 27 Å. a between the a single in the middle of TMS 6 a that is 10 to both residues at either of the membrane. However, cross-linking was with M3M, a There are a of for this example, TMS 1 and 2 not be There is a of of membrane proteins in TMSs are not helices J. G. S. Science. PubMed Scopus Google Scholar, M. H. PubMed Scopus Google Scholar). there are of TMSs 1 and 2, then residues modeled to be on of the membrane could be from the of a single TMS 2 a at about the between residues 101 and 122, that a in the there are and a in TMS these residues are not the residues that are modeled to be within a be in a loop could be and in toward 768 there is a structure in NCX to there be in the exchanger protein that TMS 6 can move depending on the of the protein. This to with residues at the intracellular or extracellular surface. A is the segment of in to membrane depolarization R. Full Text Full Text PDF PubMed Scopus Google Scholar, R. Biophys. J. Full Text PDF PubMed Scopus (76) Google Scholar, Biochemistry. PubMed Scopus Google Scholar). also is the between residues 122 and In the wild-type both of these residues are cysteines. 122 is near the between the extracellular of TMS 2 and the reentrant loop of the a of M. Nicoll D.A. Philipson K.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). There been of between and in the NCX1 (5Santacruz-Toloza L. Ottolia M. Nicoll D.A. Philipson K.D. J. Biol. Chem. 2000; 275: 182-188Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). However, a disulfide bond between residues and in the of (Fig. a between these two cysteine of the two residues is with a for TMS 6. of membrane proteins is a difficult The NCX1 protein has not been and large of purified protein are not available. our on packing of NCX1 on the mechanism of action of this and to the of additional

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