Cysteine mutagenesis of the Na+-Ca2+ exchanger supports a revised topological model placing transmembrane segment 6 in cytoplasmic loop f and suggesting segment 9 forms a P-loop-like structure.
The study proposes a revised topological model of the cardiac Na+-Ca2+ exchanger, which is critical for understanding its role in cardiac calcium homeostasis.
The current topological model of the Na+-Ca2+ exchanger consists of 11 transmembrane segments with extracellular loops a, c, e, g, i, and k and cytoplasmic loops b, d, f, h, and j. Cytoplasmic loop f, which plays a role in regulating the exchanger, is large and separates the first five from the last six transmembrane segments. We have tested this topological model by mutating residues near putative transmembrane segments to cysteine and then examining the effects of intracellular and extracellular applications of sulfhydryl-modifying reagents on exchanger activity. To aid in our topological studies, we also constructed a cysteineless Na+-Ca2+ exchanger. This mutant is fully functional in Na+gradient-dependent 45Ca2+ uptake measurements and displays wild-type regulatory properties. It is concluded that the 15 endogenous cysteine residues are not essential for either activity or regulation of the exchanger. Our data support the current model by placing loops c and e at the extracellular surface and loops d, j, and l at the intracellular surface. However, the data also support placing Ser-788 of loop h at the extracellular surface and Gly-837 of loop i at the intracellular surface. To account for these data, we propose a revision of the model that places transmembrane segment 6 in cytoplasmic loop f. Additionally, we propose that putative transmembrane segment 9 does not span the membrane, but may form a “P-loop”-like structure. The current topological model of the Na+-Ca2+ exchanger consists of 11 transmembrane segments with extracellular loops a, c, e, g, i, and k and cytoplasmic loops b, d, f, h, and j. Cytoplasmic loop f, which plays a role in regulating the exchanger, is large and separates the first five from the last six transmembrane segments. We have tested this topological model by mutating residues near putative transmembrane segments to cysteine and then examining the effects of intracellular and extracellular applications of sulfhydryl-modifying reagents on exchanger activity. To aid in our topological studies, we also constructed a cysteineless Na+-Ca2+ exchanger. This mutant is fully functional in Na+gradient-dependent 45Ca2+ uptake measurements and displays wild-type regulatory properties. It is concluded that the 15 endogenous cysteine residues are not essential for either activity or regulation of the exchanger. Our data support the current model by placing loops c and e at the extracellular surface and loops d, j, and l at the intracellular surface. However, the data also support placing Ser-788 of loop h at the extracellular surface and Gly-837 of loop i at the intracellular surface. To account for these data, we propose a revision of the model that places transmembrane segment 6 in cytoplasmic loop f. Additionally, we propose that putative transmembrane segment 9 does not span the membrane, but may form a “P-loop”-like structure. The Na+-Ca2+ exchanger NCX1 is a plasma membrane protein that exchanges three Na+ ions for one Ca2+ ion. The highest levels of exchange activity have been observed in cardiac myocytes where the NCX1.1 splice variant is expressed (1Kofuji P. Lederer W.J. Schulze D.H. Am. J. Physiol. 1992; 263: C1241-C1249Crossref PubMed Google Scholar, 2Komuro I. Wenninger K.E. Philipson K.D. Izumo S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4769-4773Crossref PubMed Scopus (128) Google Scholar, 3Nicoll D.A. Longoni S. Philipson K.D. Science. 1990; 250: 562-565Crossref PubMed Scopus (628) Google Scholar). NCX1.1 and other splice variants are present in a wide array of other cell types (1Kofuji P. Lederer W.J. Schulze D.H. Am. J. Physiol. 1992; 263: C1241-C1249Crossref PubMed Google Scholar, 2Komuro I. Wenninger K.E. Philipson K.D. Izumo S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4769-4773Crossref PubMed Scopus (128) Google Scholar, 4Goldman W.F. Yarowsky P.J. Juhaszova M. Kreuger B.K. Blaustein M.P. J. Neurosci. 1994; 14: 5834-5843Crossref PubMed Google Scholar, 5Lee S.-L. Yu A.S.L. Lytton J. J. Biol. Chem. 1994; 269: 14849-14852Abstract Full Text PDF PubMed Google Scholar, 6Nicoll D.A. Quednau B.D. Qui Z. Xia Y.-R. Lusis A.J. Philipson K.D. J. Biol. Chem. 1996; 271: 24914-24921Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 7Quednau B.D. Nicoll D.A. Philipson K.D. Am. J. Physiol. 1997; 272: C1250-C1261Crossref PubMed Google Scholar). In the myocyte, NCX1.1 is prominent in Ca2+ extrusion during cardiac relaxation (8Bers D.M. Excitation-Contraction Coupling and Cardiac Contractile Force. Kluwer Academic Publishers, Dordrecht, The Netherlands1991Google Scholar) and may also be involved in Ca2+ influx during contraction (9Kohomoto O. Levi A.J. Bridge J.H. Circ. Res. 1994; 74: 550-554Crossref PubMed Google Scholar, 10Levi A.J. Spitzer K.W. Kohmoto O. Bridge J.H. Am. J. Physiol. 1994; 266: H1422-H1433PubMed Google Scholar, 11Wasserstrom J.A. Vites A.M. J. Physiol. (Lond.). 1996; 493: 529-542Crossref Scopus (93) Google Scholar). NCX1.1 has been cloned, sequenced, and expressed inXenopus oocytes (3Nicoll D.A. Longoni S. Philipson K.D. Science. 1990; 250: 562-565Crossref PubMed Scopus (628) Google Scholar) and insect (12Li Z. Smith C.D. Smolley J.R. Bridge J.H.B. Frank J.S. Philipson K.D. J. Biol. Chem. 1992; 267: 7828-7833Abstract Full Text PDF PubMed Google Scholar) and mammalian (13Aceto J.F. Condrescu M. Kroupis C. Nelson H. Nelson N. Nicoll D. Philipson K.D. Reeves J.P. Arch. Biochem. Biophys. 1992; 298: 553-560Crossref PubMed Scopus (67) Google Scholar) cell lines. The mature NCX1 proteins, formed after cleavage of a signal peptide, have been modeled to have 11 α-helical transmembrane segments. An amino-terminal hydrophobic domain (TMS1–5) 1The abbreviations used are: TMS, transmembrane segment; MTS, methanethiosulfonate; MTSET, 2-(trimethylammonium)ethyl methanethiosulfonate bromide; MTSES, sodium (2-sulfonatoethyl) methanethiosulfonate; MTSEA, 2-(aminoethyl) methanethiosulfonate hydrobromide. is separated from a carboxyl-terminal domain (TMS6–11) by a long cytoplasmic loop, loop f (Fig. 1 A). Segment a, at the amino terminus, has been shown to be extracellular (14Cook O. Low W. Rahamimoff H. Biochim. Biophys. Acta. 1998; 1371: 40-52Crossref PubMed Scopus (34) Google Scholar, 15Hryshko L.V. Nicoll D.A. Weiss J.N. Philipson K.D. Biochim. Biophys. Acta. 1993; 1151: 35-42Crossref PubMed Scopus (62) Google Scholar), and loops b (14Cook O. Low W. Rahamimoff H. Biochim. Biophys. Acta. 1998; 1371: 40-52Crossref PubMed Scopus (34) Google Scholar, 16Doering 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) and f (14Cook O. Low W. Rahamimoff H. Biochim. Biophys. Acta. 1998; 1371: 40-52Crossref PubMed Scopus (34) Google Scholar, 17Li 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, 18Matsuoka 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 (200) Google Scholar, 19Porzig H. Li Z. Nicoll D.A. Philipson K.D Am. J. Physiol. 1993; 265: C748-C756Crossref PubMed Google Scholar) have been shown to be intracellular, but the rest of the topology of NCX1 has not yet been experimentally determined. Portions of loops b (16Doering 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) and f (18Matsuoka 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 (200) Google Scholar) are involved in regulatory properties of the exchanger, whereas the two hydrophobic domains are important in ion binding and transport (20Nicoll 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). The two hydrophobic domains share a repeated motif that has been designated α (21Schwarz E.M. Benzer S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10249-10254Crossref PubMed Scopus (181) Google Scholar). The α-1 repeat encompasses portions of TMS2 and TMS3 in the amino-terminal domain, and the α-2 repeat encompasses portions of TMS8 and TMS9 in the carboxyl-terminal domain (Fig. 1 A). The α-1 and α-2 repeats have been proposed to have similar roles in the exchange mechanism since they have similar sequences and are both modeled to be near the extracellular surface, and parallel mutations have similar effects on exchange activity (20Nicoll 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). The importance of cysteine residues in the exchanger has been examined previously. The activity of wild-type NCX1 expressed inXenopus oocytes is not inhibited by application of several sulfhydryl-modifying reagents (16Doering 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). Thus, accessible cysteines of NCX1 in the oocyte membrane are not critical for ion translocation. However, Pierce et al. (22Pierce G.N. Ward R. Philipson K.D. J. Membr. Biol. 1986; 94: 217-225Crossref PubMed Scopus Google Scholar) that the exchanger, in membrane to are present in the exchanger by and in mutant Nicoll D.A. Philipson K.D. Biophys. J. 1998; Scholar) and wild-type K.D. Longoni S. Ward R. Biochim. Biophys. Acta. PubMed Scopus Google Scholar) exchanger al. J.P. J. Biol. Chem. 1986; Full Text PDF PubMed Google Scholar) that of the exchanger activity and that the to a of We have examined the topology of with cysteines constructed and examined for to We have also examined the role of cysteine residues in the exchanger by of the cysteines and the cysteineless exchanger. The NCX1.1 (20Nicoll 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) used for in the in or the mutations by In other the constructed by the the wild-type exchanger. The cysteineless exchanger constructed by a of and The by In cysteines the cysteineless The of these are by the are in the wild-type and activity Na+gradient-dependent uptake of oocytes (16Doering 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 the effects of reagents on exchange the oocytes for with the in the or with reagents at a of (16Doering 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). data are Na+-Ca2+ exchange activity also examined exchange current the D.W. 1990; PubMed Scopus Google Scholar). to a of and with a of and To a membrane the oocyte membrane first in to the for and membrane a of the exchange current the and and or and or or 1 with by in the of to the 1992; Google Scholar). used for data and on at and at at and a of The for examining the topology of the exchanger of cysteine residues at modeled to be near the membrane surface. residues be accessible to and of the residues have functional expressed in and the effects of sulfhydryl-modifying reagents on activity The reagents and and in either in or with the to the cysteine with extracellular or intracellular This used to loop b of the exchanger to the intracellular surface (16Doering 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). the wild-type exchanger 15 this the wild-type exchanger, expressed in is not inhibited by application of of the reagents used in this (16Doering 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). This is in for MTSET, MTSES, and However, (16Doering 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), the wild-type exchanger expressed in oocytes is by intracellular application of (Fig. A). The of is a of the of exchange activity expressed in the oocyte the activity is is to the of activity. levels of is or is at by with (Fig. and of a functional exchanger since oocytes with mutant not exchange activity of not is not to intracellular and also the exchanger. by these reagents has been observed with levels of activity mutant is by intracellular The of by to the for the of of the wild-type exchanger by (Fig. the wild-type exchanger is not inhibited by sulfhydryl-modifying reagents expressed in is that with cysteine residues have that in of Thus, to aid in topological and also to the importance of cysteine a cysteineless exchanger cysteine and which to to cysteine to exchange activity. In for the in a functional exchanger (Fig. mutant the exchanger a at this of the cysteine and expressed of wild-type activity. two residues have been shown to form a Nicoll D.A. Philipson K.D. Biophys. J. 1998; Scholar). other and expressed of wild-type activity. levels of activity may the levels of at the cell surface or Thus, the cysteineless exchanger a at and at other cysteine This is designated and the of in the cysteineless are by The cysteineless exchanger near wild-type levels of exchange activity (Fig. the wild-type exchanger, by intracellular application The of of the cysteineless exchanger by intracellular also to the of exchanger activity not To the cysteineless and wild-type properties the D.W. 1990; PubMed Scopus Google Scholar). The of the cysteineless Na+-Ca2+ exchange current similar to of the wild-type current the of cytoplasmic Ca2+ present in the current the Na+ with The exchange current and then with The is D.W. Matsuoka S. Collins A. J. Physiol. 1992; PubMed Scopus Google Scholar). that the wild-type and cysteineless regulation by cytoplasmic Ca2+ D.W. Collins A. Matsuoka S. J. Physiol. 1992; PubMed Scopus Google Scholar). both the the cytoplasmic to 15 the to the regulatory Ca2+ from the and by the Ca2+ the regulatory Ca2+ from 1 to 15 The of in the for the cysteineless exchanger with both the wild-type and cysteineless effects of regulatory Ca2+ on the exchange activity and are of NCX1 D.W. Collins A. Matsuoka S. J. Physiol. 1992; PubMed Scopus Google Scholar). have been in W. Thus, the of the exchanger in the cysteineless that at five in the topology of the amino-terminal of the exchanger. cysteines at and and is modeled to be in extracellular loop c TMS2 and and is modeled to be near the of loop c and is modeled to be at the intracellular of loop and and and are modeled to be near the extracellular of and loop e (Fig. 1 A). activity present in with cysteine in loop c, d, or e, mutations in loop c activity. by Na+gradient-dependent 45Ca2+ the activity of mutant to of the expressed by the wild-type or cysteineless exchanger, and the activity of mutant (Fig. of in loop c data not also in of exchanger activity. and expressed and mutant expressed activity the wild-type exchanger. of also in loop e, in a exchanger not In of in loop e in wild-type levels of activity. To the topological of the cysteine the effects of reagents on the activity of and and inhibited by extracellular applications of the of the activity of and by intracellular application of cysteine to the during to MTSET, the not inhibited by extracellular In mutant inhibited by intracellular not application of or loop c to be extracellular and loop intracellular, and by intracellular reagents in to inhibited by extracellular we that loop e is modeled is modeled to be in near intracellular loop j, and is modeled to be in loop (Fig. cysteines at these near wild-type levels of exchange activity (Fig. inhibited by intracellular not application of (Fig. to intracellular not data support the model of the exchanger that places loop at the intracellular surface. In support of intracellular of the terminus, of the carboxyl-terminal amino to cysteine in exchanger that inhibited to activity by intracellular application of Ser-788 is modeled to be in loop h near the intracellular of However, a cysteine at in either the cysteineless or wild-type the mutant exchanger inhibited by application of (Fig. the of for the in the wild-type in the cysteineless This that this is not intracellular, be of transmembrane two loop f is to be and to a hydrophobic is to be a transmembrane segment is by It has been observed that amino near the of transmembrane in protein P. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). constructed with in hydrophobic loop f and after in a hydrophobic of loop f. and the of putative and and expressed Na+-Ca2+ exchange activity whereas activity observed in oocytes with for mutant data not This that but not putative is a transmembrane Gly-837 is modeled to be at the of extracellular loop i and TMS9 of the α-2 repeat (Fig. To the topology of loop i, mutant constructed and by This mutant a of to the wild-type exchanger (Fig. inhibited by intracellular application of either or (Fig. Thus, that Gly-837 is at the intracellular the extracellular surface, To that at the cysteine at and not at a wild-type cysteine that may have accessible the also the cysteineless expressed exchange activity of wild-type but inhibited by intracellular not application of In this amino residues to in with and 9 of the not exchange activity. residues to cysteine the cysteineless and with not mutations for the topology of the exchanger. The of a to to cysteine may with the exchanger In the where the data support the of In the of This on the of the We that MTSET, MTSES, and MTSEA, which are present at are However, has been that the is M. Y. Y. 1996; PubMed Scopus Google Scholar). Our that the oocyte membrane, does at a and to the the oocytes in a (Fig. the oocyte membrane to MTSEA, then also exchange activity. extracellular loops e, and and intracellular loops i, j, and in this of the with a in intracellular loop to intracellular applications of but extracellular applications of the with a in extracellular loop to extracellular applications of in extracellular loop c also inhibited by intracellular application of reagents (Fig. The by intracellular reagents to be to of the during since extracellular cysteine during the not e and by intracellular application of The to be similar to the of the wild-type exchanger since the of with the of expressed activity (Fig. However, we other for this of the residues from both of the of oocytes the wild-type exchanger exchanger activity by (Fig. and of the other examined also by and other and by intracellular and MTSET, (Fig. similar with the methanethiosulfonate on exchanger activity in (22Pierce G.N. Ward R. Philipson K.D. J. Membr. Biol. 1986; 94: 217-225Crossref PubMed Scopus Google Scholar). The of by intracellular reagents with the of exchange activity expressed in the oocytes (Fig. exchange the at levels of activity. The of activity to of a since be at by with (Fig. However, the does not of exchanger since is also observed with the cysteineless exchanger (Fig. is exchanger that is present at and that a to with reagents and to of the exchanger. Thus, of the exchanger is be inhibited by with the and of exchanger activity. levels of exchanger the of the exchanger that of the of of a of the and not activity. The cysteineless exchanger, 15 has similar to the wild-type exchanger. levels of observed for both the wild-type and cysteineless (Fig. However, the of current from oocytes the cysteineless mutant that of the wild-type current not It is to the activity with the two and the for this is The that the cysteineless exchanger the properties of the wild-type exchanger (Fig. The cysteineless exchanger current is by Na+ and and Thus, to be for of the endogenous cysteines in ion transport or may In where cysteines have been wild-type and cysteineless are mutant levels of exchange activity mutant mutant to be to extracellular mutant (Fig. the with cysteines by to a whereas mutant and mutant activity. Reeves et al. J.P. J. Biol. Chem. 1986; Full Text PDF PubMed Google Scholar) that the exchanger is by the of both a and proposed a mechanism for that the of has been shown that a with either or Nicoll D.A. Philipson K.D. Biophys. J. 1998; Scholar). residues and may be involved in other residues may also have a The where cysteine not be by at where exchange activity a levels of activity the cysteine to the This cysteine is in the of of one and residues in a of and that the of the cysteine be at this The model for the Na+-Ca2+ exchanger (Fig. constructed on the that of hydrophobic amino in the form transmembrane since the designated α-1 and α-2 share that they share similar and transmembrane this model in we cysteine residues modeled to be near the membrane surface and examined the effects of sulfhydryl-modifying reagents on exchange activity. The support the of loops in the and loops and l in the on the from several we propose the exchanger model in 1 The prominent of the model is that the is whereas the The terminus, segment a, is The exchanger for a signal that is L.V. Nicoll D.A. Weiss J.N. Philipson K.D. Biochim. Biophys. Acta. 1993; 1151: 35-42Crossref PubMed Scopus (62) Google J.T. Reeves J.P. Arch. Biochem. Biophys. 1991; PubMed Scopus Google Scholar), the is L.V. Nicoll D.A. Weiss J.N. Philipson K.D. Biochim. Biophys. Acta. 1993; 1151: 35-42Crossref PubMed Scopus (62) Google Scholar), extracellular of a after of the exchanger be by extracellular application of (14Cook O. Low W. Rahamimoff H. Biochim. Biophys. Acta. 1998; 1371: 40-52Crossref PubMed Scopus (34) Google Scholar). are three of that support the intracellular of loop and are both to intracellular application of intracellular reagents (16Doering 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). mutant displays regulatory properties (16Doering 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). The exchanger regulatory properties are at the intracellular surface, that is also a loop b of to that the is intracellular (14Cook O. Low W. Rahamimoff H. Biochim. Biophys. Acta. 1998; 1371: 40-52Crossref PubMed Scopus (34) Google Scholar). In this we have the of loops c is extracellular since and are inhibited by extracellular application of reagents (Fig. and loop is intracellular since mutant is inhibited by intracellular application of e is extracellular since and are inhibited by extracellular This for loop e is not since and also by intracellular We that the is to the also with the wild-type and cysteineless (Fig. extracellular for loop e has also been by the of et M. Nicoll D.A. Frank J.S. Philipson K.D. M. Biochem. Biophys. Res. PubMed Scopus Google Scholar), in which observed at a on exchanger in loop are a of data that support intracellular of loop f. The to a of loop f, the exchanger at the intracellular surface 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). and of loop f intracellular regulatory properties (18Matsuoka 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 (200) Google Scholar). on loop f have been to the intracellular surface (14Cook O. Low W. Rahamimoff H. Biochim. Biophys. Acta. 1998; 1371: 40-52Crossref PubMed Scopus (34) Google H. Li Z. Nicoll D.A. Philipson K.D Am. J. Physiol. 1993; 265: C748-C756Crossref PubMed Google Scholar). the model for the of the exchanger, of five transmembrane is by data to the of the transmembrane segments or have the and of transmembrane segments been determined. In the of the exchanger, the with two in loop h and in loop i, not from the modeled to be at the intracellular surface, but is to extracellular MTSET, and modeled to be but is to intracellular and We have also that in loop h of the wild-type exchanger is involved in with a cysteine in extracellular segment a Nicoll D.A. Philipson K.D. Biophys. J. 1998; Scholar), extracellular of loop on these TMS8 be in the to that in the model (Fig. This the for to the the exchanger protein the membrane of loop f and α-helical transmembrane then either or does not the membrane, or is a transmembrane segment loop f and We have the exchanger by but in the membrane (Fig. 1 are not to be transmembrane segments in loop f residues and since exchanger with these residues is functional (18Matsuoka 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 (200) Google Scholar). residues and are three hydrophobic two of which are putative and and the of which is a the of putative or after does not exchange but putative exchange activity. This that does the has not yet been used to transmembrane and our of and on this is not Additionally, three residues and that be important in a transmembrane and yet of of these residues does not exchange activity (20Nicoll 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). The that be to the model is to account for residues and both on the intracellular of the exchanger. Thus, the protein either not the membrane or does of two amino residues and this of the exchanger two α-helical transmembrane segments. However, TMS9 may in that to the We have modeled TMS9 in this (Fig. 1 since the the α-2 This is similar to the in the of D.A. J.M. A. J.M. R. Science. 1998; PubMed Scopus Google Scholar). The with and support placing loops and l on the intracellular et al. (14Cook O. Low W. Rahamimoff H. Biochim. Biophys. Acta. 1998; 1371: 40-52Crossref PubMed Scopus (34) Google Scholar) have also that the is k is the extracellular loop in the model for which topological data are The amino of loop k that is not of the 15 residues in loop k are and of have a on the cytoplasmic and or on the extracellular D. J. 1990; Full Text PDF PubMed Scopus Google Scholar). Thus, the of and is not yet of the model of the exchanger is that the of the membrane in the α-1 repeat is at a to Gly-837 in the α-2 and yet mutant is accessible to extracellular and to intracellular The exchanger to be the P. M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar) in that the repeated domains have with the ion which repeated domains on the of the membrane D.A. J.M. A. J.M. R. Science. 1998; PubMed Scopus Google Scholar). the form two ion domains on of the membrane or in to form a ion In either the protein that ion on one of the membrane the on the other of in the model of the exchanger is that the near the is In the of the α-1 the the membrane to form TMS2 and In the of the α-2 the the membrane to form and then the either may loop in and of the membrane shown or does not the The may form of the and the motif a loop that a for for to the We are to for on this
Nicoll et al. (Fri,) reported a other. Cysteine mutagenesis and sulfhydryl modification was evaluated on Exchanger activity and topology. Cysteine mutagenesis of the Na+-Ca2+ exchanger supports a revised topological model placing transmembrane segment 6 in cytoplasmic loop f and suggesting segment 9 forms a P-loop-like structure.