Structure-Function Analysis of CALX1.1, a Na+-Ca2+ Exchanger fromDrosophila
1998; Elsevier BV; Volume: 273; Issue: 21 Linguagem: Inglês
10.1074/jbc.273.21.12981
ISSN1083-351X
AutoresChris Dyck, Krista Maxwell, John Buchko, Michael Trac, Alexander Omelchenko, Mark Hnatowich, Larry V. Hryshko,
Tópico(s)Plant Stress Responses and Tolerance
ResumoCytoplasmic Na+ and Ca2+ regulate the activity of Na+-Ca2+ exchange proteins, in addition to serving as the transported ions, and protein regions involved in these processes have been identified for the canine cardiac Na+-Ca2+ exchanger, NCX1.1. Although protein regions associated with Na+i- and Ca2+i-dependent regulation are highly conserved among cloned Na+-Ca2+ exchangers, it is unknown whether or not the structure-function relationships characteristic of NCX1.1 apply to any other exchangers. Therefore, we studied structure-function relationships in a Na+-Ca2+ exchanger from Drosophila, CALX1.1, which is unique among characterized members of this family of proteins in that μm levels of Ca2+i inhibit exchange current. Wild-type and mutant CALX1.1 exchangers were expressed in Xenopus oocytes and characterized electrophysiologically using the giant excised patch technique. Mutations within the putative regulatory Ca2+i binding site of CALX1.1, like corresponding alterations in NCX1.1, led to reduced ability (i.e. D516V and D550I) or inability (i.e. G555P) of Ca2+i to inhibit Na+-Ca2+exchange activity. Similarly, mutations within the putative XIP region of CALX1.1, as in NCX1.1, led to two distinct phenotypes: acceleration (i.e. K306Q) and elimination (i.e. Δ310–313) of Na+i-dependent inactivation. These results indicate that the respective regulatory roles of the Ca2+i binding site and XIP region are conserved between CALX1.1 and NCX1.1, despite opposite responses to Ca2+i. We extended these findings using chimeric constructs of CALX1.1 and NCX1.1 to determine whether or not functional interconversion of Ca2+i regulatory phenotypes was feasible. With one chimera (i.e. CALX:NCX:CALX), substitution of a 193-amino acid segment, from the large intracellular loop of NCX1.1, for the corresponding 177-amino acid segment of CALX1.1 led to an exchanger that was stimulated by Ca2+i. This result indicates that the regulatory Ca2+i binding site of NCX1.1 retains function in a CALX1.1 parent transporter and that the substituted segment contains some of the amino acid sequence(s) required for transduction of the Ca2+i binding signal. Cytoplasmic Na+ and Ca2+ regulate the activity of Na+-Ca2+ exchange proteins, in addition to serving as the transported ions, and protein regions involved in these processes have been identified for the canine cardiac Na+-Ca2+ exchanger, NCX1.1. Although protein regions associated with Na+i- and Ca2+i-dependent regulation are highly conserved among cloned Na+-Ca2+ exchangers, it is unknown whether or not the structure-function relationships characteristic of NCX1.1 apply to any other exchangers. Therefore, we studied structure-function relationships in a Na+-Ca2+ exchanger from Drosophila, CALX1.1, which is unique among characterized members of this family of proteins in that μm levels of Ca2+i inhibit exchange current. Wild-type and mutant CALX1.1 exchangers were expressed in Xenopus oocytes and characterized electrophysiologically using the giant excised patch technique. Mutations within the putative regulatory Ca2+i binding site of CALX1.1, like corresponding alterations in NCX1.1, led to reduced ability (i.e. D516V and D550I) or inability (i.e. G555P) of Ca2+i to inhibit Na+-Ca2+exchange activity. Similarly, mutations within the putative XIP region of CALX1.1, as in NCX1.1, led to two distinct phenotypes: acceleration (i.e. K306Q) and elimination (i.e. Δ310–313) of Na+i-dependent inactivation. These results indicate that the respective regulatory roles of the Ca2+i binding site and XIP region are conserved between CALX1.1 and NCX1.1, despite opposite responses to Ca2+i. We extended these findings using chimeric constructs of CALX1.1 and NCX1.1 to determine whether or not functional interconversion of Ca2+i regulatory phenotypes was feasible. With one chimera (i.e. CALX:NCX:CALX), substitution of a 193-amino acid segment, from the large intracellular loop of NCX1.1, for the corresponding 177-amino acid segment of CALX1.1 led to an exchanger that was stimulated by Ca2+i. This result indicates that the regulatory Ca2+i binding site of NCX1.1 retains function in a CALX1.1 parent transporter and that the substituted segment contains some of the amino acid sequence(s) required for transduction of the Ca2+i binding signal. The identification of novel Na+-Ca2+exchange proteins has proceeded rapidly in the past 8 years. The family of Na+-Ca2+ exchangers includes transporters encoded by unique gene products (1Nicoll D.A. Longoni S. Philipson K.D. Science. 1990; 250: 562-565Crossref PubMed Scopus (627) Google Scholar, 2Li Z. Matsuoka S. Hryshko L.V. Nicoll D.A. Bersohn M.M. Burke E.P. Lifton R.P. Philipson K.D. J. Biol. Chem. 1994; 269: 17434-17439Abstract Full Text PDF PubMed Google Scholar, 3Nicoll 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 (309) Google Scholar), as well as by a variety of alternatively spliced variants (4Kofuji P. Lederer W.J. Schulze D.H. J. Biol. Chem. 1994; 269: 5145-5149Abstract Full Text PDF 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, 6Gabellini N. Iwata T. Carafoli E. J. Biol. Chem. 1995; 270: 6917-6924Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 7Quednau B.D. Nicoll D.A. Philipson K.D. Am. J. Physiol. 1997; 272: C1250-C1261Crossref PubMed Google Scholar). Promoter elements underlying the tissue-specific expression of alternatively spliced exchangers have been described (8Barnes K.V. Cheng G. Dawson M.M. Menick D.R. J. Biol. Chem. 1997; 272: 11510-11517Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 9Nicholas S.B. Yang W. Lee S.L. Zhu W. Philipson K.D. Lytton J. Am. J. Physiol. 1998; 274: H217-H232PubMed Google Scholar), and developmental changes in splice variant expression have been reported (7Quednau B.D. Nicoll D.A. Philipson K.D. Am. J. Physiol. 1997; 272: C1250-C1261Crossref PubMed Google Scholar). Moreover, exchangers have been cloned from several species and tissue types, and the opportunity now exists for detailed comparative structure-function studies of this family of transport molecules (10Philipson K.D. Nicoll D.A. Matsuoka S. Hryshko L.V. Levitsky D.O. Weiss J.N. Ann. N. Y. Acad. Sci. 1996; 779: 20-28Crossref PubMed Scopus (40) Google Scholar).Regulation of Na+-Ca2+ exchange activity by several factors has been described, including Na+iand Ca2+i, with the latter being the most thoroughly studied at the molecular level. First identified in the squid giant axon (11Baker P.F. McNaughton P.A. J. Physiol. 1976; 259: 104-114Crossref Scopus (114) Google Scholar, 12DiPolo R. J. Physiol. 1979; 73: 91-113Google Scholar), Ca2+i-dependent regulation is apparent as a stimulation of Na+-Ca2+ exchange current in response to μm levels of Ca2+i(13Hilgemann D.W. Nature. 1990; 344: 242-245Crossref PubMed Scopus (248) Google Scholar). The basis for this behavior, originally termed I2inactivation, is thought to involve entry of the exchanger protein into an I2 inactive state upon removal of Ca2+i (14Hilgemann D.W. Collins A. Matsuoka S. J. Gen. Physiol. 1992; 100: 933-961Crossref PubMed Scopus (219) Google Scholar). A high affinity Ca2+i binding site has been identified for the canine cardiac exchanger, NCX1.1, which appears to be closely associated with the Ca2+i-dependent regulatory process. This site comprises a 138-amino acid segment of the large intracellular loop of NCX1.1; mutation of specific residues within this region can lead to reductions in both45Ca2+ binding affinity to fusion proteins (15Levitsky D.O. Nicoll D.A. Philipson K.D. J. Biol. Chem. 1994; 269: 22847-22852Abstract Full Text PDF PubMed Google Scholar) and the affinity for functional Ca2+i regulation as assessed electrophysiologically (16Matsuoka 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 (204) Google Scholar).The cardiac Na+-Ca2+ exchanger also undergoes an inactivation process in response to the application of Na+i (13Hilgemann D.W. Nature. 1990; 344: 242-245Crossref PubMed Scopus (248) Google Scholar, 17Hilgemann D.W. Matsuoka S. Nagel G.A. Collins A. J. Gen. Physiol. 1992; 100: 905-932Crossref PubMed Scopus (237) Google Scholar). This mechanism, termed Na+i-dependent or I1inactivation, is analogous to ion channel gating and involves the exchanger inhibitory peptide (XIP) 1The abbreviations used are: XIP, exchanger inhibitory peptide; MOPS, 4-morpholinepropanesulfonic acid.1The abbreviations used are: XIP, exchanger inhibitory peptide; MOPS, 4-morpholinepropanesulfonic acid. region at the N terminus of the large cytoplasmic loop of NCX1.1 (18Matsuoka S. Nicoll D.A. He Z. Philipson K.D. J. Gen. Physiol. 1997; 109: 273-286Crossref PubMed Scopus (144) Google Scholar). This amino acid sequence, comprising residues 219–238, was originally identified based upon primary structural similarity with calmodulin binding sites (1Nicoll D.A. Longoni S. Philipson K.D. Science. 1990; 250: 562-565Crossref PubMed Scopus (627) Google Scholar). Exogenous application of a peptide corresponding to this amino acid sequence (i.e. XIP) to the intracellular surface of excised membrane patches produces marked inhibition of Na+-Ca2+ exchange currents (19Li 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, 20Matsuoka 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). More recent studies have shown that mutations within the XIP region of NCX1.1 are associated with substantial alterations in the rate and extent of I1 inactivation (18Matsuoka S. Nicoll D.A. He Z. Philipson K.D. J. Gen. Physiol. 1997; 109: 273-286Crossref PubMed Scopus (144) Google Scholar), lending support to the notion that the XIP region of NCX1.1 is intimately involved in the mechanism of Na+i-dependent inactivation and that exogenous application of XIP may mimic this process. Both of the ionic regulatory mechanisms (i.e.I1 and I2) can be eliminated by limited proteolysis of membrane patches with α-chymotrypsin, apparently converting NCX1.1 into a fully active, deregulated exchanger (13Hilgemann D.W. Nature. 1990; 344: 242-245Crossref PubMed Scopus (248) Google Scholar).Although the role of I1 and I2 regulation in physiological Na+-Ca2+ exchange function remains poorly understood, both processes can be readily demonstrated in intact cellular preparations (21Matsuoka S. Hilgemann D.W. J. Physiol. 1994; 476: 443-458Crossref PubMed Scopus (67) Google Scholar, 22Kimuro J. Noma A. Irisawa H. Nature. 1986; 319: 596-597Crossref PubMed Scopus (277) Google Scholar). Furthermore, substantial interaction between I1 and I2 regulation has been demonstrated in both structure-function studies and electrophysiological analyses (14Hilgemann D.W. Collins A. Matsuoka S. J. Gen. Physiol. 1992; 100: 933-961Crossref PubMed Scopus (219) Google Scholar, 16Matsuoka 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 (204) Google Scholar, 18Matsuoka S. Nicoll D.A. He Z. Philipson K.D. J. Gen. Physiol. 1997; 109: 273-286Crossref PubMed Scopus (144) Google Scholar). We have recently observed substantial differences in I1 and I2 regulation for alternatively spliced isoforms of CALX1 (23Omelchenko, A., Dyck, C., Hnatowich, M., Buchko, J., Nicoll, D. A., Philipson, K. D., and Hryshko, L. V. (1998) J. Gen. Physiol.3,in pressGoogle Scholar). The fact that alternative splicing has targeted these regulatory mechanisms at least suggests that they may play relevant physiological roles. However, it is unclear how I1 and I2 inactivation occur at the molecular level.To date, structure-function studies of the Na+-Ca2+ exchanger have been restricted to NCX1.1 (16Matsuoka 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 (204) Google Scholar, 18Matsuoka S. Nicoll D.A. He Z. Philipson K.D. J. Gen. Physiol. 1997; 109: 273-286Crossref PubMed Scopus (144) Google Scholar, 20Matsuoka 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, 24Nicoll 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 (127) Google Scholar, 25Doering 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, 26Reeves J.P. Chernaya G. Condrescu M. Ann. N. Y. Acad. Sci. 1996; 779: 73-85Crossref PubMed Scopus (6) Google Scholar), and it is unknown whether or not these findings can be extended to other members of this family of transport proteins. Thus, we combined mutagenesis, chimeric exchanger construction, and electrophysiological measurements to investigate amino acid sequences involved in ionic regulatory mechanisms of CALX1.1, a Na+-Ca2+ exchanger fromDrosophila melanogaster (27Ruknudin A. Valdivia C. Kofuji P. Lederer W.J. Schulze D.H. Am. J. Physiol. 1997; 273: C257-C265Crossref PubMed Google Scholar, 28Schwarz E. Benzer S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10249-10254Crossref PubMed Scopus (180) Google Scholar). The Drosophilaprotein was selected for study because it is unique among characterized exchangers in terms of its negative regulatory response to Ca2+i (29Hryshko L.V. Matsuoka S. Nicoll D.A. Weiss J.N. Schwarz E.M. Benzer S. Philipson K.D. J. Gen. Physiol. 1996; 108: 67-74Crossref PubMed Scopus (68) Google Scholar). Targets for mutagenesis of CALX1.1 were selected at or near regions analogous to the regulatory Ca2+i binding site and XIP region of NCX1.1, and we found striking parallels in terms of altered ionic regulatory properties between the two exchangers. Our results indicate that CALX1.1 and NCX1.1 employ an equivalent site for binding of regulatory Ca2+i. Other regions of the CALX1.1 cytoplasmic loop were identified that completely alleviate Ca2+i regulation. Mutations within the XIP region alter the pattern of Na+i-dependent inactivation, similar to that observed for NCX1.1. Our results indicate that amino acid sequences subserving Na+i- and Ca2+i-dependent regulatory processes are conserved between CALX1.1 and NCX1.1, irrespective of the fact that these exchangers are regulated by Ca2+i in opposing fashions.DISCUSSIONIn this study, we have examined ionic regulation of theDrosophila Na+-Ca2+ exchanger CALX1.1 using mutagenesis and electrophysiological techniques. Our results indicate that Ca2+i regulation in both NCX1.1 and CALX1.1 appears to employ the same regulatory Ca2+i binding site, even though these exchangers exhibit opposite patterns of Ca2+i regulation. In addition, we have examined mutations in the XIP region of CALX1.1 and obtained similar functional consequences to that observed in NCX1.1. These mutations produced the same spectrum of changes in Na+i-dependent inactivation, ranging from an acceleration to complete elimination. We have not attempted to be exhaustive in our comparisons of CALX1.1 and NCX1.1, but rather to ascertain the likelihood that identified functional domains are conserved. Our data are highly supportive of this conservation. Consequently, the unique Ca2+i regulatory phenotypes between these exchangers must reside in an as yet unidentified domain(s) of the transporters. From initial results with chimeric exchangers, we have been successful in imparting a positive Ca2+i regulatory phenotype to a CALX1.1 parent transporter by interchanging a portion of its cytoplasmic loop with the corresponding region from NCX1.1. Further delineation of this region should provide insight into the transduction of the Ca2+i binding signal.Ca2+i-dependent Regulation of CALX1.1Regulation of Na+-Ca2+ exchange currents by Ca2+i has been described in all exchangers studied to date, including the unique exchanger subtypes NCX1 (16Matsuoka 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 (204) Google Scholar), NCX2 (2Li Z. Matsuoka S. Hryshko L.V. Nicoll D.A. Bersohn M.M. Burke E.P. Lifton R.P. Philipson K.D. J. Biol. Chem. 1994; 269: 17434-17439Abstract Full Text PDF PubMed Google Scholar), and NCX3. 2C. Dyck, A. Omelchenko, M. Hnatowich, and L. V. Hryshko, unpublished observations. With the exception of CALX1 splice variants, low concentrations (i.e.μm range) of Ca2+i stimulate exchange current. CALX1.1 and CALX1.2 are unique in that exchange activity is decreased over the same range of Ca2+iconcentrations (23Omelchenko, A., Dyck, C., Hnatowich, M., Buchko, J., Nicoll, D. A., Philipson, K. D., and Hryshko, L. V. (1998) J. Gen. Physiol.3,in pressGoogle Scholar, 29Hryshko L.V. Matsuoka S. Nicoll D.A. Weiss J.N. Schwarz E.M. Benzer S. Philipson K.D. J. Gen. Physiol. 1996; 108: 67-74Crossref PubMed Scopus (68) Google Scholar). Although the primary structure of the regulatory Ca2+i binding site identified in NCX1.1 is well-conserved among all exchangers, there have been no studies to determine whether or not this site performs a similar function in other exchangers. Thus, we selected for study an exchanger, CALX1.1, with a Ca2+i regulatory phenotype opposite to that of NCX1.1 for two main reasons. First, if the Ca2+ibinding site serves a similar functional role in both NCX1.1 and CALX1.1, we anticipated that its affinity could be predictably altered by mutagenesis. Second, we speculated that if the 138-amino acid Ca2+i binding domain of NCX1.1 contained some (or all) of the amino acid determinants directly responsible for transduction of the Ca2+i binding signal, then interchanging this region between CALX1.1 and NCX1.1 presented the greatest likelihood of observing phenotypic differences in the recipient exchanger.Our results show that NCX1.1-analogous mutations within the regulatory Ca2+i binding site of CALX1.1 lead to parallel changes in the ability of Ca2+i to regulate exchange activity. That is, mutation of corresponding amino acid residues (see Fig. 2) reduces the affinity for Ca2+i regulation, irrespective of whether the regulatory effect is stimulatory (i.e. NCX1.1) or inhibitory (i.e. CALX1.1). This result strongly suggests that the function of the Ca2+i binding site is similar in both exchangers. Considering the striking degree of amino acid sequence similarity between the acidic clusters within the putative Ca2+i binding sites of all exchanger subtypes and species variants shown in Fig. 2, and our present results showing a conserved functional role for exchangers sharing only ~50% overall sequence identity and opposite Ca2+i-dependent regulatory phenotypes, it seems plausible that the regulatory Ca2+ibinding site may serve the same function in other, if not all, Na+-Ca2+ exchangers.Na+i-dependent Regulation of CALX1.1We have shown that the XIP region serves a similar role in both CALX1.1 and NCX1.1 because analogous mutations produce similar phenotypes in both exchangers. With K306Q, corresponding to K225Q in NCX1.1 (see Fig. 4) (18Matsuoka S. Nicoll D.A. He Z. Philipson K.D. J. Gen. Physiol. 1997; 109: 273-286Crossref PubMed Scopus (144) Google Scholar), current decay into (Fig. 5) and recovery from (Fig. 6) the I1 inactive state was increased ~2-fold, mirrored by a slight increase in the extent of inactivation as indicated by a lower value of Fss, the fraction of steady-state to peak exchange current. According to the two-state model for I1 inactivation proposed by Hilgemann et al.(17Hilgemann D.W. Matsuoka S. Nagel G.A. Collins A. J. Gen. Physiol. 1992; 100: 905-932Crossref PubMed Scopus (237) Google Scholar), the larger I1 recovery rate constant of K306Q compared with CALX1.1 provides a satisfactory account for the accelerated current decay rate observed with K306Q versusCALX1.1. The observation that Fss is reduced for K306Q also indicates that entry into the I1 inactive state has been accelerated. In contrast, the I1 inactivation process was apparently eliminated altogether in Δ310–313, a result identical to that obtained with the corresponding deletion mutant in NCX1.1, Δ229–232 (18Matsuoka S. Nicoll D.A. He Z. Philipson K.D. J. Gen. Physiol. 1997; 109: 273-286Crossref PubMed Scopus (144) Google Scholar), and with α-chymotrypsin-digested (i.e.deregulated) CALX1.1 (Fig. 6). Although the degree of primary structural similarity within the XIP-like regions of the various exchangers assembled in Fig. 4 is clearly less than for the acidic clusters of the Ca2+i binding site (Fig. 2), it is sufficiently prominent to tentatively conclude that this region may also share a common function in the control of Na+i-dependent inactivation of Na+-Ca2+ exchangers in general. This possibility is supported by the observation that both CALX1.1 (29Hryshko L.V. Matsuoka S. Nicoll D.A. Weiss J.N. Schwarz E.M. Benzer S. Philipson K.D. J. Gen. Physiol. 1996; 108: 67-74Crossref PubMed Scopus (68) Google Scholar) and NCX2 (2Li Z. Matsuoka S. Hryshko L.V. Nicoll D.A. Bersohn M.M. Burke E.P. Lifton R.P. Philipson K.D. J. Biol. Chem. 1994; 269: 17434-17439Abstract Full Text PDF PubMed Google Scholar), like NCX1.1, are inhibited by XIP, which appears to mimic the I1 inactivation process.Ca2+i-insensitive and Chimeric ExchangersAlthough the function of the XIP region and Ca2+i binding site appears to be conserved between CALX1.1 and NCX1.1, we have no definitive explanation for the observed differences in Ca2+i-dependent regulatory phenotypes. However, we observed that analogous mutations can render both exchangers insensitive to Ca2+i(e.g. G555P in CALX1.1 and G503P in NCX1.1) (see Fig. 3) (18Matsuoka S. Nicoll D.A. He Z. Philipson K.D. J. Gen. Physiol. 1997; 109: 273-286Crossref PubMed Scopus (144) Google Scholar, 33Hryshko L.V. Nicoll D.A. Matsuoka S. Levitsky D.O. Li Z. Weiss J.N. Philipson K.D. Dhalla N.S. Singal P.K. Takeda N. Beamish R.E. Pathophysiology of Heart Failure. Kluwer Academic Publishers, Boston1996: 331-342Google Scholar). One possibility is that Ca2+i binds to these exchangers but the transduction process has been disabled. Supporting this notion is a report by Levitsky et al. (15Levitsky D.O. Nicoll D.A. Philipson K.D. J. Biol. Chem. 1994; 269: 22847-22852Abstract Full Text PDF PubMed Google Scholar) showing that a fusion protein containing the Ca2+ibinding site of NCX1.1, but bearing a G503P equivalent mutation, binds45Ca2+i indistinguishably from the wild-type sequence. Also consistent with this hypothesis is our result with the chimeric exchanger CALX:NCX:CALX. Here, interchange of the entire Ca2+i binding site and flanking sequences of NCX1.1 with the corresponding region of CALX1.1 led to a transporter that was stimulated by regulatory Ca2+i (see Fig.8). Thus, the Ca2+i binding function appears to have been retained, and a partial interconversion of phenotypes occurred. Although extrapolation of results from fusion and chimeric proteins to the wild-type exchanger must be made with caution, the above findings suggest that Ca2+i binding per se may be restricted to the acidic clusters within the larger Ca2+i binding domain, and that transduction of the Ca2+i binding signal may occur through sequences remote or distinct from this region. Our results with NCX:CALX:NCX neither support nor refute this notion, irrespective of the fact that the chimeric protein did acquire a novel phenotype (i.e.Ca2+i-insensitive). Although there is little evidence to indicate that Ca2+i binding and transduction domains are modular or discrete, our results are at least suggestive of this possibility. Furthermore, we can conclude that the primary structural alterations associated with these two chimeras are relatively benign with respect to the transport function and Na+i-dependent regulation of the parent molecule. Complete and "symmetric" reversal of Ca2+i-dependent regulatory phenotypes between CALX1.1 and NCX1.1 may be possible through additional studies of this type. The identification of novel Na+-Ca2+exchange proteins has proceeded rapidly in the past 8 years. The family of Na+-Ca2+ exchangers includes transporters encoded by unique gene products (1Nicoll D.A. Longoni S. Philipson K.D. Science. 1990; 250: 562-565Crossref PubMed Scopus (627) Google Scholar, 2Li Z. Matsuoka S. Hryshko L.V. Nicoll D.A. Bersohn M.M. Burke E.P. Lifton R.P. Philipson K.D. J. Biol. Chem. 1994; 269: 17434-17439Abstract Full Text PDF PubMed Google Scholar, 3Nicoll 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 (309) Google Scholar), as well as by a variety of alternatively spliced variants (4Kofuji P. Lederer W.J. Schulze D.H. J. Biol. Chem. 1994; 269: 5145-5149Abstract Full Text PDF 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, 6Gabellini N. Iwata T. Carafoli E. J. Biol. Chem. 1995; 270: 6917-6924Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 7Quednau B.D. Nicoll D.A. Philipson K.D. Am. J. Physiol. 1997; 272: C1250-C1261Crossref PubMed Google Scholar). Promoter elements underlying the tissue-specific expression of alternatively spliced exchangers have been described (8Barnes K.V. Cheng G. Dawson M.M. Menick D.R. J. Biol. Chem. 1997; 272: 11510-11517Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 9Nicholas S.B. Yang W. Lee S.L. Zhu W. Philipson K.D. Lytton J. Am. J. Physiol. 1998; 274: H217-H232PubMed Google Scholar), and developmental changes in splice variant expression have been reported (7Quednau B.D. Nicoll D.A. Philipson K.D. Am. J. Physiol. 1997; 272: C1250-C1261Crossref PubMed Google Scholar). Moreover, exchangers have been cloned from several species and tissue types, and the opportunity now exists for detailed comparative structure-function studies of this family of transport molecules (10Philipson K.D. Nicoll D.A. Matsuoka S. Hryshko L.V. Levitsky D.O. Weiss J.N. Ann. N. Y. Acad. Sci. 1996; 779: 20-28Crossref PubMed Scopus (40) Google Scholar). Regulation of Na+-Ca2+ exchange activity by several factors has been described, including Na+iand Ca2+i, with the latter being the most thoroughly studied at the molecular level. First identified in the squid giant axon (11Baker P.F. McNaughton P.A. J. Physiol. 1976; 259: 104-114Crossref Scopus (114) Google Scholar, 12DiPolo R. J. Physiol. 1979; 73: 91-113Google Scholar), Ca2+i-dependent regulation is apparent as a stimulation of Na+-Ca2+ exchange current in response to μm levels of Ca2+i(13Hilgemann D.W. Nature. 1990; 344: 242-245Crossref PubMed Scopus (248) Google Scholar). The basis for this behavior, originally termed I2inactivation, is thought to involve entry of the exchanger protein into an I2 inactive state upon removal of Ca2+i (14Hilgemann D.W. Collins A. Matsuoka S. J. Gen. Physiol. 1992; 100: 933-961Crossref PubMed Scopus (219) Google Scholar). A high affinity Ca2+i binding site has been identified for the canine cardiac exchanger, NCX1.1, which appears to be closely associated with the Ca2+i-dependent regulatory process. This site comprises a 138-amino acid segment of the large intracellular loop of NCX1.1; mutation of specific residues within this region can lead to reductions in both45Ca2+ binding affinity to fusion proteins (15Levitsky D.O. Nicoll D.A. Philipson K.D. J. Biol. Chem. 1994; 269: 22847-22852Abstract Full Text PDF PubMed Google Scholar) and the affinity for functional Ca2+i regulation as assessed electrophysiologically (16Matsuoka 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 (204) Google Scholar). The cardiac Na+-Ca2+ exchanger also undergoes an inactivation process in response to the application of Na+i (13Hilgemann D.W. Nature. 1990; 344: 242-245Crossref PubMed Scopus (248) Google Scholar, 17Hilgemann D.W. Matsuoka S. Nagel G.A. Collins A. J. Gen. Physiol. 1992; 100: 905-932Crossref PubMed Scopus (237) Google Scholar). This mechanism, termed Na+i-dependent or I1inactivation, is analogous to ion channel gating and involves the exchanger inhibitory peptide (XIP) 1The abbreviations used are: XIP, exchanger inhibitory peptide; MOPS, 4-morpholinepropanesulfonic acid.1The abbreviations used are: XIP, exchanger inhibitory peptide; MOPS, 4-morpholinepropanesulfonic acid. region at the N terminus of the large cytoplasmic loop of NCX1.1 (18Matsuoka S. Nicoll D.A. He Z. Philipson K.D. J. Gen. Physiol. 1997; 109: 273-286Crossref PubMed Scopus (144) Google Scholar). This amino acid sequence, comprising residues 219–238, was originally identified based upon primary structural similarity with calmodulin binding sites (1Nicoll D.A. Longoni S. Philipson K.D. Science. 1990; 250: 562-565Crossref PubMed Scopus (627) Google Scholar). Exogenous application of a peptide corresponding to this amino acid sequence (i.e. XIP) to the intracellular surface of excised membrane patches produces marked inhibition of Na+-Ca2+ exchange currents (19Li 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, 20Matsuoka 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). More recent studies have shown that mutations within the XIP region of NCX1.1 are associated with substantial alterations in the rate and exte
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