The Molecular Determinants of Ionic Regulatory Differences between Brain and Kidney Na+/Ca2+ Exchanger (NCX1) Isoforms

The Na+/Ca2+exchanger gene NCX1 undergoes alternative splicing leading to several isoforms that differ in a small portion of the large cytoplasmic loop. This loop is involved in many regulatory processes of NCX1, including ionic regulation by the transported substrates Na+and Ca2+. High intracellular Ca2+ can alleviate intracellular Na+-dependent inactivation in exon A (NCX1.4)-containing isoforms but not in those containing the mutually exclusive exon B (NCX1.3). Giant excised patches fromXenopus oocytes expressing various NCX1 constructs were used to examine the specific amino acids responsible for these observed regulatory differences. Using a chimeric approach, the region responsible was narrowed down to the small central part of exon A (IDDEEYEKNKTF). Replacing the second aspartic acid of this sequence with arginine (the corresponding amino acid in exon B) in an exon A background completely prevented the effect of Ca2+ on intracellular Na+-dependent inactivation. Mutating the second lysine to cysteine (exon B) had a similar, but only partial, effect. The converse double mutant, but neither single mutation alone, introduced into an exon B background (arginine to aspartic acid and cysteine to lysine) was able to restore the NCX1.4 regulatory phenotype. These data demonstrate that aspartic acid 610 and lysine 617 (using the rat NCX1.4 numbering scheme) are critical molecular determinants of the unique Ca2+ regulatory properties of NCX1.4. The Na+/Ca2+exchanger gene NCX1 undergoes alternative splicing leading to several isoforms that differ in a small portion of the large cytoplasmic loop. This loop is involved in many regulatory processes of NCX1, including ionic regulation by the transported substrates Na+and Ca2+. High intracellular Ca2+ can alleviate intracellular Na+-dependent inactivation in exon A (NCX1.4)-containing isoforms but not in those containing the mutually exclusive exon B (NCX1.3). Giant excised patches fromXenopus oocytes expressing various NCX1 constructs were used to examine the specific amino acids responsible for these observed regulatory differences. Using a chimeric approach, the region responsible was narrowed down to the small central part of exon A (IDDEEYEKNKTF). Replacing the second aspartic acid of this sequence with arginine (the corresponding amino acid in exon B) in an exon A background completely prevented the effect of Ca2+ on intracellular Na+-dependent inactivation. Mutating the second lysine to cysteine (exon B) had a similar, but only partial, effect. The converse double mutant, but neither single mutation alone, introduced into an exon B background (arginine to aspartic acid and cysteine to lysine) was able to restore the NCX1.4 regulatory phenotype. These data demonstrate that aspartic acid 610 and lysine 617 (using the rat NCX1.4 numbering scheme) are critical molecular determinants of the unique Ca2+ regulatory properties of NCX1.4. Na+/Ca2+ exchanger intracellular sodium cytoplasmic calcium 2-(N-morpholino)ethanesulfonic acid 4-morpholinepropanesulfonic acid fractional steady-state current exchanger inhibitory peptide The Na+/Ca2+ exchanger (NCX)1 is an integral membrane protein found in nearly every cell type of the body where it plays an important role in Ca2+ homeostasis. Normally the NCX serves as a Ca2+ extrusion mechanism driven by the Na+ electrochemical gradient. In cardiac muscle, for example, Ca2+ entering through L-type channels during the action potential is subsequently extruded by the NCX. However, it has also been shown that Ca2+ influx through the exchanger can occur under certain conditions (1Kohmoto O. Levi A.J. Bridge J.H.B. Circ. Res. 1994; 74: 550-554Crossref PubMed Google Scholar). The NCX family consists of three genes, NCX1, NCX2, and NCX3. These genes, with the exception of NCX2, undergo alternative splicing leading to many different isoforms (2Nicoll D.A. Longoni S. Philipson K.D. Science. 1990; 250: 562-565Crossref PubMed Scopus (641) Google Scholar, 3Li 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, 4Nicoll D.A. Quednau B.D. Qiu Z. Xia Y.R. Lusis A.J. Philipson K.D. J. Biol. Chem. 1996; 271: 24914-24921Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 5Quednau B.D. Nicoll D.A. Philipson K.D. Am. J. Physiol. 1997; 272: C1250-C1261Crossref PubMed Google Scholar). In the case of NCX1, 12 different alternatively spliced isoforms have been observed, several of which are expressed in a tissue-specific manner (6Kofuji P. Lederer W.J. Schulze D.H. J. Biol. Chem. 1994; 269: 5145-5149Abstract Full Text PDF PubMed Google Scholar, 7Lee S.L., Yu, A.S. Lytton J. J. Biol. Chem. 1994; 269: 14849-14852Abstract Full Text PDF PubMed Google Scholar). To date, the prototypical canine cardiac exchanger NCX1.1 has been most widely studied with respect to structure, function, and regulation (8Nicoll D.A. Philipson K.D. Ann. N. Y. Acad. Sci. 1991; 639: 181-188Crossref PubMed Scopus (40) Google Scholar, 9Matsuoka 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 (202) Google Scholar). Alternative splicing of the NCX1 gene produces transcripts that differ in a segment corresponding to a small region of the protein in the large cytoplasmic loop, 600 amino acids from the beginning of the open reading frame (see Fig. 1). The splicing process results in the mutually exclusive use of exons denoted A or B followed by the inclusion or exclusion in any combination of the cassette exons C to F (5Quednau B.D. Nicoll D.A. Philipson K.D. Am. J. Physiol. 1997; 272: C1250-C1261Crossref PubMed Google Scholar). Cardiac muscle expresses the NCX1.1 isoform encoded by a transcript containing alternatively spliced exons A, C, D, E, and F. The two alternatively spliced isoforms used in this study are NCX1.3 and NCX1.4. NCX1.3, which is encoded by a transcript containing alternatively spliced exons B and D, is the predominant isoform expressed abundantly in the kidney and ubiquitously elsewhere. NCX1.4, encoded by transcripts including alternatively spliced exons A and D, and NCX1.5 (which contains exons A, D, and F) are the major isoforms expressed in neurons of the brain. Exon A encodes 35 amino acids (residues 601–635), while exon B encodes one less at 34 (residues 601–634). Exon D is relatively small encoding for 6 amino acids (GGFTLT). The specific physiological functions of the NCX1 alternatively spliced products are largely unknown. NCX activity is regulated in many different ways including by the ions it transports. Overall the rate of exchange is limited by the extent to which the different transport substrates saturate the ion translocation binding sites. However, NCX is also regulated allosterically by both transport substrates. Intracellular sodium (Na+i) regulates the NCX by inducing an inactive conformation in a process referred to as Na+i-dependent or I1 inactivation (10Hilgemann D.W. Matsuoka S. Nagel G.A. Collins A. J. Gen. Physiol. 1992; 100: 905-932Crossref PubMed Scopus (243) Google Scholar). In the absence of cytoplasmic Ca2+ (Ca2+i), the exchanger enters an inactive state in a process referred to as Ca2+i-dependent or I2 inactivation (11Hilgemann D.W. Collins A. Matsuoka S. J. Gen. Physiol. 1992; 100: 933-961Crossref PubMed Scopus (221) Google Scholar). These two regulatory processes show interaction. For example, in the cardiac exchanger NCX1.1, I1 inactivation is alleviated if Ca2+i is sufficiently high. Recent studies using recombinant expression systems and in vitro assays have identified functional differences in the regulatory properties of NCX1 alternatively spliced isoforms. Heet al. (12He S. Ruknudin A. Bambrick L.L. Lederer W.J. Schulze D.H. J. Neurosci. 1998; 18: 4833-4841Crossref PubMed Google Scholar) compared rat NCX1.4 (containing alternatively spliced exons A and D) and NCX1.3 (containing exons B and D) inXenopus oocytes. They observed an increase of 39% in the activity of NCX1.4 upon activation by protein kinase A that was not seen for NCX1.3. Also two alternatively spliced NCX isoforms ofDrosophila melanogaster, Calx1.1 and Calx1.2, which differ by five amino acids at a position corresponding to the alternative splicing site of mammalian NCX1, show significant differences in their regulatory responses to Ca2+i and Na+i (13Omelchenko A. Dyck C. Hnatowich M. Buchko J. Nicoll D.A. Philipson K.D. Hryshko L.V. J. Gen. Physiol. 1998; 111: 691-702Crossref PubMed Scopus (40) Google Scholar). One of our laboratories has recently identified ionic regulatory differences between the two NCX1 isoforms NCX1.4 and NCX1.3 (14Dyck C. Omelchenko A. Elias C.L. Quednau B.D. Philipson K.D. Hnatowich M. Hryshko L.V. J. Gen. Physiol. 1999; 114: 701-711Crossref PubMed Scopus (87) Google Scholar). Dycket al. (14Dyck C. Omelchenko A. Elias C.L. Quednau B.D. Philipson K.D. Hnatowich M. Hryshko L.V. J. Gen. Physiol. 1999; 114: 701-711Crossref PubMed Scopus (87) Google Scholar) used outward exchange currents measured in giant excised patches from Xenopus oocytes to demonstrate that Na+i-dependent inactivation was alleviated by high Ca2+i in NCX1.4 but not in NCX1.3. In this study, we used the rat isoforms NCX1.3 and NCX1.4 to determine which amino acids contribute to the different regulatory properties of these exchangers. Using electrophysiological recordings we compared the functional properties of different isoforms and mutants and were able to identify two residues that are critical in conferring the unique regulatory properties for each isoform. The rat NCX1.3 and NCX1.4 cDNAs were constructed as follows. The coding region of the NCX1.7 clone F1 (7Lee S.L., Yu, A.S. Lytton J. J. Biol. Chem. 1994; 269: 14849-14852Abstract Full Text PDF PubMed Google Scholar) was excised using theMunI and Bst1107I restriction sites located at nucleotides −24 and +2913, respectively, where +1 is the start of the NCX1 open reading frame. This fragment was made blunt-ended using the Klenow fragment of DNA polymerase and ligated intoSmaI-digested pBluescript SKII− (Stratagene). The region encoding the large cytoplasmic loop of kidney NCX1.3 and brain NCX1.4 was obtained using reverse transcription-coupled PCR of kidney and brain mRNA, respectively. A BclI fragment (+812 to +2093) of this large cytoplasmic loop, which contained the NCX1.3- or NCX1.4-specific alternatively spliced region, was subsequently ligated into the BclI-digested pBluescript− NCX1.7 construct. The exchanger coding region was confirmed by sequencing and moved to the pcDNA3.1+ mammalian expression vector (Invitrogen) usingHindIII and BamHI restriction sites. The chimeric constructs were by PCR The was and of the sequence from position to was the reverse of from position to The chimeric constructs and were using NCX1.4 and NCX1.3 as the A fragment in the was using the and a reverse with the exon A sequence at the site of and a of exon B The B fragment was made using the reverse and a of exon B sequence with a exon A These two were and using the The was The was using NCX1.4 and NCX1.3 as and three of The were to those The region of exon B was using the and an reverse with exon B sequence and an exon A the sequence of exon A at the of in the The small region of exon A was using a of that had that were of the exon B The fragment of the was using the and an of exon B sequence with a the exon A sequence at the of the of the were that the at the which made it to the of the three at the using five of The were used to the The contained a region of exon A the and was using the and NCX1.3 constructs as The was used with a reverse of exon A sequence with an exon B the sequence of exon B at the of the The was used with a of the exon B sequence with an the exon A sequence at the using NCX1.3 as These two were and to and for PCR followed by using the The constructs with the were by an PCR where the each with the mutation in the The were the as those of the constructs that were by PCR were using for NCX1.4 or for and ligated into the pBluescript or which were constructs were confirmed by sequencing and moved into the mammalian expression vector pcDNA3.1+ with and BamHI the constructed cDNAs in pcDNA3.1+ were by with the restriction which at a position nucleotides of the of the exchanger open reading frame in the pcDNA3.1+ the constructs were using the PCR was using the to the were in of a of Xenopus oocytes were with of the The oocytes were as (14Dyck C. Omelchenko A. Elias C.L. Quednau B.D. Philipson K.D. Hnatowich M. Hryshko L.V. J. Gen. Physiol. 1999; 114: 701-711Crossref PubMed Scopus (87) Google Scholar). oocytes were and at in the were used for electrophysiological recordings for The of the different and mutants were compared by current using the giant excised as D.W. 1990; PubMed Scopus Google Scholar, L.V. Matsuoka S. Nicoll D.A. S. Philipson K.D. J. Gen. Physiol. 1996; PubMed Scopus Google Scholar, M. Dyck C. Hnatowich M. Omelchenko A. Hryshko L.V. J. Gen. Physiol. 1997; PubMed Scopus Google Scholar). were and the were to of To increase and the were in a to the was from the oocytes by in a containing at for the had been the oocytes were into a containing at were with and the patches were by of the A was used for were and using an with and To outward exchange currents the contained 6 with at To outward exchange the were between and Na+ or to with or Ca2+ and were using Biol. 1994; PubMed Scopus Google Scholar) to of and respectively. were at are the fractional steady-state was from the of current at from current to a single to the were a used to the data to a using of with the used for were in the peptide at the of corresponding to the sequence of exon A from the and the corresponding region from exon These were peptide A and respectively. The amino acid sequence of peptide A was and of peptide B was were found to by high The were in the to the cytoplasmic of the at a of The of this study was to identify the amino acids responsible for the functional differences in ionic regulation observed between the two Na+/Ca2+ exchanger isoforms NCX1.3 and NCX1.4. regulatory Ca2+ can alleviate Na+i-dependent inactivation in exon isoforms but not in those containing exon B as shown (14Dyck C. Omelchenko A. Elias C.L. Quednau B.D. Philipson K.D. Hnatowich M. Hryshko L.V. J. Gen. Physiol. 1999; 114: 701-711Crossref PubMed Scopus (87) Google Scholar) for canine NCX1.3 and NCX1.4. we the isoforms from rat that also differ only in the region of alternative splicing to mutually exclusive exons A and These two exons and as shown in Fig. with the of amino acids in the constructs were and the corresponding was oocytes. The giant excised was used to between and Fig. the of Na+i-dependent inactivation at regulatory Ca2+ for the rat NCX1.4 but not for the NCX1.3 isoform. This the NCX1.4 high exchange activity at high regulatory These of regulatory also seen with the rat cardiac isoform NCX1.1 not The current shown are single data from several for each are compared in Fig. using the of steady-state to current The obtained for the NCX1.4 and NCX1.3 isoforms at high Ca2+i were different the obtained for NCX1.4 and NCX1.3 were significant of the for the different exchanger constructs at both and are the of steady-state to current for the NCX1 were obtained as in are and the of recordings from not less patches with a of to a different as compared with NCX1.4 under Ca2+i to a significant with as compared with NCX1.4 under a significant as compared with NCX1.3 under a significant with as compared with NCX1.3 at a significant as compared with NCX1.3 at was to determine the residues involved in this functional were found in the or of exon A. two were of the amino acids of exon A followed by the amino acids of exon and of the amino acids of exon B followed by the amino acids of exon A. The current obtained from giant patches of oocytes with these are shown in Fig. exchange activity to high both Na+i-dependent inactivation to an extent not different from NCX1.3 and that observed for NCX1.4 This at two part of each of exon A for of Na+i-dependent inactivation. the critical region at the of exon A and was in both this in a second was in which the central region of exon B was by the corresponding region of exon A. of residues from exon A by the exon B sequence (see The obtained for this was to that of NCX1.4 as seen in Fig. This is confirmed by of as shown in Fig. The for of the at different from NCX1.3 but not different from NCX1.4. was in which the central part of exon A was to only residues by the exon B residues Fig. the current were to those seen with NCX1.4, and the were These data that residues in the small central region of exon A were responsible for the NCX1.4 phenotype. are only five differences between exons A and B this region, to at position aspartic acid to arginine at position to acid at position lysine to cysteine at position and to at position mutants are from the beginning of exon A or B 1). was used to the exon A residues in the NCX1.4 to those of exon B one at a The one was the aspartic acid at position of exon A, which was to an arginine The currents obtained an NCX1.3 an NCX1.4 the of Na+i-dependent inactivation by high Ca2+i was This can seen in the current of and confirmed by as shown in Fig. The mutation was from lysine at position of exon A to a cysteine as in exon B This mutation an as seen in the of Fig. and in the that were NCX1.4 and different from NCX1.3 The in this small region of of exon A was to a acid The currents and the obtained with this were not different from those of the NCX1.4 as seen in Fig. mutants that were and of lysine in exon of these the NCX1.4 as is from the data of These results that the observed in was not to a in the of this region but was to specific residues the central small of the NCX1.4 exon A mutants had and the two or NCX1.4 to an NCX1.3 phenotype. for the aspartic acid at position and the lysine at position the converse were made in NCX1.3. The currents obtained using the arginine to aspartic acid at position were to those of NCX1.3 and the were not different from those obtained with NCX1.3 of the cysteine at position of exon B to lysine as in exon A also in currents and that were different from NCX1.3 at at However, the double in NCX1.3 a with an at to that of NCX1.4 and different from NCX1.3. two residues of exon B the of high Ca2+i to the process of Na+i-dependent inactivation. the of for at was that of NCX1.4 as can seen in Fig. was of exchanger activity in the double in which was not seen in at Ca2+i was also observed for several mutants as in Fig. we have for this it is these are the corresponding at To the that of the critical portion of exon A with site on the exchanger involved in the of Na+i-dependent two were corresponding to the small central region of exon A or B acids exon A, exon These were to the of the at of A or B to NCX1.3- or patches had effect on Na+i-dependent inactivation or on the of Ca2+i on this process not The NCX1 isoforms are regulated in several ways including by the ions that exchange (10Hilgemann D.W. Matsuoka S. Nagel G.A. Collins A. J. Gen. Physiol. 1992; 100: 905-932Crossref PubMed Scopus (243) Google Scholar, D.W. Collins A. Matsuoka S. J. Gen. Physiol. 1992; 100: 933-961Crossref PubMed Scopus (221) Google Scholar, S. Nicoll D.A. Hryshko L.V. Philipson K.D. J. Gen. Physiol. PubMed Scopus Google Scholar, K.D. Nicoll D.A. Matsuoka S. Hryshko L.V. Ann. N. Y. Acad. Sci. 1996; PubMed Scopus (40) Google Scholar, Y. A. M. Am. J. Physiol. PubMed Google Scholar). One of these ionic regulatory observed is by activity of the NCX is measured in excised activation of the exchanger by of cytoplasmic Na+ results in the activation of exchange current followed by a inactivation in steady-state current This type of inactivation is Na+i-dependent or I1 inactivation (10Hilgemann D.W. Matsuoka S. Nagel G.A. Collins A. J. Gen. Physiol. 1992; 100: 905-932Crossref PubMed Scopus (243) Google Scholar). is to the inactivation observed for many ion channels and to a of into and inactive S. S. S. J. Physiol. PubMed Scopus Google Scholar). In this study, the brain and kidney NCX1 isoforms NCX1.4 and NCX1.3, respectively, were used to the molecular determinants of the functional differences observed between Dyck al. (14Dyck C. Omelchenko A. Elias C.L. Quednau B.D. Philipson K.D. Hnatowich M. Hryshko L.V. J. Gen. Physiol. 1999; 114: 701-711Crossref PubMed Scopus (87) Google Scholar) these isoforms from and found that Na+i-dependent inactivation was alleviated by high of Ca2+i in NCX1.4 but not in NCX1.3. These differ only by the of encoded by mutually exclusive exon A or 1). is in Fig. the observed in was also in rat NCX1.4 and NCX1.3. The of regulatory differences is of a role for this A chimeric was used to down the region of exon A to of Na+i-dependent inactivation. These data an important role for the central region of exon A in this were made in this region of exon A to the residues encoded by exon the had effect on the current of the NCX1.4 as seen in Fig. while had a and completely the high Ca2+i of The converse mutants and had only on the NCX1.3 and were not to the NCX1.4 phenotype. However, the double currents to those of an NCX1.4 phenotype. The of the double that these two amino aspartic acid and lysine are critical molecular determinants for the unique Ca2+i regulatory properties by exon A. are amino acids between exons A and B in the small region of exon A in the these are three of which the at position as in is that these and residues also a role in the Ca2+i-dependent of NCX1.4. are several the large intracellular loop of NCX1 that have been studied with respect to ionic A and amino acid segment a is at the beginning of the loop. of an peptide corresponding to this sequence to the cytoplasmic of the exchanger and to the exchanger or Z. Nicoll D.A. Collins A. Hilgemann D.W. Philipson K.D. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). it has been that the sequence with region of the exchanger in an to of it has been that can with a peptide corresponding to amino acids of NCX1, it that this as studies using amino acids S. Res. 1997; PubMed Scopus Google Scholar, M. S. Qiu Z. Philipson K.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The region is to the of as which are to to inhibitory with the second exchanger region Am. J. Physiol. 1994; PubMed Google Scholar, Ann. N. Y. Acad. Sci. 1996; PubMed Scopus Google Scholar). in which the region has been by have also that this region is involved in Na+i-dependent inactivation Ann. N. Y. Acad. Sci. 1996; PubMed Scopus Google Scholar, S. Nicoll Z. Philipson K.D. J. Gen. Physiol. 1997; PubMed Scopus Google Scholar, C. 1997; PubMed Scopus Google Scholar, Z. N. Philipson K.D. J. Biol. 1997; PubMed Scopus Google Scholar). of amino acids at and in the loop are to the regulatory Ca2+i that is an for exchanger activity S. Nicoll D.A. Hryshko L.V. Philipson K.D. J. Gen. Physiol. PubMed Scopus Google Scholar, Nicoll D.A. Philipson K.D. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, C. Nicoll D.A. Philipson K.D. 1996; PubMed Scopus Google Scholar). This process has been Ca2+i-dependent or I2 regulation (11Hilgemann D.W. Collins A. Matsuoka S. J. Gen. Physiol. 1992; 100: 933-961Crossref PubMed Scopus (221) Google Scholar). of with in these Ca2+ binding has both in Ca2+ activation properties and also to Na+i-dependent inactivation Y. A. M. Am. J. Physiol. PubMed Google Scholar). ionic regulation of the exchanger is and at a functional between the and it is that a region, the alternative splicing region, plays a role in ionic study on the of Na+i-dependent inactivation. The data have two specific amino acids a of residues the of mutually exclusive exons A and of the sequence found in exon A by of with in exon B in of of Na+i-dependent inactivation. is that this region to a site in with the sites involved in regulation of the that the inhibitory between and binding site on the two amino acid in exon B the double are for NCX1.3 to of Na+i-dependent it is that the in the conformation is to an The using from the alternatively spliced region were in an to into potential for these regulatory that the of peptide A or B to the intracellular of the giant excised expressing NCX1.4 the effect of the high Ca2+ by with Ca2+ or by with by Ca2+ it was that the of peptide A or B to patches expressing NCX1.3 to an NCX1.4 phenotype. of the had any effect on the patches expressing the NCX1.4 or NCX1.3 isoforms. Nicoll D.A. Philipson K.D. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar) used of different of the NCX1 cytoplasmic loop and found that only those constructs containing the any Ca2+ One of their constructs contained the region of alternative splicing the calcium binding and this not Ca2+ under their is that the exon A to a site but not one Ca2+ binding using of the cytoplasmic loop the different potential Ca2+ binding differences in Ca2+ binding between exons A and of tissue-specific expression of NCX1 alternatively spliced isoforms that as and neurons NCX1 transcripts containing exon A NCX1.1, NCX1.4, and the as or kidney transcripts containing exon B NCX1.3 and in membrane potential and intracellular of Na+ and to the of the that the of Ca2+ and Na+ on NCX1 activity found in exon but not in exon is an to exchanger in a that large ionic

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