The sarcolemmal Na+-Ca 2+ exchanger is regulated by intracellular Ca ~+ at a high affinity Ca 2+ binding site separate from the Ca ~+ transport site. Previous data have suggested that the Ca 2+ regulatory site is located on the large intracellular loop of the Na +-Ca 2+ exchange protein, and we have identified a highaffinity 4SCa2+ binding domain on this loop (Levitsky, D. O., D. A. Nicoll, and K. D. Philipson. 1994. Journal of Biological Chemistry. 269:22847-22852). We now use electrophysiological and mutational analyses to further define the Ca ~+ regulatory site. Wild-type and mutant exchangers were expressed in Xenopus oocytes, and the exchange current was measured using the inside-out giant membrane patch technique. Ca ~+ regulation was measured as the stimulation of reverse Na+-Ca ~+ exchange (intracellular Na + exchanging for extracellular Ca ~+) by intracellular Ca ~+. Single-site mutations within two acidic clusters of the Ca 2+ binding domain lowered the apparent Ca ~+ affinity at the regulatory site from 0.4 to 1.1-1.8 ~.M. Mutations had parallel effects on the affinity of the exchanger loop for 45Ca~+ binding (Levitsky et al., 1994) and for functional Ca 2+ regulation. We conclude that we have identified the functionally important Ca ~ § binding domain. All mutant exchangers with decreased apparent affinities at the regulatory Ca 2+ binding site also have a complex pattern of altered kinetic properties. The outward current of the wild-type Na+-Ca 2 § exchanger declines with a half time (th) of 10. Ca 2+ removal, whereas the exchange currents of several mutants decline with th values of 0.7-4.3 s. Likewise, Ca 2+ regulation mutants respond more rapidly to Ca 2+ application.Study of Ca 2+ regulation has previously been possible only with the exchanger operating in the reverse mode as the regulatory Ca 2+ and the transported Ca 2+ are then on opposite sides of the membrane. The use of exchange mutants with low affinity for Ca 2+ at regulatory sites also allows demonstration of secondary Ca z+ regulation with the exchanger in the forward or Ca 2+ el:flux mode. In addition, we find that the affinity of wild-type and mutant Na+-Ca 2+ exchangers for intracellular Na + decreases at low regulatory Ca 2+. This suggests that Ca z+ regulation modifies transport properties and does not only control the fraction of exchangers in an active state.

We have examined the role of conserved regions and acidic or basic residues located in the putative transmembrane segments of the cardiac sarcolemmal Na ؉ -Ca 2؉ exchanger by site-directed mutagenesis. The ␣-1 and ␣-2 repeats are transmembrane regions of internal similarity, which are highly conserved among Na

Ion transport and regulation of Na+–Ca2+ exchange were examined for two alternatively spliced isoforms of the canine cardiac Na+–Ca2+ exchanger, NCX1.1, to assess the role(s) of the mutually exclusive A and B exons. The exchangers examined, NCX1.3 and NCX1.4, are commonly referred to as the kidney and brain splice variants and differ only in the expression of the BD or AD exons, respectively. Outward Na+–Ca2+ exchange activity was assessed in giant, excised membrane patches from Xenopus laevis oocytes expressing the cloned exchangers, and the characteristics of Na+ i- (i.e., I1) and Ca2+ i- (i.e., I2) dependent regulation of exchange currents were examined using a variety of experimental protocols. No remarkable differences were observed in the current–voltage relationships of NCX1.3 and NCX1.4, whereas these isoforms differed appreciably in terms of their I1 and I2 regulatory properties. Sodium-dependent inactivation of NCX1.3 was considerably more pronounced than that of NCX1.4 and resulted in nearly complete inhibition of steady state currents. This novel feature could be abolished by proteolysis with α-chymotrypsin. It appears that expression of the B exon in NCX1.3 imparts a substantially more stable I1 inactive state of the exchanger than does the A exon of NCX1.4. With respect to I2 regulation, significant differences were also found between NCX1.3 and NCX1.4. While both exchangers were stimulated by low concentrations of regulatory Ca2+ i, NCX1.3 showed a prominent decrease at higher concentrations (>1 μM). This does not appear to be due solely to competition between Ca2+ i and Na+ i at the transport site, as the Ca2+ i affinities of inward currents were nearly identical between the two exchangers. Furthermore, regulatory Ca2+ i had only modest effects on Na+ i-dependent inactivation of NCX1.3, whereas I1 inactivation of NCX1.4 could be completely eliminated by Ca2+ i. Our results establish an important role for the mutually exclusive A and B exons of NCX1 in modulating the characteristics of ionic regulation and provide insight into how alternative splicing tailors the regulatory properties of Na+–Ca2+ exchange to fulfill tissue-specific requirements of Ca2+ homeostasis.

The Na+/Ca2+ exchanger plays a prominent role in regulating intracellular Ca2+ levels in cardiac myocytes and can serve as both a Ca2+ influx and efflux pathway. A novel inhibitor, KB-R7943, has been reported to selectively inhibit the reverse mode (i.e., Ca2+ entry) of Na+/Ca2+ exchange transport, although many aspects of its inhibitory properties remain controversial. We evaluated the inhibitory effects of KB-R7943 on Na+/Ca2+ exchange currents using the giant excised patch-clamp technique. Membrane patches were obtained from Xenopus laevis oocytes expressing the cloned cardiac Na+/Ca2+ exchanger NCX1.1, and outward, inward, and combined inward-outward currents were studied. KB-R7943 preferentially inhibited outward (i.e., reverse) Na+/Ca2+ exchange currents. The inhibitory mechanism consists of direct effects on the transport machinery of the exchanger, with additional influences on ionic regulatory properties. Competitive interactions between KB-R7943 and the transported ions were not observed. The antiarrhythmic effects of KB-R7943 were then evaluated in an ischemia-reperfusion model of cardiac injury in Langendorff-perfused whole rabbit hearts using electrocardiography and measurements of left ventricular pressure. When 3 microM KB-R7943 was applied for 10 min before a 30-min global ischemic period, ventricular arrhythmias (tachycardia and fibrillation) associated with both ischemia and reperfusion were almost completely suppressed. The observed electrophysiological profile of KB-R7943 and its protective effects on ischemia-reperfusion-induced ventricular arrhythmias support the notion of a prominent role of Ca2+ entry via reverse Na+/Ca2+ exchange in this process.

No abstract

exchangers exhibit a common Ca 2ϩ -dependent regulatory mechanism, whereby their activity requires the presence of low concentrations of Ca 2ϩ on their intracellular surface, and their activity is augmented in parallel with elevated intracellular Ca 2ϩ levels (3). This important regulatory property may permit the timely coupling of exchange function to alterations in intracellular Ca 2ϩ concentrations to meet the continuous needs for overall Ca 2ϩ balance. The general similarities of exchange function and regulatory properties within the large NCX protein family are ascribed to their conserved structural arrangements: nine predicted transmembrane (TM) segments form the ion translocation pathway and a large loop of ϳ500 amino acid residues splits TM helix-5 and -6 on the intracellular side of the molecule (4). Ca 2ϩ -dependent regulation is attributed exclusively to Ca 2ϩ interactions on the intracellular loop (5). A pair of Ca 2ϩ binding domains (CBD1 and -2), called CALX-␤ motifs, has been identified (6). Sequence analysis revealed that CBD1 has conserved Ca 2ϩ binding sites throughout the NCX family, whereas greater sequence diversity and/or Ca 2ϩ binding capabilities occurs in CBD2 (7,27). Given that CBD1 exhibits a higher Ca 2ϩ affinity than CBD2 (8), it has been suggested that CBD1 acts as the primary sensor in the pair of CBDs. Mutations of carboxylate residues at CBD1 result in a pronounced reduction of the affinity for functional Ca 2ϩ regulation (9). The Ca 2ϩ -bound structures of CBD1 of NCX1 have recently been determined by NMR, and more recently by x-ray crystallography (8,11

No abstract

The Na+-Ca 2+ exchanger from Drosophila was expressed in Xenopus oocytes and characterized electrophysiologically using the giant excised patch technique. This protein, termed Calx, shares 49% amino acid identity to the canine cardiac Na+-Ca 2+ exchanger, NCX1. Calx exhibits properties similar to previously characterized Na+-Ca z+ exchangers including intracellular Na + affinities, current-voltage relationships, and sensitivity to the peptide inhibitor, XIP. However, the Drosophila Na+-Ca 2+ exchanger shows a completely opposite response to cytoplasmic Ca 2+. Previously cloned Na+-Ca 2+ exchangers (NCX1 and NCX2) are stimulated by cytoplasmic Ca 2+ in the micromolar range (0.1-10 jxM). This stimulation of exchange current is mediated by occupancy of a regulatory Ca 2+ binding site separate from the Ca 2+ transport site. In contrast, Calx is inhibited by cytoplasmic Ca 2+ over this same concentration range. The inhibition of exchange current is evident for both forward and reverse modes of transport. The characteristics of the inhibition are consistent with the binding of Ca 2+ at a regulatory site distinct from the transport site. These data provide a rational basis for subsequent structure-function studies targeting the intracellular Ca z+ regulatory mechanism. Key words: sodium-calcium exchange 9 calcium regulation 9 Drosophila melanogaster