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.

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