Ionic Regulatory Properties of Brain and Kidney Splice Variants of the Ncx1 Na⁺–Ca²⁺ Exchanger

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.