Ionic Regulatory Properties of Brain and Kidney Splice Variants of the NCX1 Na

Ion transport and regulation of Na 1 –Ca 2 1 exchange were examined for two alternatively spliced isoforms of the canine cardiac Na 1 –Ca 2 1 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 1 –Ca 2 1 exchange activity was assessed in giant, excised membrane patches from Xenopus laevis oocytes expressing the cloned exchangers, and the characteristics of Na 1 i (i.e., I 1 ) and Ca 2 1 i (i.e., I 2 ) 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 I 1 and I 2 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 a -chymotrypsin. It appears that expression of the B exon in NCX1.3 imparts a substantially more stable I 1 inactive state of the exchanger than does the A exon of NCX1.4. With respect to I 2 regulation, significant differences were also found between NCX1.3 and NCX1.4. While both exchangers were stimulated by low concentrations of regulatory Ca 2 1 i , NCX1.3 showed a prominent decrease at higher concentrations ( . 1 m M). This does not appear to be due solely to competition between Ca 2 1 i and Na 1 i at the transport site, as the Ca 2 1 i affinities of inward currents were nearly identical between the two exchangers. Furthermore, regulatory Ca 2 1 i had only modest effects on Na 1 i -dependent inactivation of NCX1.3, whereas I 1 inactivation of NCX1.4 could be completely eliminated by Ca 2 1 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 1 –Ca 2 1 exchange to fulfill tissue-specific requirements of Ca 2 1 homeostasis. key words: sodium–calcium exchange • regulation • alternative splicing I N T R O D U C T I O N Sodium–calcium exchangers constitute a family of ion counter transporters that play a prominent role in cellular Ca 2 1 homeostasis (Lederer et al., 1996; Philipson et al., 1996). These integral membrane-spanning proteins are generally thought to serve as Ca 2 1 efflux mechanisms that are driven by the Na 1 electrochemical gradient. Although present in many (and possibly all) tissues of the body, Na 1 –Ca 2 1 exchange has been most extensively characterized in cardiac and neuronal tissue. Three unique transporter gene products have been identified in mammals (NCX1, NCX2, and NCX3) and two have been shown to undergo alternative splicing (Quednau et al., 1997). Within the NCX1 subfamily, 12 alternatively spliced isoforms have been identified, whereas a single version of NCX2 has been cloned to date. The existence of three splice variants of NCX3 has been demonstrated (Quednau et al., 1997). The cardiac Na 1 –Ca 2 1 exchanger, NCX1.1, remains the most extensively characterized isoform with respect to structure, function, and regulation (Hryshko and Philipson, 1997; Lederer et al., 1996; Philipson et al., 1996). The consequences of alternative splicing of Na 1 –Ca 2 1 exchangers are largely unknown. One possibility is that expression of different exon combinations could provide a mechanism for directing Na 1 –Ca 2 1 exchangers to appropriate cellular locations. For example, in kidney, the exchanger may reside exclusively in the basolateral membrane of renal cells (Bindels et al., 1992; Reilly et al., 1993; Van Baal et al., 1996). Alternatively, or perhaps coincidentally, splice variants could exhibit altered functional and/ or regulatory properties appropriate for different cellular environments and Ca 2 1 homeostasis requirements. Support for this notion comes from our recent demonstration of significant functional differences in the regulatory phenotypes of alternatively spliced isoforms of the Na 1 – Ca 2 1 exchanger from Drosophila (Omelchenko et al., Address correspondence to Dr. Larry V. Hryshko, Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Tache Avenue, Winnipeg, Manitoba, Canada, R2H 2A6. Fax: 204233-6723; E-mail: [email protected] on F ebuary 0, 2013 jgp.rress.org D ow nladed fom Published October 25, 1999