Structure-function studies of the sodium-calcium exchanger isoforms, NCX1 and NCX2

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de Moissac, Danielle
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The sodium-calcium exchanger (NCX) is a countertransporter of Na+ and Ca2+ across most cell membranes. It has been identified as an essential component of Ca2+ homeostasis in physiological and disease conditions in both cardiovascular and neurological settings. The exchanger not only transports Na+ and Ca2+, but is also regulated by these ions. Although ionic regulatory profiles differ between NCX isoforms, similar regulatory domains have been identified. Previous structure-function studies have determined key residues within these domains, particularly in the eXchanger Inhibitory Peptide region (XIP) and the Ca2+ binding domains (CBD1/2), which have a direct impact on ionic regulation of the outward exchange currents. Recent structural studies of the Ca2+ binding domains of NCX1 suggest a mechanism by which Ca2+ binding would not only be essential for activation of current but may also influence Na+-dependent inactivation. The alternative splice region is located within the Ca2+ binding domain and may play a role in mediating these regulatory phenotypes. Previous studies have demonstrated that specific combinations of the mutually-exclusive and cassette exons are associated with profound effects on ionic regulation in NCX1. This study focuses on examining the mechanisms by which the alternative splice region, in combination with specific regulatory domains, modulates exchange activity in two isoforms, NCX1 and NCX2. Chimaeric and mutant constructs in the alternative splice region were expressed in Xenopus oocytes and outward Na+-Ca2+ exchange activity was assessed using the giant, excised patch clamp technique. Substitution of the region corresponding to the mutually exclusive exon in either exchanger greatly reduced the extent of Na+-dependent inactivation, independently of intracellular Ca2+ concentrations. However, replacement of both the region corresponding to the mutually exclusive exon A and the XIP region reestablishes a wild-type profile in NCX2. The first mutually exclusive exon is therefore critical in determining Na+ and Ca2+-dependent regulatory properties. Furthermore, non-conserved residues within the XIP region may be essential in maintaining the structural stability of the Na+-dependent inactive state of NCX1, and by interacting with the mutually exclusive exon, may contribute to the structure-function relationship and the distinct regulatory phenotype of each Na+-Ca2+ exchanger variant and isoform.
structure-function, NCX