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

The sodium-calcium exchanger (NCX) is a countertransporter of Na+ and Ca2+ across membranes of most cell types. 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 in the large cytoplasmic loop of the exchangers. Previous structure-function studies have determined key residues within these domains, particularly in the eXchanger Inhibitory Peptide region (XIP) and the Ca 2+ binding domains (CBD1/2), which have a direct impact on ionic regulation of the outward exchange currents, mediating Ca2+ flux. 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 Ca 2+ binding domain and may play a role in mediating these regulatory phenotypes. Alternatively spliced variants of NCX1 are often expressed in a tissue-specific manner and differ according to the level of development. Previous studies have demonstrated that specific combinations of the mutually-exclusive and cassette exons are associated with profound effects on ionic regulation in NCX1. While no alternative splicing has been identified for NCX2, the region corresponding to the alternative splice region may be a critical determinant of the unique ionic regulatory profile exhibited by this exchanger. This study focuses on examining the mechanisms by which specific regulatory domains modulate exchange activity in two isoforms, NCX1 and NCX2.;Chimaeric exchangers substituting the XIP region of NCX1.4 with the corresponding region from NCX2.1 caused an apparent loss of Na+-dependent inactivation, whereas a reduction in the extent of inactivation and a IS-fold increase in the rate of recovery from this inactivation were observed in the NCX1.3--XIP2 chimaera. Similarly, substitution of charged amino acids within the XIP region in NCX1.3 caused a slight increase in the rate of recovery, equivalent to that observed for NCX2.1. Thus non-conserved residues in the XIP region may be essential in maintaining the structural stability of the Na+-dependent inactive state of NCX1. Furthermore, the XIP region may interact with other regulatory domains of the protein, such as the mutually exclusive exon, thereby contributing to the structure-function relationship as well as the regulatory phenotype of each Na+-Ca 2+ exchanger variant and isoform. As NCX1 and NCX2 are expressed in a cell and tissue specific manner, examination of their ionic regulatory domains may help to elucidate the mechanisms by which NCX exchange activity is modulated in response to specific cellular environments, both physiologically and pathophysiologically, and may provide an explanation for the requirement of specifically regulated NCX variants in mammalian systems.;To investigate the role of the mutually exclusive exons in NCX1 (A and B), their analogous counterpart in NCX2, and their association with the XIP region, chimeric exchangers were constructed with single-exon and combination interchanges with the XIP regions from NCX1 and NCX2. Single and double amino acid substitutions were also done to determine the role of specific residues within these two regions. Chimaeric and mutant constructs were expressed in Xenopus oocytes and outward Na+-Ca2+ exchange activity was assessed using the giant, excised patch clamp technique. The Ca2+ dependance of Na+-dependent inactivation was analyzed, as well as rate of recovery from inactivation. Substitution of the region corresponding to the mutually exclusive exon from NCX2 with either exons A or B from NCX1 greatly reduced the rate and extent of Na +-dependent inactivation, independently of intracellular Ca 2+concentrations. However, replacement of both the region corresponding to the mutually exclusive exon and the XIP region from NCX2 with the analogous regions from NCX1.4 re-establishes a wild-type NCX2 profile. Moreover, this construct gains an additional phenotype of NCX1.4, that of alleviation of Na+-dependent inactivation at higher [Ca2+ i]. The first mutually exclusive exon is therefore critical in determining Na+ and Ca2+-dependent regulatory properties in NCXs and appears to interact with other regulatory regions of the cytoplasmic loop (i.e. the XIP region) to achieve distinctive regulatory phenotypes.