Structure-function studies of Drosophila NaS+-Ca2S+ exchanger proteins

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Dyck, Christopher James
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The cardiac Na$\sp{+}$-Ca$\sp{2+}$ exchanger, NCX1.1, is the most extensively studied member of this multi-gene family of ion counter-transporters. It is unknown whether the structure-function relationships of NCX1.1 apply to any other exchanger. To examine this possibility, structure-function studies targeting these regulatory domains were performed with a Drosophila Na$\sp{+}$-Ca$\sp{2+}$ exchanger, CALX1.1. Although NCX1.1. and CALX1.1. are both regulated by Na$\sp{+}\sb{i}$ and Ca$\sp{2+}\sb{i}$, an opposite response to regulatory Ca$\sp{2+}\sb{i}$ is observed between these two exchangers. That is, whereas Ca$\sp{2+}\sb{i}$ activates NCX1.1 exchange activity, CALX1.1 is inhibited. Wild-type and mutant exchangers were expressed in Xenopus laevis oocytes and characterized electrophysiologically using the giant excised patch clamp technique. The second major aspects of this thesis involves the study of two alternatively spliced isoforms of the Na$\sp{+}$-Ca$\sp{2+}$ exchanger identified in Drosophila melanogaster. These isoforms, called CALX1.1 and CALX1.2, differ by five amino acids in a region near the C-terminus of the large cytoplasmic loop. To determine whether the sequence difference observed for Drosophila isoforms influenced intrinsic regulatory properties, we examined Na$\sp{+}\sb{i}$- and Ca$\sp{2+}\sb{i}$-dependent regulation for both exchangers using the giant excised membrane patch clamp technique. (Abstract shortened by UMI.)