| Research Abstract |
We have recently developed the first selective inhibitor of Na^+/Ca^<2+> exchanger (NCX), KB-R7943, that has a therapeutic potential for clinical use. In this study, we have analyzed the topology of the cardiac NCX molecule as well as interactions of NCX with the transport substrate Ca^<2+>, KB-R7943, a nonselective inhibitor Ni^<2+>, and an activator Li^+. Based on detailed analysis by cysteine scanning and site-directed mutagenesis, we found that the highly conserved α-1 and α-2 repeat regions in NCX is involved in interactions with these inhibitors and ligands, suggesting that these regions participate in the formation of ion transport pathway in NCX.This study have also provided evidence that NCX consists of 9 transmembrane segments, with regions containing α-1 and α-2 repeats forming re-entrant membrane loops originating from the opposite sides of the membrane. On the other hand, Na^+/H^+ exchanger (NHE) is an excellent target for the study of cellular signal transduction, because it is extensively regulated by a variety of extracellular signals. We have recently analyzed interactions of calmodulin (CaM) and the calcineurin-homologous protein CHP with various NHE isoforms, and found that CaM is involved in Ca^<2+>-induced activation of NHE1, whereas CHP is an essential cofactor to support physiological activity of multiple NHE isoforms. We also analyzed membrane topology of NHE1, and found that NHE1 comprises 12 transmembrane segments with N- and C- termini in thcytosol.
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