Function and molecular mechanism of Na^+-Ca^<2+> exchange

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We studied molecular mechanism and function of cardiac Na^+-Ca^<2+> exchange. The XIP region (2 19-238) of Na^+-Ca^<2+> exchange (NCX1) has been postulated to be an autoregulatory domain, because a synthetic peptide corresponding to the region inhibits the exchange activity. To investigate the role of XIP region, site-directed mutations of the region were prepared and expressed in Xenopus oocytes. The function of these mutants was studied by the measurement of Na^+-Ca^<2+> exchange current (I_<Na-Ca>) with the inside-out giant membrane patch technique. Na^+-dependent inactivation was eliminated in mutants at lysin 229. In mutants at phenylalanine 223, tyrosine 224, and lysine 225, Na^+-dependent inactivation was conserved, but regulation by cytoplasmic Ca^<2+> was suppressed. It was suggested that the XIP region is a responsible region to Na^+-dependent inactivation and Ca^<2+> regulation.Then, to investigate interaction between Na^+-Ca^<2+> exchange and Na^+-K^+ pump in ventricular ce … More lls, amount of Na^+ which was excluded by Na^+-K^+ pump was estimated by time integral of Na^+-K^+ pump current and measurement of reversal potential of I_<Na-Ca>. The estimation of Na^+ concentration change suggested that activities of Na^+-Ca^<2+> exchange and Na^+-K^+ pump mutually depend on Na^+ in an intracellular space of approximately 14 % of total cell volume. To further study the function of sarcolemmal Na^+-Ca^<2+> exchange, we improved the inside-out patch technique to obtain a large membrane patch, "macro patch". The outward I_<Na-Ca> in the macro patch, which was excised from intact ventricular cells, showed Na^+-dependent inactivation and Ca^<2+> activation in a similar manner to I_<Na-Ca> in the giant patch from cardiac "blebs". However, reversal potentials of I_<Na-Ca>, which was isolated as XIP-sensitive current, deviated from 3 Na^+ : 1 Ca^<2+> exchange and suggested 4 Na^+ : 1 Ca^<2+> exchange.We will continue this series of studies to get further insight into the molecular mechanism of XIP region and stoichiometry of the exchanger. Less