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

我们研究了心脏Na^+-Ca^<2+>交换的分子机制和功能。Na^+-Ca^<2+>交换（NCX1）的XIP区（2 19-238）被认为是一个自调节结构域，因为该区域对应的合成肽抑制了交换活性。为了研究XIP区域的作用，制备了该区域的位点定向突变并在爪蟾卵母细胞中表达。用内外巨膜贴片技术测定Na^+-Ca^<2+>交换电流（I_<Na-Ca>），研究这些突变体的功能。Na^+依赖性失活在lysin 229突变体中被消除。在苯丙氨酸223、酪氨酸224和赖氨酸225突变体中，Na^+依赖性失活是保守的，但细胞质Ca^<2+>的调节被抑制。结果表明，XIP区是Na^+依赖性失活和Ca^<2+>调控的关键区域。然后，为了研究Na^+-Ca^<2+>交换与Na^+-K^+泵之间的相互作用，通过Na^+-K^+泵电流的时间积分和I_<Na-Ca>的逆转电位测量来估计Na^+-K^+泵排除的Na^+量。对Na^+浓度变化的估计表明，Na^+-Ca^<2+>交换和Na^+-K^+泵的活性相互依赖于约占细胞总体积14%的细胞内空间的Na^+。为了进一步研究肌层Na^+-Ca^<2+>交换的功能，我们改进了由内向外的贴片技术，获得了一个大的膜贴片，即“宏贴片”。从完整心室细胞中切除的大膜片中，外展的I_<Na-Ca>显示Na^+依赖性失活和Ca^<2+>激活，其方式与心脏“泡”巨大膜片中的I_<Na-Ca>相似。然而，I_<Na-Ca>的反转电位偏离了3na ^+: 1ca ^<2+>的交换，提示4na ^+: 1ca ^<2+>的交换。我们将继续这一系列的研究，以进一步了解XIP区域的分子机制和交换剂的化学计量学。少

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