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

Na^-Ca^<2>交换的功能及分子机制

基本信息

批准号：
09670043
负责人：
MATSUOKA Satoshi
金额：
$ 2.05万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670043/
关键词：
Na Ca Exchange XIP Heart macro patch 不活性化活性化 Na^+Ca^<2+>交換 Na^+イオン Ca^<2+>イオンパッチクランプ

项目摘要

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

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区域（2 19-238）被认为是自动调节域，因为合成肽对应于该区域抑制了交换活性。为了研究XIP区域的作用，制备了该区域的位置定向突变，并在爪蟾卵母细胞中表达。通过测量Na^+ - Ca^<2+>交换电流（I_ <a-na-Ca>）与内而外的巨型膜贴片技术的测量，这些突变体的功能是研究了。在赖氨酸229的突变体中消除了Na^+ - 依赖性灭活。在苯丙氨酸223，酪氨酸224和赖氨酸225的突变体中，抑制了细胞质Ca^<2+>的调节。有人提出，XIP区域是对Na^+ - 依赖性失活和Ca^<2+>调节的负责任区域。然后调查Na^+-ca^<2+>交换和Na^+-k^+泵的相互作用。 i_ <na-ca>。 Na^+浓度变化的估计表明，在细胞内空间中，Na^+-ca^<2+>交换和Na^+-k^+泵的活性相互依赖于Na^+，约为总细胞体积的14％。为了进一步研究Sarcolemmal Na^+-Ca^<2+>交换的功能，我们改进了内而外的贴片技术，以获得大型膜贴片“宏观贴片”。宏观斑块中的外部I_ <a-ca>非常适合完整的心室细胞，显示了Na^+ - 依赖性失活，而Ca^<2+>以与心脏“ blebs”巨大斑块中的I_ <a-ca>相似的方式激活。然而，I_ <a-na-ca>的逆转电势被分离为XIP敏感电流，偏离了3 Na^+：1 Ca^<2+>交换并建议4 Na^+：1 Ca^<2+>交换。我们将继续进行这一系列研究，以进一步了解XIP区域的分子机制和交换的分子机制。较少的