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

Na^-Ca^<2>交换的功能及分子机制

• 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

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）被认为是一个自身调节结构域，因为与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，制备该区域的定点突变并在爪蟾卵母细胞中表达。用由内而外的巨膜膜片技术测定Na ^+-Ca ^&lt;2 +&gt;交换电流（I_），研究了这些突变体的功能。<Na-Ca>在溶素229的突变体中，Na ^+依赖性失活被消除。在苯丙氨酸223、酪氨酸224和赖氨酸225的突变体中，Na ^+依赖的失活是保守的，但细胞质Ca ^&lt;2 +&gt;的调节受到抑制。结果表明，XIP区是Na ^+依赖性失活和Ca ^&lt;2 +&gt;调节的重要区域，因此，我们在心室肌细胞中研究了Na ^+-Ca ^&lt;2 +&gt;交换和Na ^+-K ^+泵的相互作用，并探讨了它们之间的相互关系。 ...更多信息 通过对Na ^+-K ^+泵电流的时间积分和I_2反转电位的测量，估算了Na ^+-K ^+泵排出的Na ^+量。<Na-Ca>对Na ^+浓度变化的估计表明，Na ^+-Ca ^&lt;2 +&gt;交换和Na ^+-K ^+泵的活性相互依赖于约占总细胞体积14%的细胞内空间中的Na ^+。为了进一步研究肌膜Na ^+-Ca ^&lt;2 +&gt;交换的功能，我们改进了由内而外的膜片技术，得到了一个大的膜片，称为"宏膜片"。从完整心室细胞中切取的大斑片的外向I_2表现出Na ^+依赖的失活和Ca ^2+激活，其方式与心脏"大泡"中的巨斑片的I_2相似。<Na-Ca><Na-Ca>然而，作为XIP敏感电流的I_2的反转电位偏离了3 Na ^+：1 Ca ^&lt;2 +&gt;交换，而是4 Na ^+：1 Ca ^&lt;2 +&gt;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。<Na-Ca>少

项目成果

Matsuoka, S.et al.: "Regulation of the cardiac Na^+-Ca^<2+> exchanger by the endogenous XIP region." Journal of General Physiology. 109. 273-286 (1997)

Matsuoka, S.et al.：“内源性 XIP 区域对心脏 Na^-Ca^2 交换器的调节”。

Fujioka, Y.et al.: "Interaction of the Na^+-K^+ pump and Na^+-Ca^<2+> exchange via [Na^+]_i in a restricted space of guinea-pig ventricular cells." Journal of Physiology. 509. 457-470 (1998)

Fujioka, Y.et al.：“在豚鼠心室细胞的有限空间中，Na^ -K^ 泵和 Na^ -Ca^<2 > 通过 [Na^ ]_i 进行交换的相互作用。”

Matsuoka,S.: "Reversal potential of the Na^+-Ca^<2+> exchange suggests 4:1 stoichiometry in “macro"patch from guinea-pig ventricular cells." Biophysical Journal. 76. A252 (1999)

Matsuoka, S.：“Na^+-Ca^<2+> 交换的逆转潜力表明豚鼠心室细胞“宏观”补片中的化学计量为 4:1。”《生物物理学杂志》。76. A252 (1999)

Fujioka, Y.: "Interaction of the Na^+-K^+ pump and Na^+-Ca^<2+> exchange via[Na^+]_i in a restricted space of guinea-pig ventricular cells." Journal of Physiology. 509. 457-470 (1998)

Fujioka, Y.：“Na^ -K^ 泵和 Na^ -Ca^<2 > 通过 [Na^ ]_i 在豚鼠心室细胞的有限空间中交换的相互作用。”

Matsuoka, S.: "Reversal potential of the Na^+-Ca^<2+> exchange suggests 4 : 1 stoichiometry in “macro" patch from guinea-pig ventricular cells." Biophysical Journal. 76. A252- (1999)

Matsuoka, S.：“Na^+-Ca^<2+> 交换的逆转潜力表明豚鼠心室细胞“宏观”斑块中的化学计量为 4:1。”《生物物理学杂志》。76. A252- (1999)