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Analysis of the polyamine-binding sites in inwardly rectifying K^+ channels.

内向整流K^通道中多胺结合位点的分析。

基本信息

批准号：
08457636
负责人：
YAMADA Mitsuhiko
金额：
$ 4.61万
依托单位：
OSAKA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

We analyzed the Mg^<2+>/polyamine sensitivity of the IRK2 (Kir2.2) channel heterologously expressed in HEK293T cells. IRK2 (Kir2.2) is believed to be the subunit of the cardiac inwardly-rectifying K^+ channel, I_<K1>. The IRK2 channels showed strong inward rectification in the cell-attached configuration of the patch-clamp method. However, they gradually lost the inward rectification in the inside-out patch membranes whose intracellular side was continuously perfused with Mg^<2+>-free solution. Mg^<2+> and spermine spplied to the internal side of the patch membrane suppressed the outward channel currents at the potential 40 mV positive to the potassium equilibration potential (E_K) with the IC_<50> of 10muM and 3 nM,respectively. Because millimolar and submillimolar Mg^<2+> and polyamine are known to exist in cytosol of most cell types, the Mg^<2+>/Polyamine block is likely to underlie the inward rectification of IRK2 channels in intact cells. Mg^<2+> caused the instantaneous rectification by inducing the subconducting level of the channel, while spermine time-dependently suppressed the open probability of the channel at potentials positive to E_K. When Mg^<2+> was further applied to the channel in the presence of spermine, the inward rectification of the channel was paradoxically attenuated compared with that in the presence of spermine alone. This phenomenon was well explained by the model in which Mg^<2+> and spermine compete with each other through binding the same binding site (s) on the channel. These data (1) indicate that the physiological inward rectification of the cardiac I_<K1> channel is determined through such competitive binding of intracellular Mg^<2+>/polyamine to the channel and (2) raise a possibility that artificial polyvalent cations introduced into cardiocytes might be utilized to pharmacologically modulate the strong inward rectification of the channels induced by intracellular polyamines.

我们分析了HEK293T细胞中异源表达的IRK2(Kir2.2)通道的Mg 2+ /多胺敏感性。 IRK2 (Kir2.2)被认为是心脏内向整流K^+通道I_<K1>的亚基。 IRK2 通道在膜片钳方法的细胞贴壁配置中显示出强烈的内向整流。然而，它们逐渐失去了由内而外的补片膜的向内整流，其细胞内侧持续灌注不含Mg 2+ 的溶液。施加到膜片内侧的Mg^2+和精胺在钾平衡电位(E_K)正40mV的电位下抑制向外通道电流，IC_<50>分别为10μM和3nM。因为已知毫摩尔和亚毫摩尔Mg 2+ 和多胺存在于大多数细胞类型的细胞质中，所以Mg 2+ /多胺阻断可能是完整细胞中IRK2通道的内向整流的基础。 Mg^<2+>通过诱导通道的亚导水平引起瞬时整流，而精胺时间依赖性地抑制通道在E_K正电势下的打开概率。当在精胺存在下将Mg 2+ 进一步施加到通道时，与单独存在精胺的情况相比，通道的向内整流相反地减弱。该现象可以通过Mg 2+ 和精胺通过结合通道上相同的结合位点而相互竞争的模型得到很好的解释。这些数据(1)表明心脏I_<K1>通道的生理向内整流是通过细胞内Mg^2+/多胺与通道的这种竞争性结合来确定的，并且(2)提出了引入心肌细胞的人工多价阳离子可用于药理调节由细胞内多胺诱导的通道的强向内整流的可能性。