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

我们分析了HEK 293 T细胞中异源表达的IRK 2（Kir2.2）通道的Mg^<2+>/多胺敏感性。IRK 2（Kir2.2）被认为是心脏内向整流钾通道（I）的亚基<K1>。IRK 2通道在膜片钳方法的细胞贴附构型中显示出强烈的向内整流。然而，在细胞内侧持续灌注无Mg^&lt;2+&gt;溶液的由内而外的膜片膜中，它们逐渐失去了向内的整流作用。在钾离子平衡电位（E_K）正向40 mV时，Mg^（2+）和精胺分别抑制膜片膜的外向通道电流，抑制浓度<50>分别为10 μ M和3 nM。因为已知在大多数细胞类型的胞质溶胶中存在毫摩尔和亚毫摩尔的Mg^2+和多胺，所以Mg^2+/多胺阻滞可能是完整细胞中IRK 2通道向内整流的基础。Mg^&lt;2+&gt;通过诱导通道的亚导能级而引起瞬时整流，精胺则随时间依赖性地抑制通道在E_K正电位下的开放概率。当在精胺存在的情况下进一步将Mg^&lt;2+&gt;加到通道上时，与单独存在精胺时相比，通道的内向整流反而减弱。这一现象可以用Mg^&lt;2+&gt;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来解释。这些数据（1）表明心脏I_通道的生理性内向整流<K1>是通过细胞内Mg^&lt;2+&gt;/多胺与通道的竞争性结合来决定的;（2）提出了一种可能性，即引入心肌细胞的人工多价阳离子可能被用来间接调节细胞内多胺诱导的通道的强内向整流。

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.