Bednarczyk Piotr, Dołowy Krzysztof, Szewczyk Adam
Laboratory of Intracellular Ion Channels, Nencki Institute of Experimental Biology, Warsaw, Poland.
J Bioenerg Biomembr. 2008 Aug;40(4):325-35. doi: 10.1007/s10863-008-9153-y. Epub 2008 Jul 31.
The ATP-regulated potassium channel is present in the inner membrane of heart mitochondria. In this study, the activity of a single channel was measured after reconstituting the myocardium inner mitochondrial membrane into a planar lipid bilayer. We provide direct evidence of vectorial pH regulation of mitoK(ATP) channels. When the matrix side was alkalized, this changed the channel conductance, the open probability, and the mean open and closed dwell time distributions. The conductance of the mitoK(ATP) channel increased from about 110 +/- 8 to 145 +/- 5 pS upon changing the pH from 7.2 to 8.2. This effect was reversed by reverting the pH to the neutral value. The mitoK(ATP) channel activity was not altered by alkalization of the cytosolic side of the planar lipid bilayer. We also observed that acidification from pH 7.2 to 6.2, in either the matrix or cytosolic compartments, decreased the open probability of the channel. This effect was reversed by perfusion with a pH 7.2 medium. Additionally, our results suggest that the mitoK(ATP) channel is regulated by multiple phosphorylation events. The channel activity was inhibited by an ATP/Mg(2+) complex, but not by ATP alone, nor by a non-hydrolysable ATP analog, e.g. AMP-PNP/Mg(2+). The mitoK(ATP) channel "run-down" was reversed by incubating with the ATP/Mg(2+) complex on both sides of the planar lipid bilayer. We conclude that both pH and ATP play an important regulatory role for the cardiac mitoK(ATP) channel with respect to the phenomenon of ischemia-reperfusion.
ATP调节的钾通道存在于心脏线粒体的内膜中。在本研究中，将心肌线粒体内膜重构到平面脂质双分子层后，测量了单通道的活性。我们提供了mitoK(ATP)通道矢量pH调节的直接证据。当基质侧碱化时，这会改变通道电导、开放概率以及平均开放和关闭驻留时间分布。将pH从7.2变为8.2时，mitoK(ATP)通道的电导从约110±8 pS增加到145±5 pS。将pH恢复到中性值可逆转此效应。平面脂质双分子层胞质侧的碱化不会改变mitoK(ATP)通道的活性。我们还观察到，无论是在基质还是胞质区室中，将pH从7.2酸化至6.2都会降低通道的开放概率。用pH 7.2的培养基灌注可逆转此效应。此外，我们的结果表明mitoK(ATP)通道受多种磷酸化事件调节。通道活性受到ATP/Mg(2+)复合物的抑制，但不受单独的ATP或不可水解的ATP类似物（如AMP-PNP/Mg(2+)）的抑制。通过在平面脂质双分子层两侧与ATP/Mg(2+)复合物孵育，可逆转mitoK(ATP)通道的“衰退”。我们得出结论，就缺血再灌注现象而言，pH和ATP对心脏mitoK(ATP)通道均起着重要的调节作用。
