Kuniyasu Akihiko, Kaneko Kazuyoshi, Kawahara Kohichi, Nakayama Hitoshi
Department of Molecular Cell Function, Graduate School of Medical and Pharmaceutical Sciences, Kumamoto University, 5-1 Ohe-Honmachi, 862-0973 Kumamoto, Japan.
FEBS Lett. 2003 Sep 25;552(2-3):259-63. doi: 10.1016/s0014-5793(03)00936-0.
Cardiac ATP-sensitive K(+) (K(ATP)) channels are proposed to contribute to cardio-protection and ischemic preconditioning. Although mRNAs for all subunits of K(ATP) channels (Kir6.0 and sulfonylurea receptors SURs) were detected in hearts, subcellular localization of their proteins and the subunit combination are not well elucidated. We address these questions in rat hearts, using anti-peptide antibodies raised against each subunit. By immunoblot analysis, all of the subunits were detected in microsomal fractions including sarcolemmal membranes, while they were not detected in mitochondrial fractions at all. Immunoprecipitation and sucrose gradient sedimentation of the digitonin-solubilized microsomes indicated that Kir6.2 exclusively assembled with SUR2A. The molecular mass of the Kir6.2-SUR2A complex estimated by sucrose sedimentation was 1150 kDa, significantly larger than the calculated value for (Kir6.2)(4)-(SUR2A)(4), suggesting a potential formation of micellar complex with digitonin but no indication of hybrid channel formation under the conditions. These findings provide additional information on the structural and functional relationships of cardiac K(ATP) channel proteins involving subcellular localization and roles for cardioprotection and ischemic preconditioning.
心脏ATP敏感性钾（K(ATP)）通道被认为有助于心脏保护和缺血预处理。尽管在心脏中检测到了K(ATP)通道所有亚基（Kir6.0和磺脲类受体SURs）的mRNA，但其蛋白质的亚细胞定位和亚基组合尚未完全阐明。我们利用针对每个亚基产生的抗肽抗体，在大鼠心脏中解决了这些问题。通过免疫印迹分析，在包括肌膜的微粒体组分中检测到了所有亚基，而在线粒体组分中根本未检测到。对洋地黄皂苷增溶的微粒体进行免疫沉淀和蔗糖梯度沉降表明，Kir6.2仅与SUR2A组装。通过蔗糖沉降估计的Kir6.2 - SUR2A复合物的分子量为1150 kDa，明显大于(Kir6.2)(4)-(SUR2A)(4)的计算值，这表明可能形成了与洋地黄皂苷的胶束复合物，但在此条件下没有杂交通道形成的迹象。这些发现为心脏K(ATP)通道蛋白的结构和功能关系提供了额外信息，涉及亚细胞定位以及对心脏保护和缺血预处理的作用。
