VOLATILE ANESTHETIC ACTION ON CARDIAC K CHANNELS

描述(由申请人提供)： 挥发性麻醉剂(VAS)已经使用了一个多世纪，但其强大的心脏保护作用直到最近才被证实。然而，麻醉剂对心脏离子通道作用的分子机制尚不清楚。VAS抗心律失常作用的一个可能靶点是缓慢延迟整流钾(IKS)通道，该通道是哺乳动物心脏主要的复极电流的基础。VAS对IKS通道的抑制将导致动作电位的延长，作用类似于III类抗心律失常药物。这项建议的总体目标是阐明VA对IKs通道的作用机制的分子模型。拟议的实验将研究VAS对iKs下面的两个亚基成分的影响：一个由KCNQ1基因编码的孔形成区域和一个辅助的调节p亚基mink。初步结果表明，异氟烷对KCNQ1单独和与水貂亚单位联合作用具有不同的作用。一种挥发性麻醉剂与通道蛋白相互作用的新模式被认为是一种新的模式，其中辅助调节亚基调节麻醉作用。总体假设是，水貂亚基减弱了VA对iKs的影响。膜片钳技术将被用来监测在哺乳动物心肌细胞系中瞬时表达的KCNQ1和水貂的功能变化。过量表达水貂的天然心肌细胞中IKS功能的变化也将被监测。此外，朗宁多夫豚鼠离体心模型将被用来确定iKs在麻醉诱导的心脏保护中的作用。需要检验的假设是： 1.水貂亚基阻碍了KCNQ1上异氟烷相互作用位点的可及性。 2.蛋白激酶A对IKs的磷酸化可抵消异氟醚的抑制作用。 3.KCNQ1+MMK通道复合体对不同亚基的麻醉效应不是KCNQ1+MMK通道所特有的。 4.在异氟醚诱导的心脏保护中，KCNQ1和水貂亚基的表达存在差异。 综上所述，这项提议将表征辅助调节亚单位水貂在易失性麻醉作用中的作用。这种麻醉剂与离子通道相互作用的新方法将在已知发生通道亚单位上调和下调的病变心肌中具有功能意义。

DESCRIPTION (provided by applicant): Volatile anesthetics (VAs) have been in use for over a century, but their potent cardioprotective effects have only recently been established. However, the molecular mechanisms of anesthetic action on cardiac ion channels are unknown. A likely target for the antiarrhythmogenic action of the VAs is the slow delayed-rectifier potassium (IKs) channel that underlies a major repolarizing current in the mammalian heart. Inhibition of the IKs channel by the VAs will result in the prolongation of the action potential, similar in action to the Class III antiarrhythmic agents. The overall goal of this proposal is to elucidate a molecular model for the mechanism of VA action on the IKs channel. The proposed experiments will investigate the effects of VAs on the two-subunit components underlying IKs: a pore-forming region encoded by the gene KCNQ1 and an auxiliary regulatory p-subunit, minK. Preliminary results show that isoflurane exerts differential effects on KCNQ1 alone and in combination with the minK subunit. A novel mode of volatile anesthetic interaction with a channel protein where an auxiliary regulatory subunit modulates anesthetic action is postulated. The overall hypothesis is that the minK subunit diminishes the effect of VA on IKs. The patch clamp technique will be used to monitor functional changes in KCNQ1 and minK transiently expressed in a mammalian cardiac cell line. Changes in IKs function in native myocytes overexpressing minK will also be monitored. In addition, the Langendorff isolated guinea pig heart model will be used to determine the role of IKs in anesthetic-induced cardioprotection. The hypotheses to be tested are: 1. The minK subunit hinders the accessibility of an isoflurane interaction site on KCNQ1. 2. Phosphorylation of IKs by protein kinase A counteracts the inhibitory effects of isoflurane. 3. The differential anesthetic effects on the subunits are not unique to the KCNQl+mmK channel complex. 4. The KCNQ1 and minK subunits are differentially expressed in isoflurane-induced cardioprotection. In summary, this proposal will characterize the role of an auxiliary regulatory subunit, minK, on volatile anesthetic action on IKs. This novel approach to anesthetic interaction with ion channels will have functional significance in the diseased myocardium where up- and downregulation of channel subunits are known to occur.