VOLATILE ANESTHETIC ACTION ON CARDIAC K CHANNELS

描述（由申请人提供）： 挥发性麻醉剂（VA）已使用了世纪，但其有效的心脏保护作用只是最近才被确定。然而，麻醉剂对心脏离子通道作用的分子机制尚不清楚。VA抗心律失常作用的一个可能靶点是哺乳动物心脏中主要复极化电流的基础慢延迟整流钾（IKs）通道。VA对IKs通道的抑制将导致动作电位延长，其作用与III类抗心律失常药相似。该提案的总体目标是阐明VA对IKs通道作用机制的分子模型。拟议的实验将调查VA对IKs的两个亚基组件的影响：由基因KCNQ1编码的成孔区和辅助调节p亚基minK。初步结果表明，异氟烷对KCNQ1单独和与minK亚基组合发挥不同的作用。一种新的模式，挥发性麻醉剂相互作用的通道蛋白，其中一个辅助调节亚基调节麻醉剂的行动被假定。总体假设是minK亚基减少VA对IKs的影响。膜片钳技术将用于监测哺乳动物心脏细胞系中瞬时表达的KCNQ1和minK的功能变化。还将监测过表达minK的天然肌细胞中IKs功能的变化。此外，Langendorff离体豚鼠心脏模型将用于确定IK在麻醉诱导的心脏保护中的作用。待检验的假设为： 1. minK亚基阻碍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烷的抑制作用。 3.对亚基的不同麻醉作用不是KCNQ1 +mmK通道复合物所独有的。 4. KCNQ1和minK亚基在异氟醚诱导的心脏保护中的差异表达 总之，本提案将描述辅助调节亚基minK在吸入麻醉剂对IKs作用中的作用。这种新的麻醉剂与离子通道相互作用的方法将在已知发生通道亚基上调和下调的患病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.