Effects of cocaine on cardiac ion channels

这项提案中概述的实验将审查可卡因滥用的医疗和健康后果，可卡因滥用已被确定为NIDA在这个国家的一个严重问题。自1998年以来，可卡因在美国的使用量有所上升，部分原因是成本低、供应充足。与这一趋势平行的是与可卡因使用和与可卡因相关的死亡相关的急诊室入院率的增加。在大多数情况下，与可卡因有关的死亡的确切原因尚不清楚，但许多人被认为是由心律失常引起的，心律失常是与可卡因滥用有关的主要死亡原因。在人类中，可卡因已知会增加心率和血压，这些效果主要归因于这种药物的交感神经刺激作用。可卡因还会引起心脏电生理的显著变化，降低传导，减缓动作电位后的复极。这些电干扰被认为是可卡因对心脏离子通道的直接麻醉作用造成的。在这个方案中，我们将研究钠和钾通道对可卡因的敏感性，这些通道对启动和终止心脏动作电位非常重要。抑制这些通道会扰乱心脏的协调电活动，这是众所周知的室性心律失常发生的危险因素。电生理学和分子生物学的结合将被用来研究这些通道的抑制机制，并识别对可卡因结合重要的位置。人类心脏钠通道(HH1)和人类乙醚-a-g-GO延迟整流钾通道(HERG)将在哺乳动物细胞中表达，并使用膜片钳技术记录产生的电流。我们的数据表明，可卡因与这些通道的结合是状态依赖的，可卡因优先与开放和失活的通道结合。将构建HH1和HERG的失活缺陷突变体，以研究失活门控对可卡因结合的贡献，并促进开放通道阻断的测量。具有第六跨膜片段的其他突变体也将被研究，这些片段可能是这些通道的麻醉剂结合部位。拟议的研究将深入了解可卡因引起的心脏电生理变化的潜在机制，以及该药物的心脏毒性效应。

The experiments outlined in this proposal will examine the medical and health consequences of cocaine abuse, which has been identified as NIDA as a serious problem in this country. Since 1998 the use of cocaine in the US has risen, in part due to the low cost and ample supply. Paralleling this trend has been an increase in the rate of emergency room admissions related to cocaine use and cocaine-related deaths. The exact causes of the cocaine-related fatalities in most cases are not known but many are believed to result from cardiac arrhythmias, the leading cause of death associated with cocaine abuse. In humans, cocaine is known to increase heart rate and blood pressure, effects that are primarily attributable to the sympathomimetic actions of this drug. Cocaine also produces significant changes in cardiac electrophysiology, decreasing the conduction and slowing the repolarization following an action potential. These electrical disturbances are believed to result from a direct anesthetic effect of cocaine on cardiac ion channels. In this proposal, we will investigate the cocaine sensitivity of sodium and potassium channels important for initiating and terminating the cardiac action potential. Inhibiting these channels disrupts the coordinated electrical activity of the heart, a well-known risk factor for the generation of ventricular arrhythmias. A combination of electrophysiology and molecular biology will be used to investigate the mechanisms of inhibition of these channels and to identity sites important for cocaine binding. The human cardiac sodium channel (hH1) and the human ether-a-g-go delayed rectifier potassium channel (HERG) will be expressed in mammalian cells and the resulting currents recorded using patch clamp techniques. Our data indicate that cocaine binding to these channels is state-dependent, with cocaine preferentially binding to the open and inactivated channels. Inactivation-deficient mutants of hH1 and HERG will be constructed to investigate the contribution of inactivation gating to cocaine binding and to facilitate the measurement of open-channel block. Additional mutants with the sixth transmembrane spanning segments, the putative anesthetic binding sites of these channels, will also be investigated. The proposed studies ill provide insight into the mechanisms underlying the cocaine-induced changes in cardiac electrophysiology and the cardiotoxic effects of this drug.