Role of Herg-like K+ Channels in G.I. Smooth Muscle

本研究旨在探讨内脏平滑肌静息膜电位的发生机制。静息电位是平滑肌兴奋性的主要决定因素之一，在很大程度上受K+离子的渗透性支配。初步研究表明，几种胃肠道平滑肌的静息电位在一定程度上是由一种独特的人类乙醚-go-go （HERG）样K+通道控制的。这种K+电流显示向内整流，似乎是常用的促动力学剂西沙必利的潜在靶标。第一个具体目的是表征胃肠道平滑肌细胞中herg样K+电流的详细生物物理特性。该电流在单个细胞中的电压依赖性动力学将使用膜片钳技术确定，其在静息电位和动作电位复极化中的生理作用将被表征。第二个具体目标是确定平滑肌herg样K+电流的药理学调节。在这些研究中，将确定III类抗心律失常化合物（已知的HERG通道阻滞剂）的作用，并研究促动力学剂西沙必利对HERG电流的作用机制。初步数据显示，西沙必利可减弱单个胃肠道平滑肌细胞的HERG电流，使肌条去极化。兴奋性神经递质乙酰胆碱对这些K电流的影响将被确定，并将评估蛋白激酶C参与的第二信使调节。在第三个特定目标中，将通过免疫荧光技术和Western blotting定位herg样通道的蛋白质表达。第四个具体目标是确定平滑肌中herg样电导与其他内整流电流的关系。在这些研究中，IKi， ATP敏感的K+通道和超极化激活的阳离子电流的作用将被检查。平滑肌中heg样K+通道的抑制性调节剂可能为平滑肌过度兴奋性疾病的治疗提供新的途径。

The objective of this proposal is to pursue studies on the genesis of the resting membrane potential in visceral smooth muscle. The resting potential is one of the major determinants of smooth muscle excitability and is largely governed by the permeability to K+ ions. Preliminary studies demonstrate that the resting potential of several gastrointestinal smooth muscle is, in part, controlled by a unique human ether-a-go-go (HERG)-like K+ channel. This K+ current demonstrates inward rectification and appears to be a potential target of the commonly used prokinetic agent, cisapride. The first specific aim is to characterize the detailed biophysical properties of the HERG-like K+ current in gastrointestinal smooth muscle cells. The voltage- dependent kinetics of this current in single cells will be determined using the patch clamp technique and its physiological role in resting potential and repolarization of the action potential will be characterized. The second specific aim is to identify the pharmacological regulation of the smooth muscle HERG-like K+ currents. in these studies, the effects of class III antiarrhythmic compounds which are known HERG channel blockers will be determined, and the mechanism of action of the prokinetic agent, cisapride on the HERG currents will be investigated. Preliminary data show that cisapride attenuates the HERG currents in single gastrointestinal smooth muscle cells and depolarizes muscle strips. The effects of the excitatory neurotransmitter, acetylcholine on these K currents will be determined and the involvement of the second messenger regulation by protein kinase C will be evaluated. In the third specific aim, the protein expression of HERG-like channels will be localized by immunofluorescence techniques and by Western blotting. The fourth specific aim is to define the relationship of the HERG-like conductance with those of the other inwardly rectifying currents in smooth muscle. In these studies the role of IKi, ATP- sensitive K+ channel and the hyperpolarization-activated cation currents will be examined. Inhibitory modulators of the HERG-ike K+ channels in smooth muscle may provide new approaches to treatment of disorders that involve smooth muscle hyperexcitability.