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

本研究的目的是探讨内脏平滑肌静息膜电位的起源。静息电位是平滑肌兴奋性的主要决定因素之一，并且在很大程度上受K+离子渗透性的控制。初步研究表明，几种胃肠平滑肌的静息电位部分由独特的人类以太-a-go-go（HERG）-样K+通道控制。该K+电流显示内向整流，似乎是常用促动力剂西沙必利的潜在靶点。第一个具体目标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。将使用膜片钳技术测定单细胞中该电流的电压依赖性动力学，并表征其在静息电位和动作电位复极化中的生理作用。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，将测定已知为HERG通道阻断剂的III类抗癫痫化合物的作用，并研究促动力剂西沙必利对HERG电流的作用机制。初步数据显示，西沙比利减弱单个胃肠道平滑肌细胞中的HERG电流并使肌肉条去极化。将确定兴奋性神经递质乙酰胆碱对这些K电流的影响，并评价蛋白激酶C对第二信使调节的参与。在第三个具体目标中，HERG样通道的蛋白质表达将通过免疫荧光技术和蛋白质印迹来定位。第四个具体的目的是确定的HERG样电导与其他内向整流电流在平滑肌的关系。在这些研究中，将检查伊基、ATP敏感性K+通道和超极化激活的阳离子电流的作用。平滑肌中HERG样K+通道的抑制性调节剂可能为治疗涉及平滑肌过度兴奋的疾病提供新的方法。

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.