This proposal requests support to study the intracellular regulation of potassium-selective (K+) ion channels in heart cells. Ion channels are intramembrane proteins giving rise to the cardiac action potential and thus govern the rate and force of contraction of the heart. Cardiac arrhythmia, a leading cause of cardiovascular deaths, are caused by disturbances in this electrical control. Even more importantly, ion channels control the beat-to-beat regulation of the heart and allow the heart to respond to hormones and neurotransmitters and meet ever changing biological demands. The present proposal focuses on K+ channels they are highly regulated by intracellular second messengers. The large diversity of K+ channels reflects their importance in controlling many aspects of cardiac function. Work over the past several years has dramatically expanded our knowledge of the number of K+ channels and their intracellular regulation. Much of this work has been accomplished with patch clamp techniques in combination with reconstitution of biomedical pathways in the patch or intracellular environment. However, many of the details of ion channel regulation cannot be elucidated with these techniques alone. This is especially true for multistep, membrane-bound second messenger systems where specific inhibitors of the steps are lacking. This application proposes a continuation of electrophysiological and biochemical reconstitution approaches to further elucidate the control of several important K+ channels, including the muscarinic-gated inward rectifier, K.ACh, and two newly discovered K+ channels (IK.AA, IK.PC) gated by lipophilic membrane constituents. In particular, the lipoxygenase pathway's participation in control of IK.ACh will be explored and its physiologic relevance tested. The participation of phospholipid metabolic pathways in the control of IK.AA and IK.PC will be examined. In addition, molecular biology techniques will be employed to determined the primary structure of cardiac potassium-selective ion channels. Three cloning strategies to sequence voltage-sensitive and relatively voltage-insensitive K+ channels will be applied and the channels expressed in Xenopus oocytes and mammalian cell lines. The ability to alter specific regulatory sites in the primary structure of K+ channels will help provide the needed specificity to dissect the regulatory processes.
