The broad aim of this project is to characterize the molecular events involved in ion permeation and block, channel gating and ion channel regulation. In particular, the goal is an understanding of the molecular and physical basis of the function of the inward rectifier channel K1 and the pacemaker channel If in cardiac cells. The channel currents will be studied in isolated guinea pig ventricular and nodal cells using patch clamp techniques. Channel permeation and block studies will be conducted for the inward rectifier channel to characterize the mechanism of rectification, particularly the effect of block by intracellular Mg2+. The interactions between permeant and impermeant ions will be examined and modeled to gain an understanding of the mechanisms of permeation and rectification, and the structure of the ion conductance pathway. Channel gating will be investigated to ascertain its role in rectification. For the pacemaker channel, ion permeation will be investigated for monovalent and divalent cations and modeled with a permeation energy profile to determine the basis of this channel's very low single-channel conductance but its low selectivity between Na+ and K+. Gating of the pacemaker channel will be studied to learn about the kinetic states that are involved in the slow activation of the current by hyperpolarizing voltage important for the pacemaker activity. For both channels, the mechanisms of cellular modulation will be examined physiologically and we will attempt the isolation and characterization of the biochemical pathways involved in channel regulation. The results of these studies will provide new information about the detailed molecular mechanisms involved in the function of the inward rectifier and pacemaker channels that will help in the understanding of basic cardiac function, regulation of the current in the heart, the role of Mg2+ in the heart, and the molecular mechanisms of ion permeation, gating and regulation of membrane channels in general. These studies are expected to have relevance to the physiology of the heart, as well as the physiology of nerve, muscle, and other excitable cells.
