Voltage-dependent, potassium ion selective, membrane proteins exist in virtually all animal cells. The physiological roles of these K channels are diverse and include the maintenance of cell resting potentials, repolarization of cell action potentials, and regulation of rhythmic electrical activity. As a consequence, these channels are involved in various pathologies and are the targets of diverse therapeutic agents including those directed toward diseases of the pancreas and the cardiovascular system. Molecular level knowledge of the structure and function of these channels will be valuable in understanding the pathologies and will lead to the design of improved therapeutic measures. The long-term goal of this research is to provide information for a molecular level view of the conformational change process in voltage- gated K channels. A combination of biochemical, molecular biological, and electrophysiological methods will be used to examine certain specific features of this process in channels of known primary structure. Previous studies of the channel conformational change process have concentrated on control by membrane voltage-ignoring weakly voltage dependent conformational changes. Our past work has implicated a histidine group in some K channels that is involved in a weakly voltage dependent step in channel opening. One specific focus of the research proposed here is to determine the location of this amino acid and confirm its role in channel gating. Another specific goal of this proposal is to identify the region of the channel protein that contains the site for modulation by external divalent cations like Ca2+. Drawing on work in the past grant period, we hypothesize two possible loci which will be tested with site specific mutagenesis and electrophysiological methods. Channel gating is substantially perturbed by divalent cations interacting with an important internal region of these proteins. Another study in this application is designed to provide information on the chemical nature of this region. In all these studies, cloned ion channels will be expressed by RNA injection in Xenopus oocytes. Voltage clamp electrophysiology techniques will be used to assay channel function and will include measurement of macroscopic and single channel currents. The functional properties that are the focus of this research are common to many K channel types in cells as diverse as lymphocytes and heart and brain cells. Thus, the knowledge gained in these studies will be of general utility in revealing the molecular properties of the physiology , pathology, and pharmacology of ion channels.
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