The long-range goal of the research program in this laboratory is to characterize the electrophysiological properties of the ionic currents in cardiac tissue and to provide an understanding of the role of intracellular second messengers in the regulation and/or modulation of these currents. The research proposal outlined here is specifically aimed at mechanistic studies of Ca2+ currents, K+ currents and gap junctions in isolated heart muscle cells and in the effects of intracellular messengers on the properties of these currents. Experiments will be peformed in which """"""""concentrtion jumps"""""""" of putative intracellular second messenges, e.g. H+, cAMP, cGMP, ATP and Ca2+, are made (from photolabile precursors) inside of cells under electrophysiological investigation. Whole-cell patch clamp recordings will be employed most extensively on isolated adult and neonatal atrial and ventricular myocytes; additional studies will also exploit conventional intracellular recording techniques. In studies of Ca2+ and K+ current regulation, single cells will be utilized; in studies of gap junctions, cell pairs, either obtained from the dissociation procedure or recontacted after dissociation, will be used. These studies should permit determination of the kinetics and concentration dependences of intracellular messenger-mediated effects on macroscopic currents. In addition, later studies are aimed at evaluation of the influence(s) of intracellular messengers on the permeability of gap junctions as well as on controlling the number of functional channels which form in a gap junction. Although likely to be beyond the scope of the present proposal, we anticipate that the approaches employed will later be readily extended to studies of the mechanistic details of intracellular messenger-mediated modulation of ion channels at the single-channel level also employing patch-clamp recording techniques. It is unlikely that any direct clinical applications would result from the proposed experiments or from our work in general. It is anticipated, however, that these studies will provide insight into how the various cardiac membrane ion channels function normally as well as how they can be (and/or are) regulated and modulated by intracellular signals.
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