Mechanisms of Current Modulation in Cardiac Myocytes

Rockefeller University, New York NY

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Abstract

A long term goal is a[unreadable] detailed understanding of the many cellular mechanisms that modulate cardiac[unreadable] "background" membrane currents, which can contribute to diastolic[unreadable] potentials, to the plateau of the cardiac action potential, and to the[unreadable] initiation and termination of cardiac arrhythmias. Sharp focus is presently[unreadable] on cardiac CFTR (Cystic Fibrosis Transmembrane conductance Regulator) Cl[unreadable] channels which, functionally, seem closely similar to the epithelial CFTR Cl[unreadable] channels that are dysfunctional (often due to defective processing) in[unreadable] cystic fibrosis patients. The specific aims are (1) to pursue detailed[unreadable] characterization of the endogenous cellular regulatory mechanisms of[unreadable] mammalian cardiac CFTR Cl conductance in intact myocytes, the (2) to[unreadable] determine the gating mechanisms of individual cardiac CFTR channels in[unreadable] excised patches of myocyte membrane. Cardiac myocytes are presently one of[unreadable] the few (if not the only) preparations in which it is possible to examine[unreadable] both the complex regulation of native CFTR channels in their natural[unreadable] physiological environment by endogenous kinase and phosphatase systems, and[unreadable] the underlying gating behavior of single channels. The work will allow a[unreadable] rigorous comparison between the functional properties of epithelial and[unreadable] cardiac CFTR channels. Two related experimental approaches are used:[unreadable] whole-cell current recording in intact myocytes that are voltage clamped and[unreadable] internally dialyzed via wide tipped patch pipettes fitted with a pipette[unreadable] perfusion device, via which nucleotides or their analogs, or specific[unreadable] inhibitors of kinases of phosphatases, can be readily applied to the cell[unreadable] interior; and the excised "giant" patch technique for recording unitary[unreadable] channel currents in large inside-out patches of sarcolemmal membrane, to the[unreadable] cytoplasmic face of which nucleotides, peptide inhibitors, and also much[unreadable] larger molecules such as purified kinases and phosphatases, can be directly[unreadable] and rapidly applied. Kinetic analyses of single-channel currents from these[unreadable] patches will advance understanding of the channel's gating mechanisms. It[unreadable] appears that, during each open-close gating cycle, a single[unreadable] highly-phosphorylated CFTR channel hydrolyzes one molecule of ATP at its[unreadable] N-terminal nucleotide binding domain to open, and then hydrolyzes a second[unreadable] ATP at its N-terminal nucleotide binding domain to close. CFTR channels[unreadable] thus offer an opportunity, unprecedented in biology, to examine individual[unreadable] ATP hydrolysis cycles in a single protein molecule, in its natural[unreadable] environment, in real time.[unreadable]