Action potentials regulate the function of the heart by determining beating rate and contractility. The action potential in turn is generated by a population of ionic channels which undergo conformational changes to alter the permeability of the membrane. Much of our present understanding of the ionic currents which regulate heart function has been acquired through measurement of macroscopic currents under voltage clamp. Researchers have recently developed a technique measuring the current from small patches of membrane containing one to a few ionic channels. The purpose of the present research is to measure single channel currents from seven-day chick embryonic heart cell aggregates and single cells. The patch clamp technique allows measurements of single channels to be made from intact and excised patches of heart cell membrane. Single cells can also be voltage clamped to compare with conventional two-electrode voltage clamp. Attention will be focused on outward currents in the plateau and pacemaker ranges of membrane potential. Specific ionic channels will be identified by analysis of (1) single channel reversal potentials, (2) time constants, (3) conductance, and (4) pharmacology. Standard two-electrode voltage clamp and single cell voltage clamp recordings in the pacemaker and plateau range will be used to define total membrane currents. The effect of ionic concentrations and channel-blocking agents will be studied in excised membrane patches. The extracellular and intracellular face of the heart cell membrane may be selectively exposed. The selectivity of putative potassium channels to varying ionic concentrations will be measured. Potassium channel blocking agents will be compared for effects on channel conductance and kinetics. By combining techniques, a more detailed understanding of cardiac membrance currents regulating the pacemaker and plateau potentials may be gained. The proposal requests support for three years.
