This project is designed to continue to investigate the properties of ion channels in the heart using electrophysiological techniques in combination with chemical probes of the channels. Some of the experiments will focus on one type (L-Type) of cardiac calcium channel and the modulation of this type of channel by 1,4 dihydropyridine (DHP) compounds. This important group; of drugs includes compounds that enhance (agonists) or inhibit (antagonists) Ca+2 influx by modulating channel gating instead of by plugging or unplugging the channel. This part of the proposal is a continuation of work begun in this laboratory during the previous funding period. The proposed experiments will focus on the influence pH on the actions of these compunds, and will include optical measurement to directly monitor the effects of membrane potential on drug binding. In addition, experiments are also planned to provide more information about the nature of the gating changes these drugs cause. Other experiments are planned to study two important potassium channels: the inward rectifier and the delayed rectifier channels. The properties of the instantaneous current/voltage (I/V) relationships of these two K channels will be characterized and compared over a wide range of voltages and in the presence of different permeant ions to provide information about their relative permeabilities. A series of quaternary ammonium molecules will be used to block the channels and provide information about their structures. The results of this set of experiments will generate new information about the channels that regulate action potential duration in the heart, and will be useful in the design of therapeutic compounds targeted to regulate specific stages of cardiac action potential repolarization. The usefulness of these drugs as potential Class IV antiarrhythmic agents is pointed out. Experiments will be carried out in enzymatically-dispersed guinea pig ventricular cells. Membrane currents will be measured with the patch clamp technique arranged in whole cell and single channel configurations. An optical procedure will be used to monitor the fluorescence of a 1,4-DHP Ca channel antagonist in a set of experiments designed to monitor the influence of membrane potential on drug binding.
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