Drugs which block transmembrane calcium flux have been known for almost 20 years and inorganic ions with the same effect have been used in biology for more than a century. However, almost nothing is known about the detailed interactions between these agents and the receptor(s) of the calcium channel.
The aim of the proposed research is to test the hypothesis that some calcium channel blockers bind to their receptors with an affinity that depends on the state of the channel, i.e., rested, activated, or inactivated. Preliminary voltage clamp experiments in isolated cardiac muscle suggest that the affinity of verapamil for its receptor is much higher when the channel is inactivated than when it is resting or activated. Furthermore, blockade appears to disappear when the membrane is resting at a relatively negative potential. Indirect evidence from the literature suggests that the block produced by nifedipine is much less dependent on membrane rest or voltage. Incomplete information regarding inorganic calcium channel blockers like cobalt, nickel and cadmium suggest that these agents are affected even less than nifedipine by membrane activity and voltage. The proposed project would systematically study the kinetics of interaction of verapamil, nifedipine, cobalt and cadmium with the cardiac calcium channel. Ferret papillary muscles would be studied with the voltage clamp, single sucrose gap technique. A mathematical model of state-dependent blockade, implemented on a laboratory minicomputer will be used to extract association and dissociation rate constants of interaction of the blockers with rested, activated, and inactivated channels. Ability of the model to fit the experimental data and the relative magnitudes of the rate constants will test the working hypothesis and permit characterization of the agents as state-dependent or not state-dependent blockers. Because ischemic and arrhythmic cardiac tissue is in a different state from normal myocardium, this information will permit a better understanding of the effects of the drugs in diseased hearts. In addition, full characterization of the blocking drugs will make them much more useful as physiological and pharmacological tools in basic research.
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