The research described in this proposal is designed to provide information about the relationship between the structure and function of cardiovascular L-type calcium channels. Functional characteristics of native and recombinant L-type channels will be studied using patch clamp procedures in combination with specific chemical compounds that interact with the channel protein and regulate channel gating. There are four specific aims of the project. The first is to use electrophysiological techniques to identify the location of the high affinity dihydropyridine (DHP) binding site on native heart L-type channels relative to the membrane surface.
The second aim i s to use an electrophysiological approach to describe the biophysical properties of recombinant channel activity expressed by Chinese Hamster Ovary cells stably transfected with cDNAs for specific heart and smooth muscle calcium channel subunits.
The third aim of the project is to investigate the modulation of recombinant channel activity by a quaternary DHP compound to identify the location of the high affinity DHP binding site in homooligomeric alpha1, and if necessary multimeric alpha1,beta channels.
The final aim i s to investigate the modulation of recombinant channels by neutral DHPs to identify structural components that underlie DHP voltage-dependent regulation of L-channel gating, and to test the hypothesis that structural differences in cardiac and smooth muscle alpha1 subunits contribute to the selective targeting of smooth muscle L-type calcium channels by this important family of drugs. The results of this work will provide insight into the molecular components that constitute the DHP binding site and regulate L-channel gating and into the molecular basis of tissue selectivity of DHP compounds. Because DHP regulation of calcium channel activity is associated with control of channel gating, this information will provide insight into the molecular mechanisms that underlie the opening and closing of these channels. In turn, this information will thus provide a framework to understand how calcium entry into heart cells is regulated in normal and diseased states, and how this regulation can be specifically controlled by therapeutic drugs.
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