The proposed experiments use the """"""""gating current"""""""" of the L-type calcium channel (L-type calcium channel) as a new tool to investigate the cellular and molecular regulation of calcium current in heart. L-type calcium channel is generated by the movement of voltage-sensor charges in the alpha1-subunit of the calcium channel protein and only recently has become available for use in heart (see Preliminary Results) developing rapidly from the measurements of total gating charge movement in heart (Field, et. al. 1988; Bean & Rios, 1989; Hadley & Lederer, 1989). L-type calcium channel provides information on molecular transitions and rearrangements of the calcium channel protein that is otherwise unavailable. As will be shown in this application, these transitions are linked to channel opening, inactivation and closing and L-type calcium channel can be used to examined these channel states. Calcium channel function is known to be modulated by voltage, intracellular calcium ([Ca2+]i), protein phosphorylation, neurohormones, metabolic blockade and specific drugs as well as many other factors. Because new information regarding calcium channel function can be obtained by measuring l_type calcium channel the actions of these modulator on L-type calcium channel will investigated. To do this adult guinea pig and rat myocytes and neonatal rat cells, will be voltage-clamped using a patch-clamp method in whole-cell configuration to measure L-type calcium channel. In parallel experiments ICa will be measured as needed. Jumps of [Ca2+]i (produced by flash photolysis of the caged calcium compounds DM Nitrophen and Nitr-5 or the caged calcium buffer, diazo-2) will be carried out to investigate the influence of [Ca2+]i on L-type calcium channel and ICa. Quantitative investigation of the action of [Ca2+]i on l_type calcium channel will be carried out while measuring [Ca2+]i with the fluorescent calcium indicator indo-1. Calcium channel interaction with the dihydropyridine (DHP) calcium channel blockers can be examined using L-type calcium channel. This is possible because of the photosensitivity of specific dihydropyridines and their ability to affect L-type calcium channel. Specific experiments are planned that use L-type calcium channel to investigate how channels are modulated under physiological conditions, during development, with metabolic insult and with drug application. Not only does the planned work offer a new approach to the investigation of the regulation of ICa, but it also provides complementary information to single channel and whole cell calcium current measurements. This work should provide important primary information that will broaden our understanding of how the calcium channel functions, how it influences normal excitability and how it may lead to spontaneous activity and ectopy during arrhythmogenesis.
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