L-type Ca channels serve many vital functions in the heart. They sustain the plateau of the action potential and help set action potential duration and refractoriness. The influx of Ca2+ through L-type Ca channels triggers Ca2+ release from the sarcoplasmic reticulum, establishing the link between electrical activity at the cell surface and the contraction of cardiac myoctes. L-type Ca channels in nodal cells contribute to pacemaker activity and to the speed of conduction in those cells. Their activity may also play an important role in the generation of very slow conduction in ischemic myocardium, and especially at the border between normal and ischemic regions. In order to understand the basis of electrical re-entry that can occur at the ischemic border zone to trigger dangerous ventricular arrhythmias, it is crucial to understand factors that modulate Ca channels. The long-term goal of this project is to characterize the molecular mechanisms responsible for the modulation of cardiac L-type Ca channels, with special attention to the conditions that exist at the ischemic border zone. Because ischemic myocardium has a depolarized resting potential, high intracellular Ca2+, and low border zone. Because ischemic myocardium has a depolarized resting potential, high intracellular Ca2+, and low intracellular and extracellular pH, and because cardiac ischemia produces large beta-adrenergic stimulation, Specific Aim 1 focuses on the regulation of L-type Ca channels by protein kinase A under depolarized, Elevated Ca2+, and acidic pH conditions. We hypothesize that PKA stimulation sustains the activity of Ca channels the might otherwise become inactivated, leading to a abnormal slow conduction. Because beta- adrenergic release of norepinephrine is accompanied by a release of ATP, Specific Aim 2 focuses on some new observations of the effects of extracellular ATP on L-type Ca channels. Lipid metabolism is affected by ischemia, so Specific Aim 3 focuses on the regulation of L-type Ca channels by lysolipids and metabolites of arachidonic acid. Finally, Specific Aim 4 focuses on the regulation of Ca channels by nitric oxide and S-nitrosothiols. The experimental approach is to characterize the regulation of single L-type Ca channels incorporated from cardiac sarcolemma into planar lipid bilayers. We will record single channel activity of the reconstituted Ca channels, and evaluate channel conductance and unitary gating events. We control the extracellular and intracellular ionic conditions on both sides of the channels, the activity of endogenous regulatory enzymes like kinases and phosphatases, and the membrane environment. Thus, we will create conditions that mimic the ischemic border zone, and make a detailed characterization of the modulation of Ca channels there.
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