L-type Ca channels serve many vital functions in the heart. The influx of Ca2+ ions through L-type Ca channels during the plateau of the cardiac action potential provides the """"""""trigger"""""""" for the release of Ca2+ from the sarcoplasmic reticulum. This establishes the link between electrical events at the cell surface and the initiation of contraction. The activation of L-type Ca channels in nodal cells contributes substantially to the pacemaker rates and to the speed of action potential propagation in those cells. Modulation of Ca channel activity is a crucial element in positive and negative inotropy and chronotropy, and may play a crucial role in the generation of very slow conduction and the development of the re-entry circuits in ischemic ventricular muscle that can lead to ventricular fibrillation. The long-term goal of this project is to characterize how changes in the intracellular environment affect L-type Ca channel activity. The objective it to understand the molecular forces that control Ca channel activity under normal conditions as well as those that are important during cardiac ischemia. Changes in the membrane potential and the intracellular concentrations of Ca2+ , Mg2+, H+, and ATP are known to result from ischemic episodes, and all impact on the availability, open probability, and gating kinetics of L-type Ca channels. In addition, the activity of intracellular proteins such as protein kinases, phosphoprotein phosphatases, lipases, and regulatory G-proteins also have strong effects on channel activity. The experimental approach is to characterize the effects of intracellular ions and regulatory proteins on L-type Ca channels incorporated from cardiac sarcolemma into planar lipid bilayers and in excised patches from giant reconstituted liposomes, where there is good experimental access to the intracellular side of the channel. We will test whether specific ionic or enzymatic modulators of Ca channels act directly on the channels or via intermediate effector systems. Endogenous regulatory components that are present in the sarcolemmal membranes will be activated or inhibited by specific drugs or ligands, and the effect of that modification on L-type Ca channel behavior will be studied. Additionally, exogenous enzymes, other proteins, ions, or small molecules will be added to the intracellular side, and the effect on channel properties recorded. Modification of Ca channel properties will be observed at the single-channel level as changes in conductance, open probability, activation or inactivation kinetics, open- and closed-time distributions, or """"""""lifespan"""""""" of the channels in the cell-free membranes. In order to dissociate any multi-protein complexes of channels and endogenous regulators, we will also study the properties of Ca channels in membrane fragments stripped of extrinsic membrane proteins by treatment with high pH or with other chaotropic agents. In addition, Ca channels will be solubilized from the sarcolemma and reconstituted into artificial membranes, providing even better control of the channel's regulatory environment.
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