Calcium channels play a crucial role in the control of cellular function. In the heart, they initiate contraction and help determine pacemaker rhythm and conduction in the nodal regions. In vascular smooth muscle they regulate contraction and therefore control vascular tone. One Ca channel type is the target for the widely used Ca anatagonist drugs and its gating is modulated by neurotransmitters and hormones. The interest in Ca channels is further enhanced by the fact that as Ca selective transport proteins, they may share many properties with other important Ca binding molecules. We propose to combine functional measurements at the level of individual channel molecules with biochemical attempts to purify the channel protein. Ca channels from cardiac and vascular smooth muscle will be investigated. Single channel recording techniques will be used for a quantitative description of the biophysical channel properties. Patch clamp measurements on intact cells will be compared with recordings from cardiac Ca channels incorporated into lipid bilayers, an approach which gives ideal control over the ionic and lipid environment of the channel. Reconstitution of the L-type cardiac Ca channel in lipid bilayers will also be used to follow the functional integrity of the channel protein through biochemical solubilization and purification. These techniques will help answer some fundamental questions about Ca channel function: What are the parameters determing an ion's rate of Ca channel entry? How many ion binding sites does the channel have? Can it be occupied by more than one ion at a time? Do ions interact within the pore? Is the channel pore functionally symmetrical? How does the composition of the membrane lipid affect channel function? What are the differences between cardiac and smooth muscle Ca channel types? In an attempt to relate channel function to structure, we will investigate the role of sugar residues for channel function and will try to create proteolytic lesions with specific functional consequences. The functional consequences of Ca channel phosphorylation will be investigated in detail. We will ask the following questions: Is phosphorylation necessary and sufficient to make a Ca channel available for activation by depolarization? What are the rates of phosphorylation and dephosphorylation? What protein kinase systems are involved? Are the regulatory systems different in heart and vascular smooth muscle?
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