Calcium channel function will be examined using electrophysiological methods. The present proposal is directed toward specific aspects of activation, inactivation, conduction in open channels and regulation of channel numbers. A complete experimental picture of Ca channel gating and conduction and regulation of Ca channel numbers is the long term goal. The combined suction pipette-microelectrode method for voltage clamp and internal perfusion of isolated snail nerve cell bodies will be used in conjunction with the cell-attached patch clamp method for recording from single channels. The patch clamp method will also be applied to PC-12 cells for whole cell voltage clamping and the study of membrane patches. Macroscopic currents will be analyzed using exponential fitting. Microscopic current fluctuations will be analyzed using noise spectra and impedance plots. The open time and frequency of opening of single channels will be measured. Correlation among the three different approaches, macroscopic currents, microscopic fluctuations and single channel openings will be made and specific models of activation, inactivation and conduction will be examined. Regulation of functional Ca channel numbers by intracellular Ca, neural transmitters, organic Ca channel blockers and divalent cations will also be investigated. The studies are being extended to PC-12 cells for several reasons: for comparative purposes, because membrane patches in a variety of configurations (inside-out, outside-out, cell-attached) are readily studied and because binding studies and other biochemical studies are being done on this cell line. Calcium channels are the link between excitation and a number of important cellular functions such as contraction and secretion. They are important in the function of the nervous and musculoskeletal systems and the endocrine systems and, therefore, may be involved in the numerous diseases that affect these systems.
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