Calcium entry via Ca channels contributes to a wide range of neuronal functions including pacemaker activity, spike electrogenesis, transmitter release and neurite outgrowth. Multiple components of Ca current have been found in neurons as well as other excitable cells. For example, our previous patch clamp recordings demonstrated that chick dorsal root ganglion (DRG) cells contain three types of Ca channels (T, N and L-type) which differ in single channel conductance, gating kinetics, and pharmacological sensitivity. This proposal describes studies of the properties, distribution and functional roles of the various types of Ca channel in cultured neurons, hippocampal slices and isolated synaptic endings. (1) We will continue development of selective agents for dissecting channel function, having seen selective effects of Bay K 8644 and wCgTX, a 27-amino acid marine snail toxin. We will analyze effects of organic drugs (verapamil, diltiazem, nifedipine, etc), peptide toxins (wCgTx, atrotoxin, B-leptinotarsin), and agents that might selectively block T-type channels. (2) We will explore differences amongst the channel types in mechanisms of ion permeation and gating. Modulation of channel activity by neurotransmitters or internal messengers will be analysed at the level of single Ca channels. (3) The distribution of various Ca channel types on cell bodies, dendrites, and growth cones of cultured neurons will be studied with on-cell, outside-out and loose patch recordings. The electrophysiological findings will be compared with localization of N- and L-type channels by autoradiography with 125I-wCgTx. (4) Participation of Ca channel types in electrical activity of the hippocampus will be studied with patch and chopped clamp recordings from hippocampal pyramidal and granule cells. We will characterize the dendritic Ca channels that underly fast pre-potentials and epileptiform activity, and look for modulatory effects of C-kinase on Ca channels that might accompany long-term potentiation. (5) The involvement of L-type orrN-type Ca channels in mediating Ca entry and transmitter release will be studied in presynaptic terminals of neuromuscular junctions, cultured neurons, brain slices, and synaptosomes. Activity of synaptosome Ca channels will also be recorded following incorporation in planar bilayers.
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