9422040 Many cellular functions are activated by calcium, which enters cells through specialized, highly regulated proteins (calcium channels) in the cell membrane. In neurons, calcium entry alters the cell's electrical activity, secretion of neurotransmitter, and growth or regeneration of neuronal processes; it also activates several calcium-dependent enzymes which in turn can generate the type of long-lasting changes in neuronal properties thought to underlie learning and memory. The proposed experiments will investigate a novel and poorly understood calcium entry pathway in neurons. This class of calcium channels can be activated experimentally by forming a small cell free patch of membrane. Although such channels are found in high density in several different neurons and muscles, their physiological activators are largely unknown. Physiological functions of the channels are also unknown, although some evidence suggests they may help mediate the large, damaging calcium influxes seen under certain pathological conditions. Recent experiments indicate that these channels may be rapidly activated by Nerve Growth Factor, a protein which causes neuronal differentiation and neurite outgrowth in several types of neurons. The proposed experiments will examine the biochemical mechanisms by which Nerve Growth Factor and related molecules activate this class of calcium channel. Additional experiments will examine a possible role of the channels in neurite outgrowth, and will attempt to isolate specific peptide blockers for these channels. The primary techniques used are electrical measurement of the currents flowing through the channels, and rapid measurement of intracellular calcium concentration in single cells using fluorescent indicators. These two techniques can be used simultaneously in a single cell. The ability to study these pathways at a single channel level, coupled with direct measurement of one of their primary physiological effects (elevation of intracellular calcium), should contribute to the understanding of this widespread but poorly understood calcium influx pathway. The experiments will also contribute to an understanding of the actions of peptide growth factors.
