This project is aimed at elucidating the regulation and functional role of voltage-gated calcium (Ca) channels in the peripheral nervous system of vertebrates. Voltage-gated Ca channels are essential for the normal function of the sympathetic nervous system. Ca ions, entering the neuron via voltage-gated channels that open in response to depolarizing stimuli, regulate a diverse spectrum of functions including neurotransmitter release, neurite outgrowth, neuronal excitability and gene expression. Several different types of Ca channels have been identified and each is likely to be specialized for regulating particular Ca-dependent processes. This proposal focuses on studying N and L type Ca channels of sympathetic neurons and on determining their relative importance in the control of neurosecretion and neuronal excitability. Single Ca channel currents will be recorded with the use of patch-clamp technique from sympathetic neurons isolated from adult rats and frogs. The behavior of N and L channels will be studied in high resolution single channel recordings in order to characterize the gating pattern of each component. A direct comparison of neurons from adult frog and rat will be essential in addressing possible species-based differences in the behavior of Ca channels. Receptor-mediated inhibition of Ca channel currents is important in pre-synaptic inhibition of transmitter release. The effect of norepinephrine (NE) on the gating of single Ca channel currents will be studied in detail to determine the mechanism of inhibition. The consequences of directly activating various second messenger systems proposed to be involved in transducing the effects of NE and other neurotransmitters on Ca channel function will also be determined. Neurotransmitter release in isolated sympathetic neurons will be monitored by measuring, in the same patch, both the changes in membrane capacitance and the properties of the resident Ca channel currents. This will be achieved with a modified version of the patch clamp recording technique. The location of specialized zones of transmitter release will be determined in cell-attached patches obtained from cell bodies, axons and growth cones of sympathetic neurons. The hypothesis that N channels cluster within specialized transmitter release zones will be tested by monitoring single Ca channel currents and capacitance changes in the same cell-attached patches. The functional role of different classes of Ca channels in activating two distinct Ca-activated K channel currents (KCa and KAHP) in sympathetic neurons will be determined. Cell-attached patches from various cellular regions will establish whether a particular type of Ca channel co-localizes with a specific Ca-activated K channel. A better appreciation of the inter-relationship between different classes of Ca and K channel currents will provide new insights into the mechanisms by which neuronal excitability and neurotransmitter release are regulated.
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