CALCIUM CHANNEL DYSFUCTION IN LAMBERT-EATON SYNDROME

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In Lambert-Eaton syndrome (LES), the motor nerve terminals release an insufficient number of acetylcholine quanta in response to a nerve impulse. This disorder is frequently associated with cancer, particularly small-cell carcinoma (SCC) of the lung. The long-term objective of the present project is to understand the molecular basis of the presynaptic impairment in LES and to determine the role of SCC cells in the autoimmune etiology and pathogenesis of this disorder. It is hypothesized that the pathologically active IgG produced by this disease binds to a voltage-dependent calcium channel (VDCC), inhibits its function and causes a permanent loss of the channel by inducing antigenic source for the production of circulating LES autoantibodies resides in SCC cells. To test these hypotheses, the following specific aims will be pursued: 1) further characterize the LES IgG-induced blockade of VDCCs in bovine adrenal chromaffin (BAC) cells; 2) determine the mechanism of action of LES antibodies mediating a direct functional inhibition of VDCCs; 3) demonstrate antigenic modulation and accelerated Ca2+ channel degradation induced by LES IgG; 4) determine relative contribution of direct inhibition of VDCC function and antigenic modulation to overall LES pathophysiology; 5) demonstrate specific binding of LES IgG to an antigenic recognition site in a Ca2+ channel-complex; 6) determine the specificity of action of LES antibodies on T-, N- and L-type VDCCs; 7) demonstrate the existence of VDCCs in SCC cells and determine their channel types; 8) determine cross-reactivity of LES IgG with VDCCs in SCC cells; 9) develop an autoimmune animal model of the syndrome by immunizing animals with crude membrane extracts and purified LES antigens from SCC and BAC cells: 10) characterize and establish the validity of the autoimmune animal model; and (11) evaluate synaptic efficacy and pharmacological sensitivity of the neuromuscular junctions in murine passively transferred LES> This project will employ electrophysiological, biochemical and immunological techniques including: 1) whole-cell and single- channel patch-clamp recording techniques; 2) electrical assay of exocytosis by membrane capacitance monitoring; 3) fura-2 measurement of free intracellular Ca2+ concentration; 4) protein purification; and 5) IgG antibody isolation, purification and fragmentation.