CALCIUM CHANNEL DYSFUCTION IN LAMBERT-EATON SYNDROME

在Lambert-Eaton综合征（LES）中，运动神经末梢释放 乙酰胆碱量子数量不足， 神经冲动 这种疾病通常与癌症有关， 特别是肺的小细胞癌（SCC）。 长期 本项目的目的是了解分子 LES中突触前损害的基础，并确定 SCC细胞在自身免疫病因学和发病机制中的作用 这种混乱。 据推测，病理活性 这种疾病产生的IgG与电压依赖性钙离子结合， 通道（VDCC），抑制其功能并导致永久性损失 通过诱导抗原源来产生 循环LES自身抗体存在于SCC细胞中。 为了检验这些假设，将实现以下具体目标： 继续：1）进一步表征LES IgG诱导的阻断 牛肾上腺嗜铬（BAC）细胞中的VDCC; 2）确定 LES抗体介导直接功能性免疫应答的作用机制 VDCC抑制; 3）证明抗原调节， LES IgG诱导的加速Ca 2+通道降解; 4） 测定直接抑制VDCC的相对贡献 功能和抗原调节整体LES病理生理学; 5)证明LES IgG与抗原的特异性结合 识别位点; 6）确定Ca 2+通道复合物中的识别位点; LES抗体对T-、N-和L-型VDCC作用的特异性; 7)证明SCC细胞中VDCC的存在，并确定 8）确定LES IgG与它们的通道类型的交叉反应性; 9）开发SCC细胞中VDCC的自身免疫动物模型; 通过用粗制膜提取物免疫动物， 从SCC和BAC细胞纯化的LES抗原：10）表征和 建立自身免疫动物模型的有效性;和（11） 评估突触的功效和药理学敏感性， 小鼠被动转移LES中的神经肌肉接头> 该项目将采用电生理学、生物化学和 免疫学技术包括：1）全细胞和单细胞- 通道膜片钳记录技术; 2）电分析 膜电容监测胞吐作用; 3）fura-2 细胞内游离Ca ~（2+）浓度的测定; 4）蛋白质 5）IgG抗体分离、纯化和纯化; 碎片化

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.