CHEMOSENSORY TRANSDUCTION

化学感应传导

• 批准号: 6379285
• 负责人: JUDITH L VAN HOUTEN
• 金额: $ 32.32万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-09-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6379285
• 关键词: Paramecium; acetates; affinity chromatography; antibody formation; binding proteins; biological signal transduction; biotin; calcium transporting ATPase; chemoreceptors; cyclic AMP; denaturing gradient gel electrophoresis; electrophysiology; fluorescent dye /probe; glutamates; glycosylphosphatidylinositols; membrane permeability; membrane transport proteins; voltage /patch clamp

This is an application for renewal of funding study chemical sensing in Paramecium. This organism is amenable to multiple techniques for dissecting chemosensory transduction mechanisms in chemoreceptor cells: biochemistry, molecular biology, transmission genetics, electrophysiology, behavioral analysis of populations and individual cells. After we disrupt the cell and study its transduction components in vitro, we can often return our study to the level of the intact cell and ask whether our findings have relevance for the physiology of the cell. Also, we know the identity of stimuli, which bind to distinct receptors. In the past funding period, we have accomplished most and gone beyond some of our original specific aims which were to apply molecular, immunological, biochemical and fluorescent dye techniques to the study of the cAMP receptor and the plasma membrane calcium pump, which appears to participate in transduction pathways. We have partial clones of the cAMP receptor gene, and the full length gene for the plasma membrane calcium pump. We have subcloned and studied the pump's calmodulin binding domain. Highlights include There are not 1 but at least 3 transduction pathways, 2 of which involve the calcium pump; attractant stimulus NH4CI has no receptor and rapidly alkalinizes the cell; while all attractant stimuli hyperpolarize the cell, only glutamate increases cAMPI and very rapidly; some chemoreceptors probably are GPI anchored and not integral membrane proteins; biotin is an attractant and its receptor likely is GPI anchored cloned; attractants hyperpolarize cells and the conductances initiated by biotin and acetate have been partially characterized. There is a sustained outward conductance (possibly the pump current) and a large inward conductance for the off-response in biotin; there is an initial inward calcium and outward K conductance in acetate with a small inward conductance for the acetate off-response. The calcium pump appears to sustain the hyperpolarization in both biotin and acetate. We used mutants with conductance defects to test these ideas. We propose to test three hypotheses that arise from our studies. 1) Chemoreceptors for biotin and glutamate are GPI anchored proteins. 2) Chemosensory transduction in 2 pathways in modulates a calcium pump to generate a hyperpolarizing conductance. Gene structure suggests that these modulations involve PKA, PKC or calmodulin. 3) Biotin and acetate induce distinct sets of Conductances, which we will test using voltage clamp analysis of normal, mutant and transformed cells. We will use electrophysiological, biochemical, molecular biology, and immuno-techniques in a collaborative effort between two laboratories. These studies should inform the research of others who study sensory transduction in olfaction, taste, chemotaxis and other signaling stems.

这是续签资金研究化学传感的应用。 该生物适合于化学感受器细胞中的化学感应转导机制的多种技术：生物化学，分子生物学，传播遗传学，电生理学，人群和单个细胞的行为分析。 在干扰细胞并在体外研究其转导组件后，我们通常可以将研究恢复到完整的细胞水平，并询问我们的发现与细胞的生理学是否相关。 同样，我们知道与不同受体结合的刺激的身份。在过去的资金期间，我们已经完成了最大的成就，并且超出了我们的一些原始特定目的，这些目的是将分子，免疫学，生化和荧光染料技术应用于CAMP受体和质膜钙泵的研究，这似乎参与了转导途径。 我们有cAMP受体基因的部分克隆，以及质膜钙泵的全长基因。 我们已经取代并研究了泵的钙调蛋白结合域。 亮点包括没有1个，但至少有3个转导途径，其中2条涉及钙泵。吸引力刺激NH4CI没有受体，并迅速碱化细胞。虽然所有吸引刺激刺激超极化，但只有谷氨酸会增加CAMPI，并且非常迅速。一些化学感受器可能是GPI锚定的，而不是整体膜蛋白。生物素是一种吸引剂，其受体可能是锚定克隆的GPI。吸引剂超极化细胞和生物素和乙酸产生的电导已部分表征。 生物素中有持续的向外电导（可能是泵电流）和大量的内向电导。乙酸盐中有一个初始的内向钙和向外的K电导，乙酸反响应的内向电导率很小。 钙泵似乎可以维持生物素和乙酸盐中的超极化。 我们使用具有导电缺陷的突变体来测试这些想法。 我们建议测试我们的研究产生的三个假设。 1）生物素和谷氨酸的化学感受器是GPI锚定蛋白。 2）在2个途径中的化学气体转导调节钙泵以产生超极化电导。 基因结构表明这些调节涉及PKA，PKC或钙调蛋白。 3）生物素和醋酸盐会诱导不同的电导集，我们将使用对正常，突变和转化细胞的电压夹具分析进行测试。 我们将在两个实验室之间的协作工作中使用电生理，生化，分子生物学和免疫技术。 这些研究应告知其他研究嗅觉，味道，趋化性和其他信号茎的其他人的研究。