DINOFLAGELLATE TOXINS AS TOOLS FOR STUDYING MEMBRANE CHOLINERGIC RECEPTORS

乙酰胆碱(ACh)受体是最具特性的 膜蛋白参与兴奋性现象。这个 这种受体的功能特征得到了 发现具有新作用和高受体的非常特定的毒素- 有约束力的亲和力。我们最近证实了胆碱能活动。 在雪卡毒素梭鱼的提取物中，以及在提取物中 与从Ostreopsis中获得的层析分离的部分相同 豆状--一种与雪卡特拉有关的底栖甲藻 加勒比海。甲醇提取物抑制突触电位和 神经松解或沐浴引起的宫缩 青蛙缝纫肌肉中的乙酰胆碱。这种影响似乎是 由甲藻物质中的极性部分引起的 能降低烟碱型ACh受体的离子电导 培养的鸡胚肌细胞中的通道。其他研究表明， 提取物和第二个层析组份显示毒扁豆碱 活性，与氚毒碱配体的结合竞争 对大鼠脑膜和青蛙表现出激动剂样作用 胃肌肌。此应用程序旨在发现 甲藻毒素的胆碱能作用机制 使用各种电生理学、药理学和 生化技术。建议进行实验，以实现 具体目标如下：a)层析分离 加勒比海红藻有毒提取物的级分 反相高效液相色谱法测定豆状棘豆的含量 胆碱活性成分的化学性质和结构；及 C)确定孤立者的活动地点和作用机制 胆碱能受体活性化合物。将使用的研究策略 在后一个阶段是：i)使用神经元的结合实验 膜和膜从鱼雷电器官中检验 放射性标记配体的置换及与之竞争 不同受体亚型；II)突触的细胞内记录 青蛙电机端板的电位及其变化的测量 活性组分引起的震级和时间特征； 以及iii)记录膜离子电流，使用单一的 通道和全细胞版本的膜片钳技术 培养鸡胚胎交感、睫状神经细胞和神经细胞 和背根神经节。一旦确定并表征了这些新的 甲藻毒素应该是这项研究有用的分子探针 中枢和外周神经系统的胆碱能受体。 参加这些学习的少数民族学生将学习现代 神经化学和电生理方法以及组织 培养技术。

The acetylcholine (ACh) receptor is one of the best characterized of the membrane proteins involved in excitability phenomena. The functional characterization of this receptor has been helped by the discovery of very specific toxins with novel actions and high receptor- binding affinities. We have recently demonstrated cholinergic activity in extracts obtained from ciguatoxic barracuda and in extracts, as well as in chromatographically isolated fractions, obtained from Ostreopsis lenticularis - the benthic dinoflagellate associated with ciguatera in the Caribbean. Methanolic extracts inhibited synaptic potentials and the contractions elicited by nerve-released or bath-applied acetylcholine in frog sartorious muscles. This effect appeared to be caused by a polar fraction in the dinoflagellate material that was capable of reducing the ionic conductance of nicotinic ACh receptor channels in cultured chick embryo myocytes. Other studies showed that extracts and a second chromatographic fraction displayed muscarinic activity, competing with the binding of tritiated muscarinic ligands to rat brain membranes and exhibiting an agonist-like action in frog gastric muscularis muscles. This application aims to discover the mechanism of the cholinergic effects of these dinoflagellate toxins using a variety of electrophysiological, pharmacological and biochemical techniques. Experiments are proposed to attain the following specific aims: a) the chromatographic isolation of the fractions from toxic extracts of the Caribbean dinoglagellate Ostreopsis lenticularis using reverse phase HPLC; b) the determination of the chemical nature and structure of cholinoactive components; and c) identification of the sites and mechanisms of action of the isolated cholinergic - receptor active compounds. Research strategies to be used in this latter phase are: i) binding experiments using neuronal membranes and membranes from Torpedo electric organ to examine displacement of and competition with radiolabeled ligands of the different receptor subtypes; ii) intracellular recording of synaptic potentials in frog motor end plates and measurement of changes in their magnitude and temporal characteristics caused by the active fractions; and iii) recording of membrane ionic currents, using both the single channel and the whole cell versions of the patch clamp technique in cultured chick embryo myocytes and neurons from sympathetic, ciliary and dorsal root ganglia. Once identified and characterized these new dinoflagellate toxins should be useful molecular probes for the study of cholinergic receptors in the central and peripheral nervous systems. Minority students participating in these studies will learn modern neurochemical and electrophysiological methods as well as tissue culture techniques.