RECONSTITUTION OF CALCIUM CHANNELS IN PLANAR BILAYERS

该项目利用平面磷脂双层膜来研究这种影响。 脂质、离子和生化探针对传导和门控的影响 骨骼肌横管钙通道的特性。 鉴于T型管的巨大潜力，人们对它有相当大的兴趣 组织提供钙的纯化和分子克隆 通道蛋白。平面双层膜在这个项目中的优势是 三重：它允许记录来自通道的单一钙电流 存在于纯化的T形小管中；它允许实验控制 围绕通道的双层磷脂；它允许控制 海峡两岸的解决方案。一个具体的目标是理解 表面电荷对钙通道的定量调制作用 传导和门控动力学。这里的兴趣在于区分 依赖于脂质极性头部基团的电荷效应之间 由通道中存在的电荷控制的组成和效果 蛋白质本身。为此，已经找到了稳定的条件 两种极限脂蛋白中T管钙通道的活性 环境：在由中性脂质组成的双层中 磷脂酰乙醇胺或带负电荷的脂类双层 磷脂酰丝氨酸。这两种脂质成分将有助于确定 钙通道中脂蛋白相互作用的性质和程度。一个 第二个具体目标是了解Na、BA和Ca 影响i)通道如何激活和停用；ii) 开度-电压曲线；以及iii)选择性(Ba，Ca)或损耗 明渠中的选择性(Na)。这里感兴趣的是确定 化学特异度、脂类敏感性和部位的物理位置 (即靠近栅极或在孔内)对于两者中的电流载流子， 细胞内侧和胞外侧。这些地点的地图也是如此 作为渠道内功能域的位置，将实现 通过使用有机阳离子作为传导探针，特定的钙 作为门控探针的经络毒素(阿托毒素、毒素)和单抗 抗体作为功能通道亚单位组成的探针。

The project makes use of planar phospholipid bilayers to study the effects of lipids, ions, and biochemical probes on the conduction and gating characteristics of calcium channels of skeletal muscle transverse tubules. T-tubules are of considerable interest given the great potential that this tissue offers for the purification and molecular cloning of the calcium channel protein. The advantage of planar bilayers for this project is three-fold: it allows recording of unitary calcium currents from channels present in purified t-tubules; it allows experimental control of the bilayer phospholipids surrounding the channel; and it allows control of solutions on both sides of the channel. One specific aim is to understand quantitatively the modulatory role of surface charge on calcium channel conduction and gating kinetics. The interest here is to draw a distinction between charge effects that depend on the lipid polar head group composition and effects controlled by charges present in the channel protein itself. For this purpose, conditions have been found to stabilize the activity of t-tubule calcium channels in two limiting lipid environments: in bilayers composed of the neutral lipid phosphatidylethanolamine or in bilayers of the negatively charged lipid phosphatidylserine. These two lipid compositions will help to define the nature and extent of lipid-protein interactions in calcium channels. A second specific aim is to understand the ways in which Na, Ba, and Ca affect i) how the channel activates and inactivates; ii) the probability of opening vs. voltage curve; and iii) selectivity (Ba, Ca) or loss of selectivity (Na) in the open channel. The interest here is to determine the chemical specificity, lipid sensitivity, and physical location of sites (i.e., close to the gates or within the pore) for current carriers in both, the intracellular and extracellular sides. Mapping of these sites, as well as the location of functional domains within the channel, will be achieved by the use of organic cations as conduction probes, specific calcium channel toxins (atrotoxin, taicatoxin) as probes of gating, and monoclonal antibodies as probes of the subunit composition of functional channels.