GATING AND TRANSLOCATION IN THE DIPHTHERIA TOXIN CHANNEL

这项研究的主要目的是考察这两个基本问题 离子通道中的门控和移位过程 微生物毒素。白喉毒素(DT)通道是 这类渠道的原型。将对渠道进行研究 使用脂质双层、脂泡和膜片钳技术。1. DT门控的药理作用。细胞内第二信使 1，4，5-三磷酸肌醇(IP3)与DT结合并刺激通道 从膜的反面形成。剂量-反应 IP3、其他肌醇磷酸盐和其他DT配体的曲线 包括刺激阻滞剂(ApUp，ATP)将被构建。 我们将研究这些配体的作用机制。 在功能上处于宏观电流和单通道级别， 在结构上，通过观察配体对DT的影响 突变体、片段和相关毒素，如破伤风和 假单胞菌外毒素A(PSA)。肌醇磷酸盐对人血清白蛋白的影响 DT通道的选择性，尤其是相对于 钙，将被检查。2.dt突变体的生理学--门控 这意味着什么。几个DT突变体(称为CRM)用于杂交 反应材料和碎片被很好地表征 在结构上并且已知形成通道(CRM45，CRM50， CRM197、CB1(溴化氰片段))。只有CRM45有 对电生理学有很好的研究，并表现出惊人的 与DT频道的属性不同。电压 和pH依赖的性质，DT，其各种碎片和 突变体和相关的通道形成毒素(PSA)将是 定量描述的。特定部位的突变体将是 用于尝试改变DT的选通属性 通过已知的结构变化。3.易位机制。 这些实验将探索蛋白质的分子机制。 跨膜转位。DT的孔半径 使用脂类中的非电解质筛分来确定通道 小泡，以查看DT的大小是否明显不同 取自CRM45(d约18A)。利用内切酶和内切酶进行蛋白水解酶 DT通道顺侧和反侧的胞外肽酶 将尝试双分子层来确定氨基的位置 和相对于膜的DT的羧基末端。 还将寻求打开和关闭状态拓扑的差异。 膜片钳实验将尝试记录DT通道 Vero细胞穿过细胞膜。抑制剂的作用 的DT条目，如SITS和低Ca++将被检查以 阐明DT受体在DT易位中的作用。

The broad aim of this study is to examine the two fundamental processes of gating and translocation in ionic channels formed by microbial toxins. The diphtheria toxin (DT) channel is the prototype for this class of channels. Channels will be studied using lipid bilayer, lipid vesicle, and patch clamp techniques. 1. Pharmacology of DT gating. The intracellular second messenger 1,4,5-inositol-triphosphate (IP3) bind to DT and stimulates channel formation from the trans side of the membrane. Dose-response curves for IP3, other inositol phosphates and other DT ligands including blockers of stimulation (ApUp, ATP) will be constructed. The mechanism of action of these ligands will be examined functionally at the macroscopic current and single channel level, and structurally through observing the effects of ligands on DT mutants, fragments and related toxins such as tetanus and Pseudomonas exotoxin A (PsA). Effects of inositol phosphates on the selectivity of the DT channel, especially with respect to calcium, will be examined. 2. Physiology of DT mutants-gating implications. Several DT mutants (called CRMs) for cross- reacting material and fragments are well characterized structurally and are known to form channels (CRM45, CRM50, CRM197, CB1 (cyanogen bromide fragment)). Only CRM45 has been well studied electrophysiologically and exhibits striking differences from the properties of the DT channel. The voltage and pH dependent properties of DT, its various fragments and mutants, and the related channel forming toxin (PsA) will be quantitatively characterized. Site specific mutants will be constructed in an attempt to alter the gating properties of DT via known structural changes. 3. Mechanism of translocation. These experiments will probe the molecular mechanism of protein translocation across membranes. The pore radius of the DT channel will be determined using non-electrolyte sieving in lipid vesicles in order to see if the size of DT is markedly different from CRM45 (d about 18A). Proteolytic digestion using endo- and exopeptidases of DT channels from cis and trans sides of the bilayer will be attempted to determine the location of the amino and carboxy terminals of DT with respect to the membrane. Differences in open and closed state topology will also be sought. Patch clamp experiments will attempt to record DT channels in Vero cells as they cross the cell membrane. Effects of inhibitors of DT entry such as SITs, and low Ca++ will be examined to elucidate the role of the DT receptor in DT translocation.