GATING AND TRANSLOCATION IN THE DIPHTHERIA TOXIN CHANNEL

这项研究的主要目的是研究两个基本的 离子通道的门控和转运过程， 微生物毒素 白喉毒素（DT）通道是 这类频道的原型。 将研究渠道 使用脂质双层、脂质囊泡和膜片钳技术。 1. DT门控的药理学。 细胞内第二信使 1，4，5-肌醇三磷酸（IP 3）与DT结合并刺激通道 从膜的反侧形成。 剂量反应 IP 3、其它肌醇磷酸和其它DT配体的曲线 包括刺激阻断剂（ApUp、ATP）。 这些配体的作用机制将被检查 在宏观电流和单沟道水平上， 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 DT通道的选择性，特别是相对于 钙，将被检查。 2. DT突变体的生理学-门控 影响 几个DT突变体（称为CRM）的交叉- 反应材料和碎片被很好地表征 结构上，并已知形成通道（CRM 45，CRM 50， CRM 197，CB 1（溴化氰片段））。 只有CRM 45有 在电生理学上得到了很好的研究， 与DT通道的属性不同。 的电压 和DT的pH依赖性，其各种片段和 突变体，以及相关的通道形成毒素（PsA）将被 定量表征。 位点特异性突变体将 试图改变DT的门控特性 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 跨膜转运 DT的孔隙半径 将使用脂质中的非电解质筛分来确定通道 囊泡，以查看DT的大小是否明显不同 从CRM 45（d约18 A）。 使用内切酶和 DT通道的顺式和反式端的外肽酶 双层将试图确定氨基的位置， 和DT相对于膜的羧基末端。 还将寻求开放和闭合状态拓扑的差异。 膜片钳实验将试图记录DT通道， 当它们穿过细胞膜时。 抑制剂的作用 DT条目如SIT和低Ca++将被检查， 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.