REGULATION OF INTESTINAL ION CHANNELS

旅行者腹泻通常是由大肠杆菌引起的， 热稳定毒素（STa）增加环GMP（cGMP）。 STa生成cGMP 通过激活膜结合鸟苷酸环化酶从GTP中分离。 这 一项提案旨在确定哪些氯离子通道参与了 水盐平衡，并研究其调节STa和 cGMP。 培养的结肠细胞系T84，一种跨上皮细胞模型， 氯化物分泌，表现出STa刺激的短路电流， 测量跨上皮转运。 氯离子的STa调节 在培养的T84细胞的通道，将探讨平行的研究 回肠细胞上。 使用T84细胞，第一个目的是研究生物物理特性， 使用全细胞贴片的STa诱导的氯电流的性质 钳夹技术，并使用切除的膜片确定是否有STa激活 氯离子通道受cGMP依赖性蛋白激酶调节。 的 第二个目的是确定STa激活的 氯电流和由其他氯分泌激活的电流 激动剂如血管活性肠肽和卡巴胆碱。 第三个目标 是为了增加我们对回肠中氯离子通道的了解， 研究它们的性质和控制机制。 全细胞电流和单通道波动都将是 测定了 全细胞电流模式的优点是， 可以与其它伴随信号隔离地测试信号。 单通道记录确定是否声称的分泌 通道位于顶膜上。 这项工作的长期目标是隔离参与的通道 控制肠道中的盐和液体运动， 记录其在卫生和卫生保健方面的监管控制。

Traveller's diarrhea is often caused by Escherichia coli which releases a heat-stable toxin (STa) increasing cyclic GMP (cGMP). STa generates cGMP from GTP by activation of a membrane bound guanylate cyclase. This proposal is designed to identify which chloride channels are involved in fluid and salt balance and to investigate their regulation by STa and cGMP. The cultured colonic cell line, T84, a model for transepithelial chloride secretion, exhibits STa-stimulated short circuit current, a measure of transepithelial transport. STa regulation of chloride channels in cultured T84 cells, will be explored in parallel with studies on ileal cells. Using T84 cells, the first aim is to investigate the biophysical properties of STa-induced chloride currents using the whole cell patch clamp technique and to determine using excised patches if STa-activated chloride channels are modulated by cGMP-dependent protein kinase. The second aim is to determine the interrelationship between STa-activated chloride currents and those activated by other chloride secretory agonists like vasoactive intestinal peptide and carbachol. The third aim is to increase our understanding of chloride channels in the ileum by investigating their properties and control mechanisms. Both whole-cell currents and single channel fluctuations will be measured. The whole-cell current mode has the advantage that individual signals can be tested in isolation from other concomitant signals. Single channel recordings determine whether the purported secretory channels are located on the apical membrane. The long term goal of this work is to isolate channels which participate in the control of salt and fluid movements in the intestine and to document their regulatory control in health and in diarrheal states.