MOLECULAR MECHANISMS OF GATING IN IONIC CHANNELS

建议研究门控和调节的分子方面。 大鼠肌球电压依赖性钠、钾离子通道的过程， 背根神经节细胞和海马神经元。牢房将会是 保持在原代组织培养中。膜片钳技术将是 用于从整个细胞膜和从小的膜片进行记录 只包含几个离子通道的。用于测试钠的失活是否 通道在动力学上与它们的激活联系在一起，钠的小集合 将使用设计用于测试的电压协议来刺激通道 通道开放对通道失活率的影响。我们会 检查激活门控的开启/关闭步骤是否缓慢 关于其他激活步骤，如果通道打开，则可能会预期 涉及到配置方面的重大更改。后者为单通道 将对已失活的钠通道进行测量 被化学物质堵塞或减慢。K的失活和非失活形式 渠道将在单一渠道层面上进行表征；微观 他们的门控动力学方面将与发现的Na 频道。维持膜电压的调节作用将是 在单独的钠和钾通道中进行检测。各种离子(用于 细胞内解决方案)，以及可能具有调控作用的化学品 效应(cAMP、ATP和硫胺素衍生物)将被引入细胞 它们对钠和钾通道功能的可能影响将是 特色化的。切除的经络生存的必要条件 将探索膜贴片。我们将测试钠通道和钾通道 我们的准备，就像有髓神经的K通道，经历了 门控行为的自发变化。远景目标是 了解可兴奋性膜上的Na和K通道是如何被 膜电位的变化，从长远来看，是通过调节 流程。

It is proposed to study molecular aspects of gating and regulatory processes in voltage-dependent Na and K ion channels of rat myoballs, dorsal root ganglion cells and hippocampal neurons. The cells will be maintained in primary tissue culture. The patch clamp technique will be used to record from whole cell membranes and from small membrane patches containing only a few ion channels. For testing whether inactivation of Na channels is kinetically linked to their activation, small ensembles of Na channels will be stimulated with voltage protocols designed to test for effects of channel opening on the rate of channel inactivation. We will examine whether the opening/closing step of activation gating is slow with regard to other activation steps, as might be expected if channel opening involves a major change in configuration. The latter single-channel measurements will be done on Na channels whose inactivation has been chemically blocked or slowed. Inactivating and non-inactivating forms of K channel will be characterized at the single channel level; microscopic aspects of their gating kinetics will be compared to those found for Na channels. Regulatory effects of maintained membrane voltages will be examined in individual Na and K channels. Various ions (to be used for intracellular solutions), as well as chemicals with possible regulatory effects (cAMP, ATP, and thiamine derivatives) will be introduced into cells and their possible effects on the function of Na and K channels will be characterized. Conditions essential for survival of channels in excised membrane patches will be explored. We will test whether Na and K channels of our preparations, like K channels of myelinated nerve, undergo spontaneous changes of gating behaviour. The long range goal is to understand how Na and K channels in excitable membranes are controlled by changes of the membrane potential and, in the long term, by modulatory processes.