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Na和K离子通道的过程， 背根神经节细胞和海马神经元。 将细胞 保持在原代组织培养物中。 膜片钳技术将是 用于记录整个细胞膜和小的膜斑 只含有几个离子通道。 用于检测Na+是否灭活 通道在动力学上与它们的激活有关，Na+的小集合 将使用电压协议刺激通道，该电压协议旨在测试 通道开放对通道失活速率的影响。 我们将 检查激活门控的打开/关闭步骤是否缓慢， 关于其他激活步骤，如果通道打开， 涉及配置的重大变化。 后一种单通道 将对失活的Na通道进行测量， 化学阻塞或减慢。 K的失活和非失活形式 通道将在单通道水平表征;微观 他们的门控动力学方面将比较那些发现钠 渠道 维持膜电压的调节作用将是 检查单个Na和K通道。 各种离子（用于 细胞内溶液），以及具有可能的调节 作用（cAMP，ATP和硫胺素衍生物）将被引入细胞 以及它们对Na和K通道功能的可能影响， 表征了 切除的血管中通道存活的必要条件 将探索膜贴片。 我们将测试Na和K通道 就像有髓神经的K通道一样， 门控行为的自发变化。 长期目标是 了解兴奋膜中的Na和K通道是如何被 膜电位的变化，从长远来看，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.