MOLECULAR MECHANISMS OF GATING IN IONIC CHANNELS

我们建议研究离子的分子机制。 氯离子通道和钾离子通道的通透性和门控。这些实验 使用胚胎或成年大鼠的中枢神经元(海马体、脊髓 原代组织培养中保持的，或通过急性培养获得的 解离。膜片钳技术将用于记录 切除的膜球的宏观膜电流 细胞胞体，并研究来自单个通道的电流 小的膜片。我们将分析离子的作用机理。 两种C_1通道的选择性和通透性 静息膜上的通道打开，GABA或甘氨酸- 激活了c1通道。阳离子/阴离子之间的相互作用 将研究静息的c1通道，以测试 以前工作中提出的阴阳离子联合渗透作用。这个 用锌离子阻断，并研究pH的影响。《离子》 发送器激活的C1通道的选择性将为 检查并与静息的C1通道进行比较，以确定 无论这些小电导的C1通道是否实现离子 通过相同的基本机制实现选择性。分子通道 作为快速和缓慢失活的组件的基础 电压激活的K电流将被识别。我们将进一步 研究这些渠道的门控，评估它们在塑造中的作用 体细胞的兴奋性，并检查它们的差异 对K通道阻滞剂敏感。我们将表演动能 零下温度下单通道门控的测量。 大的钙激活钾通道的行为将是 其特点是温度低至零下30摄氏度 方法有望极大地扩大动力学研究的范围 使用离子通道是可能的。

We propose to study the molecular mechanisms underlying ionic permeability and gating of C1 and K ion channels. The experiments use embryonic or adult rat central neurons (hippocampus, spinal cord) maintained in primary tissue culture, or obtained by acute dissociation. The patch clamp technique will be used to record macroscopic membrane currents from membrane spheres excised from the cell soma, and to study currents from individual channels in small membrane patches. We will analyze the mechanism of ionic selectivity and permeability of two varieties of C1 channel: C1 channels open in the resting membrane and GABA- or glycine- activated C1 channels. Cation/anion interactions inside the resting C1 channel will be investigated to test a mechanism of joint anion/cation permeation proposed from previous work. The block by Zn ion, and effects of pH will be studied. The ionic selectivity of the transmitter-activated C1 channels will be examined and compared to that of resting C1 channels, to determine whether or not these small-conductance C1 channels achieve ionic selectivity by the same basic mechanism. Molecular channels underlying the rapidly and slowly inactivating components of voltage-activated K current will be identified. We will further study the gating of these channels, assess their role in shaping the somatic excitability, and examine their differential sensitivity to K channel blockers. We will perform kinetic measurements on single-channel gating at subzero temperatures. The behavior of the large Ca-activated K channel will be characterized for temperatures down to -30 degrees C. This new method is expected to greatly enhance the range of kinetic studies possible with ion channels.