MOLECULAR MECHANISMS OF GATING IN IONIC CHANNELS

我们建议研究离子的分子机制 渗透性和C1和K离子通道的门控。 实验 使用胚胎或成年大鼠中枢神经元（海马、脊髓 脐带）维持在原代组织培养物中，或通过急性 清白的. 膜片钳技术将用于记录 从细胞膜上切下的膜球的宏观膜电流 细胞索马，并研究电流从个别渠道， 小的膜补丁。 我们将分析离子的机理 两种C1通道的选择性和渗透性：C1 通道在静息膜中打开，GABA或甘氨酸 激活C1通道。 阳离子/阴离子相互作用 静息C1通道将被研究，以测试 联合阴离子/阳离子渗透提出了从以前的工作。 的 通过Zn离子阻断，并将研究pH的影响。 离子 发射机激活的C1通道的选择性将是 检查并与静息C1通道进行比较，以确定 这些小电导C1通道是否实现离子 通过相同的基本机制进行选择。 分子通道 在快速和缓慢灭活的成分下， 将识别电压激活的K电流。 我们将进一步 研究这些通道的门控，评估它们在塑造 躯体兴奋性，并检查其差异 对K通道阻滞剂敏感。 我们将执行动能 在零度以下温度下对单通道门控进行测量。 大钙激活钾通道的行为将是 其特点是温度低至-30摄氏度。 这个新 方法有望大大提高动力学研究的范围 可能与离子通道有关。

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.