MOLECULAR MECHANISMS OF GATING IN IONIC CHANNELS

生物物理研究是关于(L)的机制提出的。 兴奋性和抑制性配体门控离子通道在阳离子之间的选择 和阴离子，(2)大电导K通道的栅极机制 零下的温度。这些实验使用的是胚胎大鼠的中枢神经元 (海马体、脊髓)和肌管维持在初级组织中 文化。膜片钳技术将被用来研究 切除的膜片中的单个通道。 结构上同系离子的兴奋或抑制效应 由神经递质ACh、GABA或甘氨酸操作的通道是 由通道通过电荷选择离子的能力决定 两极。在AChR通道中，几种处理(高离子强度， PH和羧基试剂)将用于中和基团 负净电荷，以评估它们在阳离子/阴离子中的重要性 选择。在GABA-R和Gly-R通道中，细胞内和 胞外二价阳离子在建立高选择性中的作用 将研究阴离子(通过通道的大量阳离子泄漏 二价盐暴露与阴离子的部分修复 观察到了外部钙的选择性)。需求中的二价体 将被确定，并将应用测试来区分 二价阳离子的结构和直接静电效应。 一个新的水平的动力学现象背后的大-门 电导钙激活的钾通道将在较低温度下进行研究 使用我们实验室开发的技术将温度降至零下30摄氏度。这些 实验将调查分子门的操作，通过它 控制通道孔的离子电流。在室温下 这个门的作用就像一个即时开关，但在非常低的温度下 跃迁变慢到离子电流的节奏改变的程度 可以测量实际浇口的行为 推断出来的。 这些研究解决了与离子通道有关的基本问题 神经元和肌肉，因此应该提供重要的基本信息 以了解这些组织的正常和受损功能。

Biophysical studies are proposed concerning (l) the mechanisms by which excitatory and inhibitory ligand-gated ion channels select between cations and anions, (2) the gate mechanism of a large-conductance K channel at subzero temperatures. The experiments use embryonic rat central neurons (hippocampus, spinal cord) and myotubes maintained in primary tissue culture. The patch clamp technique will be used to study currents from individual channels in excised membrane patches. The excitatory or inhibitory effects of the structurally homologous ion channels operated by the neurotransmitters ACh, GABA, or glycine are determined by the channels' ability to select permeant ions by charge polarity. In the AChR channel, several treatments (high ionic strength, pH, and a carboxyl reagent) will be used to neutralize groups with negative net charges, to assess their importance in the cation/anion selection. In GABA-R and Gly-R channels, the roles of intracellular and extracellular divalent cations in establishing a high selectivity for anions will be studied (a substantial cation leakage through channels exposed to divalent-free salines and partial restoration of anion selectivity by external Ca have observed). The divalent in requirements will be determined and tests will be applied to distinguish between structural and direct electrostatic effects of divalent cations. A new level of kinetic phenomena underlying the gating of large- conductance Ca-activated K channels will be studied at temperatures down to -30 degrees C using techniques developed in our laboratory. These experiments will investigate the operation of the molecular gate by which the ionic current of the channel pore is controlled. At room temperature this gate acts like an instant switch, but at very low temperature channel transitions are slowed to an extent that a cadence of ion current changes can be measured from which the behavior of the actual gate can be inferred. These studies address fundamental questions concerning ionic channels of neurons and muscle, and thus should provide basic information important for understanding normal and impaired function in these tissues.