Mechanisms of Neurotransmitter-gated Ion Channels

描述（由申请人提供）：肌肉烟碱乙酰胆碱受体通道（AChR）是一种配体门控离子通道，在脊椎动物神经肌肉交界处介导快速突触传递。除了在正常神经传递过程中明显相关的作用外，AChR在我们对突触嗜离子受体的理解中发挥了关键作用。这一建议涵盖了配体门控离子通道的两个主要功能方面，即结合-门控-脱敏和离子渗透。关于三联体结合-门控-脱敏，我们的长期目标是了解这三种相互交织的现象如何通过肌肉尼古丁乙酰胆碱受体（AChR）形成突触传递。关于离子渗透，我们的长期目标是了解AChR孔域两侧的六个酸性/碱性氨基酸残基环的作用。虽然很明显，它们一定以某种方式调节了孔末端阳离子的浓度，但这个问题的全部复杂性仍远未被理解。通过对异源表达重组小鼠受体的细胞进行单通道和宏观电流膜片钳记录的定量分析，提出了表征AChR功能的这些方面的实验。第一个目标是测试当通道打开时乙酰胆碱会从结合位点脱落的假设。这是变构蛋白活化理论的基本原则。第二个目标是了解从激动剂到激动剂的封闭和开放状态的配体亲和力如何变化，从而产生一系列的功效。第三个目标是估计在病理条件下（慢通道先天性肌无力综合征）进入脱敏状态对神经肌肉突触传递的贡献程度。第四个目标是了解酸性/碱性残基的孔侧环如何影响离子传导，以及它们的pka值如何被通道的蛋白质环境调节。

DESCRIPTION (provided by applicant): The muscle nicotinic acetylcholine receptor channel (AChR) is a ligand-gated ion channel that mediates fast synaptic transmission at the vertebrate neuromuscular junction. Other than its obviously relevant role during normal neurotransmission, the AChR has played a pivotal role in our understanding of synaptic ionotropic receptors. This proposal covers facets of the two major functional aspects of ligand-gated ion channels, namely, binding-gating-desensitization and ion permeation. With regard to the triad binding-gating-desensitization, our long-term goal is to understand how these three intertwined phenomena shape synaptic transmission through the muscle nicotinic acetylcholine receptor (AChR). With regard to ion permeation, our long-term goal is to understand the role of the six rings of acidic/basic amino-acid residues that flank the pore domain of the AChR. Although it is clear that they must somehow modulate the concentration of cations at the ends of the pore, the problem is still far from being understood in its full complexity. Experiments are proposed to characterize these aspects of the AChR's function through quantitative analysis of single-channel and macroscopic-current patch-clamp recordings performed on cells that heterologously express recombinant mouse receptors. The first goal is to test the hypothesis that acetylcholine can fall off of the binding sites when the channel is open. This is a fundamental tenet of theories of allosteric protein activation. The second goal is to understand how the ligand affinities for the closed and open states change from agonist to agonist to give rise to a range of efficacies. The third goal is to estimate the extent to which entry into desensitization contributes to neuromuscular synaptic transmission in pathological conditions (slow-channel congenital myasthenic syndromes). The fourth goal is to understand how the pore-flanking rings of acidic/basic residues affect ion conduction, and how their pKa-values are modulated by the channel's protein environment.