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，介导脊椎动物神经肌肉接头处的快速突触传递。AChR除了在正常神经传递过程中起着明显的作用外，在我们对突触离子型受体的理解中也起着关键作用。该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烟碱乙酰胆碱受体（AChR）形成突触传递。关于离子渗透，我们的长期目标是了解AChR孔结构域两侧的酸性/碱性氨基酸残基的六个环的作用。虽然很明显，它们必须以某种方式调节孔端阳离子的浓度，但这个问题的复杂性还远未得到充分理解。建议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来表征AChR功能的这些方面。第一个目标是检验当通道打开时乙酰胆碱可以从结合位点脱落的假设。这是变构蛋白激活理论的基本原则。第二个目标是了解封闭和开放状态的配体亲和力如何从激动剂到激动剂发生变化，以产生一系列功效。第三个目标是评估在病理条件下（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/碱性残基的孔侧翼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.