Mechanisms of Neurotransmitter-gated Ion Channels

描述(申请人提供)：肌肉烟碱型乙酰胆碱受体通道(AChR)是一种配体门控离子通道，在脊椎动物神经肌肉接头处介导快速突触传递。除了在正常神经传递过程中的明显相关作用外，AChR在我们对突触趋性离子受体的理解中也发挥了关键作用。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个相互交织的现象如何通过肌肉烟碱型乙酰胆碱受体(AChR)影响突触传递。关于离子渗透，我们的长期目标是了解AChR孔区两侧的六个酸性/碱性氨基酸残基的作用。尽管很明显，它们必须以某种方式调节毛孔末端的阳离子浓度，但这个问题的全部复杂性仍然远未被理解。通过对异源表达重组小鼠受体的细胞进行的单通道和宏观电流膜片钳记录的定量分析，提出了实验来表征AChR的这些方面的功能。第一个目标是测试当通道打开时，乙酰胆碱可以从结合部位脱落的假设。这是变构蛋白活化理论的一个基本原理。第二个目标是了解闭合和开放状态的配基亲和力如何从激动剂变化到激动剂，从而产生一系列的效果。第三个目标是评估在病理条件下进入脱敏状态对神经肌肉突触传递的影响程度(慢通道先天性肌无力综合征)。第四个目标是了解酸性/碱性残基的孔边环如何影响离子传导，以及它们的pKa值是如何被通道的蛋白质环境调制的。

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.