Desensitization of Nicotinic Acetylcholine Receptors

神经-肌肉突触的烟碱型乙酰胆碱受体(AChRs)进入和恢复 D(敏感)状态，在神经递质持续存在的情况下保持不活跃。 受体脱敏下调细胞反应，并编码长期活动模式。 我们的目标是在分子水平上了解AChR的脱敏过程。最重要的 D态如何连接到C(丢失的)和O(PEN)选通态的问题仍然没有解决。 我们将研究两个假设：i)D、O和C在闭合循环中相连，而ii)D不相连 连接到C或O，而不是连接到短暂的门控中间状态。我们将测试 第二个假设是通过使用突变和电压来移动点 门控和减敏构象变化通路之间的分叉，指向C to 在恢复时产生开口或朝向O方向，以防止开口。许多氨基酸会发生变化 在AChR门控和脱敏转变期间的结构，以及这些微观结构中的每一个 重排有一个相关的，而且主要是局部的自由能变化。我们将衡量这些指标 AChR跨膜区两个区域脱敏的能量变化：i)a M_1和II)疏水性M_2门中的Pro扭结。尽管进行了密集的调查，但 AChR脱敏的分子基础自AChR脱敏第一次以来一直不清楚 描述于60年前。这项提案中的实验将填补我们在 了解受体的工作原理。

Nicotinic acetylcholine receptors (AChRs) at the nerve-muscle synapse enter and recover from D(esensitized) states that remain inactive in the continuous presence of neurotransmitter. Receptor desensitization down-regulates cell responses and encodes long-term activity patterns. Our goal is to understand the AChR desensitization process at a molecular level. The essential problem of how D states are connected to C(losed) and O(pen) gating states remains unsolved. We will investigate two hypotheses: i) D, O and C are connected in a closed cycle and ii) D is not connected either to C or O but rather to a short-lived gating intermediate state. We will test the second hypothesis experimentally by using mutations and voltage to shift the point of bifurcation between gating and desensitization conformational-change pathways, towards C to produce openings upon recovery or towards O to prevent openings. Many amino acids change structure during the AChR gating and desensitization transitions, and each of these microscopic rearrangements has an associated and mostly-local free energy change. We will measure these energy changes in desensitization at two regions in the AChR transmembrane domain: i) a proline kink in M1 and ii) the hydrophobic M2 gate. Despite intensive investigation, the molecular basis of AChR desensitization has remained obscure since the process was first described 60 years ago. The experiments in this proposal will fill this substantial gap in our understanding of receptor operation.