5-羟色胺门控离子通道(5-HT3R)介导肠道和大脑中的兴奋性神经元通讯。它是SETRONS的目标,一类广泛用作止吐药的竞争性拮抗剂,并参与了几种神经系统疾病。5-HT3R与5-羟色胺或setrons复合的低温电子显微镜(cryo-EM)显示,该蛋白质具有广泛的构象景观。然而,将已知的高分辨率结构分配给有助于生理反应的实际状态仍然是一个挑战。在本研究中,我们使用电压钳荧光法(VCF)同时测量,对于在细胞膜上表达的5-HT3R,荧光和电生理学通道开放的构象变化。通过突变筛选确定的四个位置报告了通过掺入半胱氨酸束缚的罗丹明染料而在5-羟色胺结合位点周围和外部的运动,其中有或没有附近的猝灭色氨酸。VCF记录显示,5-HT3R可以访问四个具有独特荧光特征的构象家族:没有配体的“静息样”,\'类似抑制\'与setrons,“pre-active-like”与部分激动剂,和带有部分和强激动剂的“活性样”(开放通道)。数据与低温EM结构非常一致,分别匹配apo的荧光伴侣,塞创绑定,5-HT封闭,和5-HT结合的开放构象。数据显示,强激动剂在激活过程中促进所有荧光标记传感器的协调运动,而部分激动剂,特别是当功能缺失突变被设计时,稳定活性和前活性构象。总之,VCF,尽管监测电生理沉默的构象变化,阐明了通过重要的生理和临床效应物促进信号转导及其差异调节的变构机制。
The serotonin-gated ion channel (5-HT3R) mediates excitatory neuronal communication in the gut and the brain. It is the target for setrons, a class of competitive antagonists widely used as antiemetics, and is involved in several neurological diseases. Cryo-electron microscopy (cryo-EM) of the 5-HT3R in complex with serotonin or setrons revealed that the protein has access to a wide conformational landscape. However, assigning known high-resolution structures to actual states contributing to the physiological response remains a challenge. In the present study, we used voltage-clamp fluorometry (VCF) to measure simultaneously, for 5-HT3R expressed at a cell membrane, conformational changes by fluorescence and channel opening by electrophysiology. Four positions identified by mutational screening report motions around and outside the serotonin-binding site through incorporation of cysteine-tethered rhodamine dyes with or without a nearby quenching tryptophan. VCF recordings show that the 5-HT3R has access to four families of conformations endowed with distinct fluorescence signatures: \'resting-like\' without ligand, \'inhibited-like\' with setrons, \'pre-active-like\' with partial agonists, and \'active-like\' (open channel) with partial and strong agonists. Data are remarkably consistent with cryo-EM structures, the fluorescence partners matching respectively apo, setron-bound, 5-HT bound-closed, and 5-HT-bound-open conformations. Data show that strong agonists promote a concerted motion of all fluorescently labeled sensors during activation, while partial agonists, especially when loss-of-function mutations are engineered, stabilize both active and pre-active conformations. In conclusion, VCF, though the monitoring of electrophysiologically silent conformational changes, illuminates allosteric mechanisms contributing to signal transduction and their differential regulation by important classes of physiological and clinical effectors.