Crnjar Alessandro, Mesoy Susanne M, Lummis Sarah C R, Molteni Carla
Physics Department, King's College London, London, United Kingdom.
Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.
Front Mol Biosci. 2021 Apr 30;8:644720. doi: 10.3389/fmolb.2021.644720. eCollection 2021.
Pentameric ligand-gated ion channels (pLGICs) mediate fast synaptic transmission and are crucial drug targets. Their gating mechanism is triggered by ligand binding in the extracellular domain that culminates in the opening of a hydrophobic gate in the transmembrane domain. This domain is made of four α-helices (M1 to M4). Recently the outer lipid-facing helix (M4) has been shown to be key to receptor function, however its role in channel opening is still poorly understood. It could act through its neighboring helices (M1/M3), or via the M4 tip interacting with the pivotal Cys-loop in the extracellular domain. Mutation of a single M4 tyrosine (Y441) to alanine renders one pLGIC-the 5-HT receptor-unable to function despite robust ligand binding. Using Y441A as a proxy for M4 function, we here predict likely paths of Y441 action using molecular dynamics, and test these predictions with functional assays of mutant receptors in HEK cells and oocytes using fluorescent membrane potential sensitive dye and two-electrode voltage clamp respectively. We show that Y441 does not act via the M4 tip or Cys-loop, but instead connects radially through M1 to a residue near the ion channel hydrophobic gate on the pore-lining helix M2. This demonstrates the active role of the M4 helix in channel opening.
五聚体配体门控离子通道（pLGICs）介导快速突触传递，是重要的药物靶点。它们的门控机制由细胞外结构域中的配体结合触发，最终导致跨膜结构域中疏水门的打开。该结构域由四个α螺旋（M1至M4）组成。最近，面向外侧脂质的螺旋（M4）已被证明是受体功能的关键，但对其在通道开放中的作用仍知之甚少。它可能通过其相邻螺旋（M1/M3）起作用，或者通过M4末端与细胞外结构域中的关键半胱氨酸环相互作用。将单个M4酪氨酸（Y441）突变为丙氨酸会导致一种pLGIC——5-羟色胺受体——尽管配体结合牢固但无法发挥功能。我们以Y441A作为M4功能的替代物，利用分子动力学预测Y441可能的作用路径，并分别使用荧光膜电位敏感染料和双电极电压钳在HEK细胞和卵母细胞中对突变受体进行功能测定来验证这些预测。我们发现Y441并非通过M4末端或半胱氨酸环起作用，而是通过M1径向连接到孔道内衬螺旋M2上离子通道疏水门附近的一个残基。这证明了M4螺旋在通道开放中的积极作用。
