A Single Mutation in the Outer Lipid-Facing Helix of a Pentameric Ligand-Gated Ion Channel Affects Channel Function Through a Radially-Propagating Mechanism.
A Single Mutation in the Outer Lipid-Facing Helix of a Pentameric Ligand-Gated Ion Channel Affects Channel Function Through a Radially-Propagating Mechanism.
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DOI：
10.3389/fmolb.2021.644720
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发表时间：
2021
影响因子：
5
通讯作者：
Molteni C
中科院分区：
文献类型：
--
作者：
Crnjar A;Mesoy SM;Lummis SCR;Molteni C
五聚体配体门控离子通道（pLGIC）介导快速突触传递，并且是关键的药物靶标。它们的门控机制由胞外结构域中的配体结合触发，其最终在跨膜结构域中的疏水门的打开中达到高潮。该结构域由四个α螺旋（M1至M4）组成。最近，外脂螺旋（M4）已被证明是受体功能的关键，但其在通道开放中的作用仍然知之甚少。它可以通过其相邻的螺旋（M1/M3），或通过M4尖端与细胞外结构域中的关键Cys环相互作用来发挥作用。单个M4酪氨酸（Y 441）突变为丙氨酸使得一个pLGIC-5-HT 3A受体-尽管与配体结合稳健但不能发挥功能。使用Y 441 A作为M4功能的代理，我们在这里预测可能的途径Y 441行动使用分子动力学，并测试这些预测与功能测定突变受体在HEK细胞和非洲爪蟾卵母细胞分别使用荧光膜电位敏感染料和双电极电压钳。我们发现，Y 441不通过M4尖端或Cys环起作用，而是通过M1径向连接到孔衬螺旋M2上离子通道疏水门附近的残基。这证明了M4螺旋在通道开放中的积极作用。
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-HT3A 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 Xenopus 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.