Transient receptor potential canonical (TRPC) channels, as important membrane proteins regulating intracellular calcium (Ca2+i) signaling, are involved in a variety of physiological and pathological processes. Activation and regulation of TRPC are more dependent on membrane or intracellular signals. However, how extracellular signals regulate TRPC6 function remains to be further investigated. Here, we suggest that two distinct small molecules, M085 and GSK1702934A, directly activate TRPC6, both through a mechanism of stimulation of extracellular sites formed by the pore helix (PH) and transmembrane (TM) helix S6. In silico docking scanning of TRPC6 identified three extracellular sites that can bind small molecules, of which only mutations on residues of PH and S6 helix significantly reduced the apparent affinity of M085 and GSK1702934A and attenuated the maximal response of TRPC6 to these two chemicals by altering channel gating of TRPC6. Combing metadynamics, molecular dynamics simulations, and mutagenesis, we revealed that W679, E671, E672, and K675 in the PH and N701 and Y704 in the S6 helix constitute an orthosteric site for the recognition of these two agonists. The importance of this site was further confirmed by covalent modification of amino acid residing at the interface of the PH and S6 helix. Given that three structurally distinct agonists M085, GSK1702934A, and AM-0883, act at this site, as well as the occupancy of lipid molecules at this position found in other TRP subfamilies, it is suggested that the cavity formed by the PH and S6 has an important role in the regulation of TRP channel function by extracellular signals.

中文翻译：

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GSK1702934A和M085通过刺激由孔螺旋和跨膜螺旋S6形成的细胞外腔的机制直接激活TRPC6。


瞬时受体电位经典通道（TRPC）作为调节细胞内钙（Ca2+i）信号传导的重要膜蛋白，参与多种生理和病理过程。 TRPC的激活和调节更依赖于膜或细胞内信号。然而，细胞外信号如何调节TRPC6功能仍有待进一步研究。在这里，我们建议两种不同的小分子 M085 和 GSK1702934A 直接激活 TRPC6，两者都是通过刺激由孔螺旋 (PH) 和跨膜 (TM) 螺旋 S6 形成的细胞外位点的机制。 TRPC6的计算机对接扫描发现了三个可以结合小分子的胞外位点，其中仅PH和S6螺旋残基上的突变显着降低了M085和GSK1702934A的表观亲和力，并通过改变通道减弱了TRPC6对这两种化学物质的最大反应TRPC6 的门控。结合元动力学、分子动力学模拟和诱变，我们揭示了 PH 中的 W679、E671、E672 和 K675 以及 S6 螺旋中的 N701 和 Y704 构成了识别这两种激动剂的正构位点。通过对 PH 和 S6 螺旋界面上的氨基酸进行共价修饰，进一步证实了该位点的重要性。鉴于三种结构不同的激动剂 M085、GSK1702934A 和 AM-0883 在该位点起作用，以及在其他 TRP 亚家族中发现的脂质分子占据该位置，表明由 PH 和 S6 形成的空腔具有在细胞外信号调节 TRP 通道功能中发挥重要作用。