Rudolf Daniel, Pombeiro Stein Inês C A, Sturhahn Toni, Wunder Julian, Hage Axel, Leffler Andreas
Department of Anesthesiology and Intensive Care Medicine, Hannover Medical School, 30625 Hannover, Germany.
Int J Mol Sci. 2025 Aug 26;26(17):8275. doi: 10.3390/ijms26178275.
Several transient receptor potential vanilloid (TRPV) ion channels are proton-sensitive, and recent structural studies have identified poorly conserved mechanisms for the proton sensitivity of TRPV1, TRPV2 and TRPV5. While such detailed studies are lacking for TRPV3, three distinct intracellular motifs were suggested to be required for a direct channel activation by cytosolic acidification. In this study, we investigated if these mechanisms are also relevant for the activation of TRPV3 by weak acids. Wildtype (WT) and several mutants of human TRPV3 transiently expressed in HEK 293T cells were investigated by whole-cell patch clamp electrophysiology. Cells expressing TRPV3-WT generated membrane currents induced by acetic acid (HOAc), formic acid and carbonic acid at pH 5.0. Activation induced by HOAc was concentration-dependent and increased with decreasing pH values. HOAc also strongly potentiated TRPV3-mediated responses to carvacrol and heat. Among the three suggested motifs for the binding of intracellular protons, only the mutant TRPV3-Asp512Ala exhibited an almost complete loss of HOAc sensitivity. The mutation of two C-terminal charged residues (Gln689/Asp727) even resulted in a clear gain of function for both HOAc and heat, and the mutation of the 2-APB-binding site His426 did not significantly abrogate HOAc sensitivity. Finally, insertion of the recently identified binding site in TRPV2 for the weak acid probenecid into TRPV3 (Glu216His) resulted in an increased HOAc sensitivity. To conclude, our data confirm that TRPV3 is sensitized and activated by several weak acids. While Asp512 appears to be a critical intracellular proton-modulating site, a more profound understanding of the mechanisms dictating the proton sensitivity of TRPV3 may require structural studies.
几种瞬时受体电位香草酸（TRPV）离子通道对质子敏感，最近的结构研究已经确定了TRPV1、TRPV2和TRPV5质子敏感性的保守性较差的机制。虽然缺乏对TRPV3的此类详细研究，但有人提出，胞质酸化直接激活通道需要三个不同的细胞内基序。在本研究中，我们调查了这些机制是否也与弱酸激活TRPV3有关。通过全细胞膜片钳电生理学研究了在HEK 293T细胞中瞬时表达的野生型（WT）和几种人TRPV3突变体。表达TRPV3-WT的细胞在pH 5.0时产生由乙酸（HOAc）、甲酸和碳酸诱导的膜电流。HOAc诱导的激活具有浓度依赖性，并随着pH值的降低而增加。HOAc还强烈增强了TRPV3介导的对香芹酚和热的反应。在三个建议的细胞内质子结合基序中，只有突变体TRPV3-Asp512Ala几乎完全丧失了对HOAc的敏感性。两个C末端带电残基（Gln689/Asp727）的突变甚至导致对HOAc和热的功能明显增强，而2-APB结合位点His426的突变并没有显著消除HOAc敏感性。最后，将最近在TRPV2中确定的弱酸丙磺舒结合位点插入TRPV3（Glu216His）导致HOAc敏感性增加。总之，我们的数据证实TRPV3被几种弱酸致敏并激活。虽然Asp512似乎是一个关键的细胞内质子调节位点，但要更深入地了解决定TRPV3质子敏感性的机制可能需要进行结构研究。
