Wulffraat Grace C, Mamathasateesh Sanjana, Hudson Rose, He Benjamin, Jara-Oseguera Andrés, Senning Eric N
Department of Neuroscience, University of Texas at Austin, Austin, Texas, United States of America.
Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas, United States of America.
PLoS One. 2025 Sep 2;20(9):e0331224. doi: 10.1371/journal.pone.0331224. eCollection 2025.
The Transient Receptor Potential Vanilloid sub-type 1 (TRPV1) is an ion channel that is activated by heat, extracellular protons, oxidation, and it is implicated in various aspects of inflammatory pain. In this study, we uncover that residue M308 in the TRPV1 ankyrin repeat domain (ARD) stands out from most other buried ARD residues because of the greater number of human missense variants at this position while maintaining a high degree of conservation across species and TRPV channel subtypes. We use mutagenesis and electrophysiology to examine this apparent discrepancy and show that substitutions at position M308 that preserve or reduce side-chain volume have no effect on channel function, whereas substitutions with larger or more polar residues increase channel activity in response to capsaicin or temperature. Substitution of M308 with a histidine bestows channels with pH-dependence that is different from wild type, consistent with the side-chain at position 308 exerting an influence on channel gating. We speculate that M308 is highly conserved because its side-chain could serve as a target for oxidation-dependent modification. On the other hand, we show that a previously described splice variant of TRPV1 that relies on M308 as a start codon diminishes surface expression of co-transfected full-length TRPV1 in HEK293 cells. Together, our findings reveal a functionally important conserved site within the ARD of TRPV1 that could have roles in oxidation-dependent channel regulation as well as tuning the number of active channels in the membrane by enabling expression of a shorter dominant-negative splice variant.
瞬时受体电位香草酸亚型1（TRPV1）是一种离子通道，可被热、细胞外质子、氧化激活，并与炎性疼痛的各个方面有关。在本研究中，我们发现TRPV1锚蛋白重复结构域（ARD）中的M308残基与大多数其他埋藏的ARD残基不同，因为该位置的人类错义变体数量更多，同时在物种和TRPV通道亚型中保持高度保守。我们使用诱变和电生理学来研究这种明显的差异，并表明在M308位置进行的保留或减小侧链体积的取代对通道功能没有影响，而用更大或更具极性的残基进行取代会增加通道对辣椒素或温度的反应活性。用组氨酸取代M308赋予通道与野生型不同的pH依赖性，这与308位的侧链对通道门控产生影响一致。我们推测M308高度保守是因为其侧链可作为氧化依赖性修饰的靶点。另一方面，我们表明先前描述的依赖M308作为起始密码子的TRPV1剪接变体减少了HEK293细胞中共转染的全长TRPV1的表面表达。总之，我们的研究结果揭示了TRPV1的ARD内一个功能上重要的保守位点，该位点可能在氧化依赖性通道调节中发挥作用，以及通过允许表达较短的显性负性剪接变体来调节膜中活性通道的数量。
