Abstract：
TRPV1 acts as a unique polymodal ion channel having distinct structure and gating properties. In this context, TRPV1-R575D represents a special mutant located at the inner lipid-water-interface (LWI) region that has less possibility of interaction with membrane cholesterol. In control conditions, this lab-generated mutant of TRPV1 shows no \"ligand-sensitivity\", reduced surface expression, reduced localization in the lipid rafts, yet induces high cellular lethality. Notably, the cellular lethality induced by TRPV1-R575D expression can be rescued by adding 5\'I-RTX (a specific inhibitor of TRPV1) or by introducing another mutation in the next position, i.e. in TRPV1-R575D/D576R. In this work we characterized TRPV1-R575D and TRPV1-R575D/D576R mutants in different cellular conditions and compared with the TRPV1-WT. We report that the \"ligand-insensitivity\" of TRPV1-R575D can be rescued in certain conditions, such as by chelation of extracellular Ca2+, or by reduction of the membrane cholesterol. Here we show that Ca2+ plays an important role in the channel gating of TRPV1-WT as well as LWI mutants (TRPV1-R575D, TRPV1-R575D/D576R). However, chelation of intracellular Ca2+ or depletion of ER Ca2+ did not have a significant effect on the TRPV1-R575D. Certain properties related to channel gating of mutant TRPV1-R575D/D576R can be rescued partially or fully in a context -dependent manner. Cholesterol depletion also alters these properties. Our data suggests that lower intracellular basal Ca2+ acts as a pre-requisite for further opening of TRPV1-R575D. These findings enable better understanding of the structure-function relationship of TRPV1 and may be critical in comprehending the channelopathies induced by other homologous thermosensitive TRPVs.
摘要：
TRPV1充当具有不同结构和门控性质的独特多峰离子通道。在这种情况下，TRPV1-R575D代表位于内部脂质-水界面（LWI）区域的特殊突变体，与膜胆固醇相互作用的可能性较小。在控制条件下，这个实验室产生的TRPV1突变体没有显示“配体敏感性”，减少表面表达，减少脂筏中的定位，但诱导高细胞致死率。值得注意的是,TRPV1-R575D表达诱导的细胞致死性可以通过添加5'I-RTX（TRPV1的特异性抑制剂）或通过在下一个位置引入另一个突变来挽救，即在TRPV1-R575D/D576R中。在这项工作中，我们在不同的细胞条件下表征了TRPV1-R575D和TRPV1-R575D/D576R突变体，并与TRPV1-WT进行了比较。我们报告说，TRPV1-R575D的“配体不敏感性”可以在某些条件下得到拯救，例如通过细胞外Ca2+的螯合，或通过减少膜胆固醇。在这里，我们表明Ca2+在TRPV1-WT以及LWI突变体的通道门控中起着重要作用(TRPV1-R575D，TRPV1-R575D/D576R)。然而,细胞内Ca2的螯合或ERCa2的消耗对TRPV1-R575D没有显着影响。与突变体TRPV1-R575D/D576R的通道门控相关的某些特性可以以上下文依赖性方式部分或完全挽救。胆固醇消耗也改变了这些性质。我们的数据表明，较低的细胞内基础Ca2充当进一步打开TRPV1-R575D的先决条件。这些发现使人们能够更好地了解TRPV1的结构-功能关系，并且对于理解其他同源热敏TRPVs诱导的通道病至关重要。
