Kuzhikandathil E V, Wang H, Szabo T, Morozova N, Blumberg P M, Oxford G S
Department of Cell and Molecular Physiology and the Neuroscience Center, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
J Neurosci. 2001 Nov 15;21(22):8697-706. doi: 10.1523/JNEUROSCI.21-22-08697.2001.
The recently cloned vanilloid receptor subtype 1 (VR1) is a ligand-gated channel that is activated by capsaicin, protons, and heat. We have attempted to develop a dominant negative isoform by targeting several mutations of VR1 at highly conserved amino acids or at residues of potential functional importance and expressing the mutants in Chinese hamster ovary cells. Mutation of three highly conserved amino acid residues in the putative sixth transmembrane domain disrupts activation of the VR1 receptor by both capsaicin and resiniferatoxin. The vanilloid binding site in this mutant is intact, although the affinity for [(3)H]resiniferatoxin (RTX) is diminished by nearly 40-fold. Interestingly, this mutant retains a significant but diminished response to protons, supporting the existence of multiple gating mechanisms for different stimuli. The mutant appears to function by interfering with the gating induced by vanilloids rather than the expression level or permeability of the receptor. In addition, this mutant was found to function as a strong dominant negative mutation when coexpressed with wild-type VR1, providing functional evidence that the VR1 receptor forms a multimeric complex. Analysis of both current density and [(3)H]RTX affinity in cells cotransfected with different ratios of wild-type and mutant VR1 is consistent with tetrameric stoichiometry for the native capsaicin receptor.
最近克隆的香草酸受体亚型1（VR1）是一种配体门控通道，可被辣椒素、质子和热激活。我们试图通过针对VR1在高度保守氨基酸或潜在功能重要性残基处的几个突变来开发一种显性负性异构体，并在中国仓鼠卵巢细胞中表达这些突变体。假定的第六个跨膜结构域中三个高度保守氨基酸残基的突变破坏了辣椒素和树脂毒素对VR1受体的激活。尽管该突变体对[³H]树脂毒素（RTX）的亲和力降低了近40倍，但其香草酸结合位点仍然完整。有趣的是，该突变体对质子仍保留显著但减弱的反应，这支持了针对不同刺激存在多种门控机制的观点。该突变体似乎通过干扰香草酸诱导的门控而发挥作用，而不是通过受体的表达水平或通透性。此外，当与野生型VR1共表达时，发现该突变体作为一种强显性负性突变发挥作用，提供了VR1受体形成多聚体复合物的功能证据。对共转染不同比例野生型和突变型VR1的细胞中的电流密度和[³H]RTX亲和力进行分析，与天然辣椒素受体的四聚体化学计量一致。
