Welch J M, Simon S A, Reinhart P H
Departments of Neurobiology and Anesthesiology, Duke University Medical Center, Durham, NC 27710, USA.
Proc Natl Acad Sci U S A. 2000 Dec 5;97(25):13889-94. doi: 10.1073/pnas.230146497.
The recently cloned rat vanilloid receptor, VR1, can be activated by capsaicin, acid, and heat. To determine the molecular mechanisms facilitating channel opening in response to these stimuli, VR1 and six channels containing charge neutralization point mutations surrounding the putative channel pore domain were expressed and characterized in Xenopus laevis oocytes. Steady-state dose-response relationships, current-voltage relationships, ionic selectivities, and single-channel properties were recorded using voltage-clamp techniques. Three of the mutant channels are significantly more sensitive to capsaicin than is wild-type VR1, whereas none differed in their activation by acidic pH or temperature. Furthermore, one of the mutants has lost all positive cooperativity for capsaicin activation (Hill coefficient congruent with 1, VR1 congruent with 2), is much more selective for Ca(2+), and exhibits a lower efficacy for acid than for capsaicin activation. Single-channel recordings show that capsaicin- and acid-activated channels have the same conductance, that the three mutants with increased capsaicin sensitivity exhibit higher open probabilities at submaximal capsaicin concentrations, and that the gating properties of capsaicin activation differ from those of acid activation. These data indicate that VR1 undergoes conformational changes upon capsaicin binding that it does not undergo in response to activation by protons or thermal stimuli. Furthermore, these structural rearrangements include the putative pore domain and reveal the location of an intracellular domain that contributes to the positive cooperativity seen for capsaicin activation.
最近克隆出的大鼠香草酸受体VR1可被辣椒素、酸和热激活。为了确定响应这些刺激促进通道开放的分子机制，在非洲爪蟾卵母细胞中表达并鉴定了VR1以及六个围绕假定通道孔结构域含有电荷中和点突变的通道。使用电压钳技术记录稳态剂量-反应关系、电流-电压关系、离子选择性和单通道特性。其中三个突变通道对辣椒素的敏感性明显高于野生型VR1，而在酸性pH或温度激活方面没有差异。此外，其中一个突变体对辣椒素激活失去了所有正协同性（希尔系数等于1，VR1等于2），对Ca(2+)的选择性更高，并且对酸的激活效力低于对辣椒素的激活效力。单通道记录表明，辣椒素和酸激活的通道具有相同的电导，三个对辣椒素敏感性增加的突变体在亚最大辣椒素浓度下表现出更高的开放概率，并且辣椒素激活的门控特性与酸激活的不同。这些数据表明，VR1在辣椒素结合时会发生构象变化，而在质子或热刺激激活时不会发生这种变化。此外，这些结构重排包括假定的孔结构域，并揭示了一个细胞内结构域的位置，该结构域有助于辣椒素激活时出现的正协同性。
