The activation mechanism of rat vanilloid receptor 1 by capsaicin involves the pore domain and differs from the activation by either acid or heat
The activation mechanism of rat vanilloid receptor 1 by capsaicin involves the pore domain and differs from the activation by either acid or heat
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DOI：
10.1073/pnas.230146497
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发表时间：
2000-12-05
影响因子：
11.1
通讯作者：
Reinhart, PH
中科院分区：
文献类型：
--
作者：
Welch, JM;Simon, SA;Reinhart, PH
最近克隆的大鼠香草素受体，VR 1，可以被辣椒素，酸和热激活。为了确定促进通道开放的分子机制，在这些刺激，VR 1和6个通道含有电荷中和点突变周围的推定的通道孔域表达和非洲爪蟾卵母细胞的特点。使用电压钳技术记录稳态剂量-响应关系、电流-电压关系、离子选择性和单通道特性。三个突变通道是显着更敏感的辣椒素比野生型VR 1，而没有不同的激活酸性：pH值或温度。此外，突变体之一已经失去了辣椒素激活的所有正协同性（希尔系数等于1，VR 1等于2），对Ca 2+的选择性更高，并且表现出对酸的功效低于辣椒素激活。单通道记录表明，辣椒素和酸激活的通道具有相同的电导，增加辣椒素敏感性的三个突变体表现出较高的开放概率在次最大辣椒素浓度，和辣椒素激活的门控特性不同的酸激活。这些数据表明，VR 1经历的构象变化后，辣椒素结合，它不经历在响应质子或热刺激激活。此外，这些结构重排包括推定的孔结构域，并揭示了细胞内结构域的位置，这有助于辣椒素活化所见的正协同性。
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 to 1, VR1 congruent to 2), is much more selective for Ca2+, 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.