Xiao Rui, Tang Jisen, Wang Chunbo, Colton Craig K, Tian Jinbin, Zhu Michael X
Department of Neuroscience and Center for Molecular Neurobiology, Biophysics Graduate Program, Ohio State University, 1060 Carmack Road, Columbus, OH 43210, USA.
J Biol Chem. 2008 Mar 7;283(10):6162-74. doi: 10.1074/jbc.M706535200. Epub 2008 Jan 4.
Transient receptor potential channels are involved in sensing chemical and physical changes inside and outside of cells. TRPV3 is highly expressed in skin keratinocytes, where it forms a nonselective cation channel activated by hot temperatures in the innocuous and noxious range. The channel has also been implicated in flavor sensation in oral and nasal cavities as well as being a molecular target of some allergens and skin sensitizers. TRPV3 is unique in that its activity is sensitized upon repetitive stimulations. Here we investigated the role of calcium ions in the sensitization of TRPV3 to repetitive stimulations. We show that the sensitization is accompanied by a decrease of Ca(2+)-dependent channel inhibition mediated by calmodulin acting at an N-terminal site (amino acids 108-130) and by an acidic residue (Asp(641)) at the pore loop of TRPV3. These sites also contribute to the voltage dependence of TRPV3. During sensitization, the channel displayed a gradual shift of the voltage dependence to more negative potentials as well as uncoupling from voltage sensing. The initial response to ligand stimulation was increased and sensitization to repetitive stimulations was decreased by increasing the intracellular Ca(2+)-buffering strength, inhibiting calmodulin, or disrupting the calmodulin-binding site. Mutation of Asp(641) to Asn abolished the high affinity extracellular Ca(2+)-mediated inhibition and greatly facilitated the activation of TRPV3. We conclude that Ca(2+) inhibits TRPV3 from both the extracellular and intracellular sides. The inhibition is sequentially reduced, appearing as sensitization to repetitive stimulations.
瞬时受体电位通道参与感知细胞内外的化学和物理变化。TRPV3在皮肤角质形成细胞中高度表达，在其中形成一个非选择性阳离子通道，该通道由无害和有害范围内的高温激活。该通道还与口腔和鼻腔的味觉感知有关，并且是一些过敏原和皮肤致敏剂的分子靶点。TRPV3的独特之处在于其活性在重复刺激后会增强。在此，我们研究了钙离子在TRPV3对重复刺激的致敏作用中的作用。我们发现，致敏作用伴随着由钙调蛋白作用于N端位点（氨基酸108 - 130）以及TRPV3孔环处的一个酸性残基（Asp(641)）介导的Ca(2+)依赖性通道抑制作用的减弱。这些位点也对TRPV3的电压依赖性有贡献。在致敏过程中，通道的电压依赖性逐渐向更负的电位偏移，并且与电压传感解偶联。通过增加细胞内Ca(2+)缓冲强度、抑制钙调蛋白或破坏钙调蛋白结合位点，对配体刺激的初始反应增强，对重复刺激的致敏作用减弱。将Asp(641)突变为Asn消除了高亲和力细胞外Ca(2+)介导的抑制作用，并极大地促进了TRPV3的激活。我们得出结论，Ca(2+)从细胞外和细胞内两侧抑制TRPV3。这种抑制作用依次减弱，表现为对重复刺激的致敏作用。
