Distinct properties of Ca2+-calmodulin binding to N- and C-terminal regulatory regions of the TRPV1 channel.
Distinct properties of Ca2+-calmodulin binding to N- and C-terminal regulatory regions of the TRPV1 channel.
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DOI：
10.1085/jgp.201210810
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发表时间：
2012-11
期刊：
影响因子：
--
通讯作者：
Gaudet R
中科院分区：
文献类型：
--
作者：
Lau SY;Procko E;Gaudet R
瞬时受体电位(Trp)香草素1(TRPV1)是一种分子痛觉受体，属于Trp非选择性阳离子通道超家族。作为一种多通道受体，TRPV1对热和多种化学刺激都有反应。通道激活后钙离子的内流是导致TRPV1脱敏的负反馈机制。细胞内钙传感器钙调蛋白(CaM)可能参与了TRPV1的脱敏作用。在TRPV1中发现了两个CaM结合位点：N端Ankyrin重复结构域(ARD)和远端短C端(CT)片段。在这里，我们提出了钙结合CaM(Ca~(2+)-CaM)与TRPV1-CT片段的络合物的晶体结构，测定到1.95°分辨率。Ca~(2+)-CaM的两个叶以反平行的方向缠绕在一个螺旋的TRPV1-CT片段上，两个疏水锚定W787和L796分别与Ca~(2+)-CaM的C叶和N叶接触。这种结构类似于典型的钙-CaM-多肽复合体，尽管TRPV1不包含经典的CaM识别序列基序。利用结构和突变研究，我们在分离的TRPV1 C末端和全长TRPV1中都建立了TRPV1 C末端作为高亲和力的钙-CaM结合位点。虽然先前已经假设了CaM、TRPV1-ARD和TRPV1-CT的三元复合体，但我们没有发现这种复合体的生化证据。在电生理学研究中，TRPV1-ARD上的Ca~(2+)-CaM结合位点的突变取消了重复使用辣椒素后的脱敏反应，而TRPV1-CT上的Ca~(2+)-CaM结合位点的突变导致了更微妙的减慢和降低TRPV1脱敏的表型。综上所述，我们的结果表明，TRPV1-ARD是TRPV1脱敏的重要介质，而TRPV1-CT对CaM具有更高的亲和力，可能参与了不同的调节机制。
Transient receptor potential (TRP) vanilloid 1 (TRPV1) is a molecular pain receptor belonging to the TRP superfamily of nonselective cation channels. As a polymodal receptor, TRPV1 responds to heat and a wide range of chemical stimuli. The influx of calcium after channel activation serves as a negative feedback mechanism leading to TRPV1 desensitization. The cellular calcium sensor calmodulin (CaM) likely participates in the desensitization of TRPV1. Two CaM-binding sites are identified in TRPV1: the N-terminal ankyrin repeat domain (ARD) and a short distal C-terminal (CT) segment. Here, we present the crystal structure of calcium-bound CaM (Ca2+–CaM) in complex with the TRPV1-CT segment, determined to 1.95-Å resolution. The two lobes of Ca2+–CaM wrap around a helical TRPV1-CT segment in an antiparallel orientation, and two hydrophobic anchors, W787 and L796, contact the C-lobe and N-lobe of Ca2+–CaM, respectively. This structure is similar to canonical Ca2+–CaM-peptide complexes, although TRPV1 contains no classical CaM recognition sequence motif. Using structural and mutational studies, we established the TRPV1 C terminus as a high affinity Ca2+–CaM-binding site in both the isolated TRPV1 C terminus and in full-length TRPV1. Although a ternary complex of CaM, TRPV1-ARD, and TRPV1-CT had previously been postulated, we found no biochemical evidence of such a complex. In electrophysiology studies, mutation of the Ca2+–CaM-binding site on TRPV1-ARD abolished desensitization in response to repeated application of capsaicin, whereas mutation of the Ca2+–CaM-binding site in TRPV1-CT led to a more subtle phenotype of slowed and reduced TRPV1 desensitization. In summary, our results show that the TRPV1-ARD is an important mediator of TRPV1 desensitization, whereas TRPV1-CT has higher affinity for CaM and is likely involved in separate regulatory mechanisms.
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DOI：
10.1016/j.str.2008.07.010
发表时间：
2008-10-08
期刊：
Structure (London, England : 1993)
影响因子：
--
作者：
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10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
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