The TRPM8 ion channel comprises direct Gq protein-activating capacity
The TRPM8 ion channel comprises direct Gq protein-activating capacity
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DOI：
10.1007/s00424-012-1098-7
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发表时间：
2012-06-01
影响因子：
4.5
通讯作者：
Wetzel, Christian H.
中科院分区：
文献类型：
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作者：
Klasen, Katharina;Hollatz, Dominik;Wetzel, Christian H.
瞬时受体电位(Trp)家族的离子通道由多种物理和化学刺激所激活的受体组成。Trp通道以一种复杂的方式与几个细胞内信号级联相互作用，包括受体激活的上下游。研究冷刺激和薄荷醇受体TRPM8下游的级联反应，我们发现了TRPM8与GαQ在功能和结构上的相互作用的证据。我们证明了在细胞外无钙条件下，薄荷醇引起的细胞内钙增加，这可被PLC抑制剂U73122或EDELFOSIN阻断。这种代谢性钙信号也可以在表达通道死亡(即非传导)或氯离子传导的TRPM8孔突变体的细胞中观察到。然而，在GαQ缺乏的细胞或在显性阴性GαQx存在的情况下，不能检测到这种细胞内代谢性钙信号。受体漂白实验证实了TRPM8-CFP和GαQ-YFP之间的FRET，证明了TRPM8-CFP和GαQ-YFP的物理相互作用所需的密切空间接近的证据。GαQ-YFP的迁移率分析(FRAP)显示，当TRPM8固定在质膜上时，GαQ-YFP的扩散受到限制。此外，通过FRET实验监测GαQ-YFP从Gβ/Gγ-CFP复合体的解离，以及通过放射性[S-35]GTPγS与国内生产总值的交换，可以证明薄荷醇诱导和TRPM8介导的G蛋白激活。我们的观察导致了这样一种观点，即TRPM8受体的工作范围从其作为纯离子通道的功能扩展到具有额外代谢能力的分子开关。
The transient receptor potential (TRP) family of ion channels comprises receptors that are activated by a vast variety of physical as well as chemical stimuli. TRP channels interact in a complex manner with several intracellular signaling cascades, both up- and downstream of receptor activation. Investigating cascades stimulated downstream of the cold and menthol receptor TRPM8, we found evidence for both, functional and structural interaction of TRPM8 with G alpha q. We demonstrated menthol-evoked increase in intracellular Ca2+ under extracellular Ca2+-free conditions, which was blocked by the PLC inhibitors U73122 or edelfosine. This metabotropic Ca2+ signal could be observed also in cells expressing a channel-dead (i.e. non-conducting) or a chloride-conducting TRPM8 pore mutant. However, this intracellular metabotropic Ca2+ signal could not be detected in G alpha q deficient cells or in the presence of dominant-negative G alpha qX. Evidence for a close spatial proximity necessary for physical interaction of TRPM8 and G alpha q was provided by acceptor bleaching experiments demonstrating FRET between TRPM8-CFP and G alpha q-YFP. A G alpha q-YFP mobility assay (FRAP) revealed a restricted diffusion of G alpha q-YFP under conditions when TRPM8 is immobilized in the plasma membrane. Moreover, a menthol-induced and TRPM8-mediated G protein activation could be demonstrated by FRET experiments monitoring the dissociation of G alpha q-YFP from a G beta/G gamma-CFP complex, and by the exchange of radioactive [S-35]GTP gamma S for GDP. Our observations lead to a view that extends the operational range of the TRPM8 receptor from its function as a pure ion channel to a molecular switch with additional metabotropic capacity.