Bhave Gautam, Hu Hui-Juan, Glauner Kathi S, Zhu Weiguo, Wang Haibin, Brasier D J, Oxford Gerry S, Gereau Robert W
Division of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA.
Proc Natl Acad Sci U S A. 2003 Oct 14;100(21):12480-5. doi: 10.1073/pnas.2032100100. Epub 2003 Oct 1.
Protein kinase C (PKC) modulates the function of the capsaicin receptor transient receptor potential vanilloid 1 (TRPV1). This modulation manifests as increased current when the channel is activated by capsaicin. In addition, studies have suggested that phosphorylation by PKC might directly gate the channel, because PKC-activating phorbol esters induce TRPV1 currents in the absence of applied ligands. To test whether PKC both modulates and gates the TRPV1 function by direct phosphorylation, we used direct sequencing to determine the major sites of PKC phosphorylation on TRPV1 intracellular domains. We then tested the ability of the PKC-activating phorbol 12-myristate 13-acetate (PMA) to potentiate capsaicin-induced currents and to directly gate TRPV1. We found that mutation of S800 to alanine significantly reduced the PMA-induced enhancement of capsaicin-evoked currents and the direct activation of TRPV1 by PMA. Mutation of S502 to alanine reduced PMA enhancement of capsaicin-evoked currents, but had no effect on direct activation of TRPV1 by PMA. Conversely, mutation of T704 to alanine had no effect on PMA enhancement of capsaicin-evoked currents but dramatically reduced direct activation of TRPV1 by PMA. These results, combined with pharmacological studies showing that inactive phorbol esters also weakly activate TRPV1, suggest that PKC-mediated phosphorylation modulates TRPV1 but does not directly gate the channel. Rather, currents induced by phorbol esters result from the combination of a weak direct ligand-like activation of TRPV1 and the phosphorylation-induced enhancement of the TRPV1 function. Furthermore, modulation of the TRPV1 function by PKC appears to involve distinct phosphorylation sites depending on the mechanism of channel activation.
蛋白激酶C（PKC）可调节辣椒素受体瞬时受体电位香草酸亚型1（TRPV1）的功能。这种调节表现为当该通道被辣椒素激活时电流增加。此外，研究表明PKC介导的磷酸化可能直接控制该通道的开启，因为激活PKC的佛波酯在未施加配体的情况下可诱导TRPV1产生电流。为了测试PKC是否通过直接磷酸化来调节和控制TRPV1的功能，我们采用直接测序法来确定TRPV1胞内结构域上PKC磷酸化的主要位点。然后，我们测试了激活PKC的佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）增强辣椒素诱导电流以及直接开启TRPV1的能力。我们发现，将S800突变为丙氨酸可显著降低PMA诱导的辣椒素诱发电流增强以及PMA对TRPV1的直接激活。将S502突变为丙氨酸可降低PMA对辣椒素诱发电流的增强作用，但对PMA直接激活TRPV1没有影响。相反，将T704突变为丙氨酸对PMA增强辣椒素诱发电流没有影响，但显著降低了PMA对TRPV1的直接激活。这些结果，结合药理学研究表明无活性的佛波酯也能微弱激活TRPV1，提示PKC介导的磷酸化调节TRPV1，但不直接控制通道的开启。相反，佛波酯诱导的电流是由TRPV1的弱直接配体样激活与磷酸化诱导的TRPV1功能增强共同作用的结果。此外，PKC对TRPV1功能的调节似乎取决于通道激活机制，涉及不同的磷酸化位点。
