Phillips Elsa, Reeve Alison, Bevan Stuart, McIntyre Peter
Novartis Institutes of Biomedical Research, Novartis Institute for Medical Sciences, 5 Gower Place, London WC1E 6BN, United Kingdom.
J Biol Chem. 2004 Apr 23;279(17):17165-72. doi: 10.1074/jbc.M313328200. Epub 2004 Feb 11.
The vanilloid receptor 1 (VR1 or TRPV1) ion channel is activated by noxious heat, low pH and by a variety of vanilloid-related compounds. The antagonist, capsazepine is more effective at inhibiting the human TRPV1 response to pH 5.5 than the rat TRPV1 response to this stimulus. Mutation of rat TRPV1 at three positions in the S3 to S4 region, to the corresponding human amino acid residues I514M, V518L, and M547L decreased the IC(50) values for capsazepine inhibition of the pH 5.5 response from >10,000 nm to 924 +/- 241 nm in Ca(2+) assays and increased capsazepine inhibition of the capsaicin response to levels seen for human TRPV1. We have previously noted that phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV) is a strong agonist of rat TRPV1 but not human TRPV1 in Ca(2+) assays (1). Mutation of methionine 547 in S4 of rat TRPV1 to leucine, found in human TRPV1 (M547L), reduced the ability of PPAHV to activate TRPV1 by approximately 20-fold. The reciprocal mutation of human TRPV1 (L547M) enabled the human receptor to respond to PPAHV. These mutations did not significantly affect the agonist activity of capsaicin, resiniferatoxin (RTX) or olvanil in Ca(2+) assays. Introducing the equivalent mutation into guinea pig TRPV1 (L549M) increased the agonist potency of PPAHV by > 10-fold in the Ca(2+) assay and increased the amplitude of the evoked current. The rat M547L mutation reduced the affinity of RTX binding. Thus, amino acids within the S2-S4 region are important sites of agonist and antagonist interaction with TRPV1.
香草酸受体1（VR1或TRPV1）离子通道可被有害热、低pH值以及多种与香草酸相关的化合物激活。拮抗剂辣椒平抑制人类TRPV1对pH 5.5的反应比抑制大鼠TRPV1对该刺激的反应更有效。大鼠TRPV1在S3至S4区域的三个位置突变为相应的人类氨基酸残基I514M、V518L和M547L，在[Ca(2+)]i测定中，辣椒平抑制pH 5.5反应的IC(50)值从>10,000 nm降至924±241 nm，并使辣椒平对辣椒素反应的抑制作用增加到人类TRPV1的水平。我们之前注意到，在[Ca(2+)]i测定中，佛波醇12-苯乙酸13-乙酸20-高香草酸酯（PPAHV）是大鼠TRPV1的强激动剂，但不是人类TRPV1的强激动剂（1）。大鼠TRPV1的S4区域中的甲硫氨酸547突变为人类TRPV1中发现的亮氨酸（M547L），使PPAHV激活TRPV1的能力降低了约20倍。人类TRPV1的反向突变（L547M）使人类受体能够对PPAHV作出反应。这些突变在[Ca(2+)]i测定中并未显著影响辣椒素、树脂毒素（RTX）或奥伐尼尔的激动剂活性。将等效突变引入豚鼠TRPV1（L549M），在[Ca(2+)]i测定中使PPAHV的激动剂效力增加了>10倍，并增加了诱发电流的幅度。大鼠M547L突变降低了RTX结合的亲和力。因此，S2-S4区域内的氨基酸是激动剂和拮抗剂与TRPV1相互作用的重要位点。
