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Title: Molecular bases underlying the sensitivity of the capsaicin receptor TRPV1
Author: Paule, Cleoper
ISNI:       0000 0004 2693 838X
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2010
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Individual human beings report highly variable pain experiences following exposure to the same noxious stimulus, including noxious heat. A series of missense single nucleotide polymorphisms (SNPs) have been found in the human noxious heat transducer, transient receptor potential vanilloid type 1 ion channel (hTRPV1), which responds to, exogenous, and endogenous, vanilloids, protons, and depolarisation. The aim of this project was to examine the effect of SNPs on the sensitivity of hTRPV1 to certain activators. The three most frequently occurring SNPs (I315M, T469I, V585I) were used to generate 4 haplotypes: hTRPV1112 (V585I); hTRPV1121 (T469I); hTRPV1211 (I315M); and hTRPV1222 (I315M, T469I, V585I). The responses of these haplotypes to these activators were compared to the responses of the “wild type” hTRPV1 (hTRPV1111, I315, T469, V585) using whole-cell patch-clamp recordings from HEK293 cells which transiently-expressed the relevant ion channels. Site-directed mutagenesis was used to confirm the role of the SNPs in altering the sensitivity of hTRPV1 to the activators applied. In addition, in control experiments, several important collateral issues were investigated, namely: (a) the effect of the solvent, dimethyl sulphoxide (DMSO), on untransfected, and transfected, cells, respectively; and (b) the extent to which acid-sensing ion channels, constitutively-expressed by HEK293 cells, compromised the assessment of the proton-evoked responses of hTRPV1. The techniques employed included: the co-transfection of the cells with vector carrying the coding region of the green fluorescent protein (GFP) gene; the reverse transcriptase polymerase chain reaction; immunocytochemistry; the cobalt uptake assay; and whole-cell patch-clamp recordings. The pharmacological and biophysical properties of the DMSO-evoked, and capsaicin-evoked, responses were similar. Furthermore, DMSO desensitised TRPV1. Acid-sensing ion channels were sensitive to the diuretic, amiloride (30μM). However, amiloride increased hTRPV1-mediated responses evoked by noxious heat but not capsaicin. hTRPV1222 showed a higher sensitivity than hTRPV1111 to capsaicin, and heat. Thus, when capsaicin was applied, the EC50 of hTRPV1111 and hTRPV1222 was 817 nM and 89 nM, respectively. The activation thresholds when heat was applied to hTRPV1111 and hTRPV1222 were 44.5±0.31 oC, and 42.3±045 oC, respectively). However, hTRPV1111 was more sensitive to depolarisation than hTRPV1222, with the V1/2 at 37oC for hTRPV1111 and hTRPV1222 being 42.3 mV and 83.3 mV, respectively. Furthermore, no differences were found between the proton-and anandamide- sensitivity of hTRPV1111 and hTRPV1222. The sensitivities of hTRPV1121 to heat and depolarization were similar to those of hTRPV1222, but different from those of hTRPV1111. Furthermore, the sensitivity of hTRPV1112 and hTRPV1211 were similar to that of hTRPV1111, but different from that of hTRPV1222 and hTRPV1121. Charge-neutralising and conserving mutations at position 469 produced clones with similar sensitivities to hTRPV1121 and hTRPV1111, respectively. The studies undertaken established, first, that DMSO is able to activate TRPV1. Second, the non-synonymous SNP at position 469 in hTRPV1 alters the sensitivity of the ion channel to vanilloids, heat, and depolarisation. These findings suggest that missense SNPs in hTRPV1 may contribute to the development of different pain experience in humans in response to the same noxious stimulus. Accordingly, regard must be had to SNPs found in hTRPV1 when attempting to develop analgesics for pain which depends on TRPV1 activation and in treating that pain. Finally, the finding that the T469I mutation increased the sensitivity of the molecule to heat, but reduced its sensitivity to depolarisation, suggests that the various sensors in TRPV1 may be coupled allosterically rather than directly.
Supervisor: Nagy, Istvan Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral