GPCR modulation of TRPM3 and other TRPM ion channels

Student thesis: Doctoral ThesisDoctor of Philosophy

Abstract

The transient receptor potential (TRP) superfamily is composed of 28 non-selective cation channels, which have been implicated in a myriad of physiological processes ranging from temperature detection and sensory transduction to insulin release, gene transcription and ionic homeostasis. G-protein coupled receptors (GPCRs) are the largest family of signalling proteins involved in all physiological processes and are targeted by many established pharmacotherapies. TRP channels are often effectors activated or modulated by GPCR signalling. The aim of this thesis is to functionally characterise the TRP melastatin 3 (TRPM3) channel, its modulation by GPCRs and examine GPCR regulation of other members of the TRPM subfamily, specifically TRPM2, TRPM7 and TRPM8 channels. The ionic current characteristics produced by the two selective TRPM3 agonists, pregnenolone sulphate (PS) and CIM0216, were investigated using patch-clamp electrophysiology and Ca2+-imaging techniques in a heterologous expression system. The findings demonstrate that activation of TRPM3 by CIM0216 but not PS strongly desensitises in the presence of extracellular Ca2+. Application of CIM0216 results in a biphasic TRPM3 current waveform and in two open states in single-channel recordings. TRPM3 activation by both agonists was fully inhibited at acidic pH (pH 5) but unaffected by alkaline pH (pH 8). Moreover, TRPM3 activity both in vitro and in vivo was found to be inhibited by increasing DMSO concentrations. Activation of TRPM3 in heterologous and sensory neurons was promiscuously and robustly inhibited by GPCRs coupled to any of Gαi, Gαq and Gαs subunits. Investigations of GPCR signalling cascades on TRPM3 demonstrated that GPCR-mediated inhibition of TRPM3 is due to the direct interaction of liberated Gβγ subunits with the channel. Behavioural investigations in vivo demonstrated that activation of GPCRs that are targeted by analgesic drugs as well as by the proinflammatory mediators prostaglandin E2 and bradykinin inhibit TRPM3-mediated nociception and inflammatory heat hypersensitivity. The effect of GPCR activation was also examined on other members of the TRPM subfamily. My results demonstrate that GPCRs coupled to Gαi/o-, Gαq- and Gαs do not appear to influence heat- or H2O2-evoked TRPM2 activity. In contrast, TRPM7 was activated reversibly and transiently following stimulation of Gαs-coupled receptors, whereas TRPM8 was exclusively inhibited by stimulation of Gαq-coupled receptors. In conclusion, this thesis demonstrates that TRPM3 channels are thus far the only TRPM family member to be promiscuously inhibited by GPCRs that couple to Gαi/o, Gαq and Gαs.
Date of Award1 May 2020
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorDavid Andersson (Supervisor) & Stuart Bevan (Supervisor)

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