TRPM3 is inhibited by activation of Gi-protein coupled receptors

T. Quallo, C. Gentry, D. A. Andersson, S. Bevan. Wolfson Centre for Age-Related Diseases, King's College London, London, UNITED KINGDOM.

Introduction: The non-selective cation channel TRPM3 is expressed in mammalian peripheral sensory neurons, where it has been proposed to play a role in thermosensation and nociception. In these studies we have examined modulation of TRPM3 by activation of Gαi/o-coupled opioid receptors.

Method: The sensitivity of mouse isolated dorsal root ganglion (DRG) neurons to the TRPM3 agonist, pregnenolone sulphate (PS), was examined. Neurons were loaded with the ratiometric calcium indicator dye Fura-2 and changes in [Ca2+]i were measured using a microscope based imaging system.

Results: A subpopulation of DRG neurons responded to PS (20µM) with robust, reversible increases in [Ca2+]i. These were relatively repeatable and exhibited only a small amount of desensitisation. In order to examine whether activation of opioid receptors expressed on sensory neurons can modulate natively expressed TRPM3 channels, the effects of the prototypical opioid receptor agonist morphine on the PS-evoked [Ca2+]i responses of DRG neurons were investigated. Isolated DRG neurons were exposed to two consecutive PS challenges (20µM). The second PS challenge took place either in the presence or absence of 10µM morphine. In experiments where neurons were perfused with 10µM morphine for 2 minutes before and during the second PS challenge, the response amplitude was strikingly reduced to 14 ± 2% (of the first PS response; mean ± SEM; n= 659 neurons, n= 7 experiments) from 62 ± 2% in control experiments (mean ± SEM; n= 392 neurons, n= 7 experiments). In the majority of neurons (56%, n= 372/659) application of morphine completely abolished PS-evoked [Ca2+]i responses(<5% of first PS response), in contrast, complete loss of the second PS response only occurred in 2% (n= 9/392) of neurons in control experiments. This inhibitory effect was reduced by pre-treatment with pertussis toxin (PTX) but unaffected by the presence of a cell permeable cAMP analog (8-bromo-cAMP). In separate experiments we examined whether this effect was specific to opioid receptors or could be extended to other Gi/o-coupled receptors expressed on sensory neurons. Interestingly, we found that pre-treatment of dorsal root ganglion neurons for 2 minutes with a GABAB- selective agonist, baclofen (100µM) or a neuropeptide Y (NPY) receptor agonist (PYY) also lead to inhibited PS responses.

Conclusion: We have shown that TRPM3 activity can be inhibited by activation of multiple Gi-coupled receptors by their cognate ligands in isolated sensory neurons.