Low concentrations of tricyclic antidepressants stimulate TRPC4 channel activity by acting as an opioid receptor ligand

Traditionally prescribed for mood disorders, tricyclic antidepressants (TCAs) have shown promising therapeutic effects on chronic neuralgia and irritable bowel syndrome. However, the mechanism by which these atypical effects manifest is unclear. Among the proposed mechanisms is the well-known pain-related inhibitory G-protein coupled receptor, namely the opioid receptor (OR). Here, we confirmed that TCA indeed stimulates OR and regulates the gating of TRPC4, a downstream signaling of the Gi-path-way. In an ELISA to quantify the amount of intracellular cAMP, a downstream product of OR/Gi-pathway, treatment with amitripty-line (AMI) showed a decrease in [cAMP]i similar to that of the mu OR agonist. Next, we explored the binding site of TCA by modeling the previously revealed ligand-bound structure of mu OR. A conserved aspartate residue of ORs was predicted to partici-pate in salt bridge interaction with the amine group of TCAs, and in aspartate-to-arginine mutation, AMI did not decrease the FRET-based binding efficiency between the ORs and Gai2. As an alternative way to monitor the downstream signaling of Gi-path-way, we evaluated the functional activity of TRPC4 channel, as it is well known to be activated by Gai. TCAs increased the TRPC4 current through ORs, and TCA-evoked TRPC4 activation was abolished by an inhibitor of Gai2 or its dominant-negative mutant. As expected, TCA-evoked activation of TRPC4 was not observed in the aspartate mutants of OR. Taken together, OR could be proclaimed as a promising target among numerous binding partners of TCA, and TCA-evoked TRPC4 activation may help to explain the nonopioid analgesic effect of TCA.NEW & NOTEWORTHY Endogenous opioid systems modulate pain perception, but concerns about opioid-related substance misuse limit their use. This study has raised TRPC4 channel as a candidate target for alternative analgesics, tricyclic antidepres-sants (TCAs). TCAs have been shown to bind to and activate opioid receptors (ORs), leading to downstream signaling pathways involving TRPC4. The functional selectivity and biased agonism of TCA towards TRPC4 in dependence on OR may provide a better understanding of its efficacy or side effects.