Department of Physiology, Seoul National University College of Medicine, Seoul, South Korea.
Department of Physiology, Chosun University College of Medicine, Gwangju, South Korea.
Am J Physiol Cell Physiol. 2023 Jun 1;324(6):C1295-C1306. doi: 10.1152/ajpcell.00535.2022. Epub 2023 May 8.
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 G-pathway. In an ELISA to quantify the amount of intracellular cAMP, a downstream product of OR/G-pathway, treatment with amitriptyline (AMI) showed a decrease in [cAMP] similar to that of the μOR agonist. Next, we explored the binding site of TCA by modeling the previously revealed ligand-bound structure of μOR. A conserved aspartate residue of ORs was predicted to participate 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 Gα. As an alternative way to monitor the downstream signaling of G-pathway, we evaluated the functional activity of TRPC4 channel, as it is well known to be activated by Gα. TCAs increased the TRPC4 current through ORs, and TCA-evoked TRPC4 activation was abolished by an inhibitor of Gα 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. 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 antidepressants (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.
三环类抗抑郁药（TCAs）传统上用于治疗情绪障碍，已显示出对慢性神经痛和肠易激综合征有良好的治疗效果。然而，这些非典型作用的机制尚不清楚。在提出的机制中，有一个众所周知的与疼痛相关的抑制性 G 蛋白偶联受体，即阿片受体（OR）。在这里，我们证实 TCA 确实能刺激 OR 并调节 G 通路下游信号 TRPC4 的门控。在一种酶联免疫吸附试验（ELISA）中定量测量 OR/G 通路下游产物细胞内 cAMP 的量，阿米替林（AMI）的处理显示 [cAMP] 减少类似于 μOR 激动剂。接下来，我们通过模拟先前揭示的 μOR 配体结合结构来探索 TCA 的结合位点。OR 的保守天冬氨酸残基预测与 TCAs 的胺基形成盐桥相互作用，并且在天冬氨酸到精氨酸突变中，AMI 不会降低 OR 和 Gα 之间基于 FRET 的结合效率。作为监测 G 通路下游信号的替代方法，我们评估了 TRPC4 通道的功能活性，因为众所周知它被 Gα 激活。TCAs 通过 OR 增加了 TRPC4 电流，并且 Gα 的抑制剂或其显性失活突变体可消除 TCA 诱发的 TRPC4 激活。正如预期的那样，在 OR 的天冬氨酸突变体中未观察到 TCA 诱发的 TRPC4 激活。总之，OR 可以被宣布为 TCA 众多结合伙伴中的一个有前途的靶点，而 TCA 诱发的 TRPC4 激活可能有助于解释 TCA 的非阿片类镇痛作用。内源性阿片系统调节疼痛感知，但对阿片类相关物质滥用的担忧限制了它们的使用。这项研究提出了 TRPC4 通道作为替代镇痛剂，三环类抗抑郁药（TCAs）的候选靶点。已经表明 TCA 与阿片受体（OR）结合并激活它们，导致涉及 TRPC4 的下游信号通路。TCA 对 TRPC4 的功能选择性和偏向激动作用可能依赖于 OR，这可能有助于更好地理解其疗效或副作用。
