The Role of TRPM3 Ion Channels in Opioid-Induced Analgesia and Pruritus

项目摘要 吗啡（一种强效阿片类药物）的脊髓给药通常用于治疗术后疼痛。 然而，期望的阿片样物质诱导的镇痛一致地具有阿片样物质诱导的镇痛的负面副作用。 瘙痒症，导致已经痛苦的患者的衰弱性瘙痒。一线治疗是阿片类拮抗剂， 但这可能会与最初的阿片类药物治疗竞争，并导致患者在不必要的疼痛之间做出选择， 或者痒这两种感觉是密切相关的，但阿片类药物如何引起瘙痒和 镇痛仍然不清楚。 吗啡作用于μ-阿片受体（莫尔）以抑制痛觉神经元的活性。莫尔是一个 G蛋白偶联受体（GPCR）偶联到抑制性G i异源三聚体蛋白（G α i和G β γ）， 引起下游作用以抑制神经元的去极化。这种抑制的一种机制是 通过G β γ对瞬时受体电位melastatin 3（TRPM 3）离子通道的直接抑制， 神经元上表达的温度敏感性和伤害性阳离子通道。最近，一种自然发生的剪接 鉴定了不与G β γ结合的TRPM 3变体。有了这些知识， 产生的（TRPM3DEx17），其中所有TRPM3通道都表达该同种型。利用TRPM3DEx17 小鼠行吗啡诱导的瘙痒和镇痛的初步行为学研究。 数据显示TRPM 3DEx17小鼠在鞘内注射后经历显著更少的瘙痒 与WT相比，吗啡。在足底切口模型中，TRPM 3DEx17小鼠的吗啡浓度没有变化 在热和机械伤害感受中鞘内注射时的镇痛。吗啡的镇痛作用 通过抑制兴奋性（Vglu2+）神经元，而瘙痒通过抑制抑制性（Vgat+）神经元发生 脊髓中的神经元或抑制解除。有趣的是，TRPM 3和莫尔更频繁地共表达 与疼痛兴奋性Vgat 2+神经元相比，对瘙痒抑制性Vgat+神经元的作用，支持我们的研究。 行为发现。因此，该提案的目的1将通过原位杂交探索， TRPM3DEx17小鼠脊髓中Vglu2+和Vgat+神经元中的TRPM3和MOR，并使用两个报告基因 用GFP标记的兴奋性和抑制性神经元的小鼠系进行脊髓的全细胞膜片钳 脊髓切片以比较每个神经元群体中的吗啡抑制。目标2将试图克服 通过在WT小鼠中鞘内共注射TRPM 3激动剂硫酸双烯醇酮对TRPM 3的莫尔抑制 使用吗啡，观察在维持镇痛的同时是否能缓解阿片类药物引起的瘙痒。这些 结果提示TRPM 3是负责去抑制的主要去极化离子通道， 瘙痒，可作为缓解阿片类药物治疗患者吗啡诱导瘙痒的潜在靶点 治疗而不影响所需的镇痛。

PROJECT SUMMARY Spinal administration of morphine, a potent opioid, is commonly used to treat post-operative pain. However, the desired opioid-induced analgesia consistently has a negative side effect of opioid-induced pruritus, causing debilitating itch in the already distressed patient. The first line treatment is opioid antagonists, but this may compete with the initial opioid treatment and cause the patient to choose between unwanted pain or itch. Both sensations are closely related, but the mechanism of how opioids can cause both pruritus and analgesia remains unclear. Morphine acts on mu-opioid receptors (MOR) to inhibit the activity of pain-sensing neurons. MOR is a G-protein coupled receptor (GPCR) coupled to the inhibitory Gi-heterotrimeric proteins (Gαi and Gβγ) which cause downstream actions to inhibit depolarization of the neuron. One mechanism by which this inhibition occurs is by direct inhibition by Gβγ on the Transient Receptor Potential melastatin 3 (TRPM3) ion channel, a thermosensitive and nociceptive cation channel expressed on neurons. Recently, a naturally occurring splice variant of TRPM3 that does not bind to Gβγ was identified. With this knowledge, a novel mouse line was generated (TRPM3DEx17) in which all the TRPM3 channels express this isoform. Utilizing the TRPM3DEx17 mouse line we performed preliminary behavioral studies on morphine induced pruritus and analgesia. Data showed that the TRPM3DEx17 mice experienced significantly less pruritus upon intrathecal injection of morphine compared to WT. In the plantar incision model, TRPM3DEx17 mice had no change in morphine analgesia when injected intrathecally in heat and mechanical nociception. Analgesia by morphine occurs through inhibition of excitatory (Vglu2+) neurons, while pruritus occurs by inhibition of inhibitory (Vgat+) neurons, or disinhibition, in the spinal cord. Interestingly, TRPM3 and MOR are more frequently coexpressed on the itch-inhibitory Vgat+ neurons compared to the pain-excitatory Vglut2+ neurons, supporting our behavioral findings. Thus, Aim 1 of the proposal will explore via in-situ hybridization, the co-expression of TRPM3 and MOR in Vglu2+ and Vgat+ neurons in the TRPM3DEx17 mouse spinal cord, and use two reporter mouse lines with GFP tagged excitatory and inhibitory neurons to perform whole-cell patch clamping of spinal cord slices to compare morphine inhibition in each neuronal population. Aim 2 will attempt to overcome the MOR inhibition of TRPM3 by co-injecting a TRPM3 agonist, pregnenolone sulfate, in the WT mice intrathecally with morphine, and observe if opioid-induced pruritus can be alleviated while analgesia is maintained. These results may suggest that TRPM3 is the main depolarizing ion channel responsible for the disinhibition of pruritus and can be a potential target for alleviating morphine induced pruritus in patients undergoing opioid therapy without affecting the desired analgesia.