Ruland Julia G, Kirchhofer Sina B, Klindert Sebastian, Bailey Chris P, Bünemann Moritz
Department of Pharmacology and Clinical Pharmacy, Philipps-University, Marburg, Germany.
Department of Pharmacy and Pharmacology, University of Bath, Bath, UK.
Br J Pharmacol. 2020 Aug;177(15):3489-3504. doi: 10.1111/bph.15070. Epub 2020 May 19.
Various GPCRs have been described as being modulated in a voltage-dependent manner. Opioid analgesics act via activation of μ receptors in various neurons. As neurons are exposed to large changes in membrane potential, we were interested in studying the effects of depolarization on μ receptor signalling.
We investigated potential voltage sensitivity of μ receptors in heterologous expression systems (HEK293T cells) using electrophysiology in combination with Förster resonance energy transfer-based assays. Depolarization-induced changes in signalling were also tested in physiological rat tissue containing locus coeruleus neurons. We applied depolarization steps across the physiological range of membrane potentials.
Studying μ receptor function and signalling in cells, we discovered that morphine-induced signalling was strongly dependent on the membrane potential (V ). This became apparent at the level of G-protein activation, G-protein coupled inwardly rectifying potassium channel (K 3.X) currents and binding of GPCR kinases and arrestin3 to μ receptors by a robust increase in signalling upon membrane depolarization. The pronounced voltage sensitivity of morphine-induced μ receptor activation was also observed at the level of K 3.X currents in rat locus coeruleus neurons. The efficacy of peptide ligands to activate μ receptors was not (Met-enkephalin) or only moderately ([D-Ala , N-Me-Phe , Gly -ol]-enkephalin) enhanced upon depolarization. In contrast, depolarization reduced the ability of the analgesic fentanyl to activate μ receptors.
Our results indicate a strong ligand-dependent modulation of μ receptor activity by the membrane potential, suggesting preferential activity of morphine in neurons with high neuronal activity.
多种G蛋白偶联受体（GPCRs）已被描述为以电压依赖性方式受到调节。阿片类镇痛药通过激活各种神经元中的μ受体发挥作用。由于神经元会经历膜电位的大幅变化，我们对研究去极化对μ受体信号传导的影响感兴趣。
我们在异源表达系统（HEK293T细胞）中，结合基于福斯特共振能量转移的检测方法，利用电生理学研究μ受体的潜在电压敏感性。还在含有蓝斑神经元的生理大鼠组织中测试了去极化诱导的信号变化。我们在膜电位的生理范围内施加去极化步骤。
在细胞中研究μ受体功能和信号传导时，我们发现吗啡诱导的信号传导强烈依赖于膜电位（V）。这在G蛋白激活、G蛋白偶联内向整流钾通道（K3.X）电流以及GPCR激酶和抑制蛋白3与μ受体的结合水平上变得明显，膜去极化时信号传导显著增加。在大鼠蓝斑神经元的K3.X电流水平上也观察到了吗啡诱导的μ受体激活的明显电压敏感性。去极化时，肽配体激活μ受体的效力未增强（甲硫氨酸脑啡肽）或仅适度增强（[D-丙氨酸，N-甲基苯丙氨酸，甘氨酸-醇]-脑啡肽）。相比之下，去极化降低了镇痛药芬太尼激活μ受体的能力。
我们的结果表明膜电位对μ受体活性有强烈的配体依赖性调节，提示吗啡在神经元活动高的神经元中具有优先活性。
