BackgroundChronic Fatigue Syndrome/ Myalgic Encephalomyelitis (CFS/ME) is a complex multifactorial disorder of unknown cause having multi-system manifestations. Although the aetiology of CFS/ME remains elusive, immunological dysfunction and more particularly reduced cytotoxic activity in natural killer (NK) cells is the most consistent laboratory finding. The Transient Receptor Potential (TRP) superfamily of cation channels play a pivotal role in the pathophysiology of immune diseases and are therefore potential therapeutic targets. We have previously identified single nucleotide polymorphisms in TRP genes in peripheral NK cells from CFS/ME patients. We have also described biochemical pathway changes and calcium signaling perturbations in NK cells from CFS/ME patients. Notably, we have previously reported a decrease of TRP cation channel subfamily melastatin member 3 (TRPM3) function in NK cells isolated from CFS/ME patients compared with healthy controls after modulation with pregnenolone sulfate and ononetin using a patch-clamp technique. In the present study, we aim to confirm the previous results describing an impaired TRPM3 activity in a new cohort of CFS/ME patients using a whole cell patch-clamp technique after modulation with reversible TRPM3 agonists, pregnenolone sulfate and nifedipine, and an effective TRPM3 antagonist, ononetin. Indeed, no formal research has commented on using pregnenolone sulfate or nifedipine to treat CFS/ME patients while there is evidence that clinicians prescribe calcium channel blockers to improve different symptoms.MethodsWhole-cell patch-clamp technique was used to measure TRPM3 activity in isolated NK cells from twelve age- and sex-matched healthy controls and CFS/ME patients, after activation with pregnenolone sulfate and nifedipine and inhibition with ononetin.ResultsWe confirmed a significant reduction in amplitude of TRPM3 currents after pregnenolone sulfate stimulation in isolated NK cells from another cohort of CFS/ME patients compared with healthy controls. The pregnenolone sulfate-evoked ionic currents through TRPM3 channels were again significantly modulated by ononetin in isolated NK cells from healthy controls compared with CFS/ME patients. In addition, we used nifedipine, another reversible TRPM3 agonist to support the previous findings and found similar results confirming a significant loss of the TRPM3 channel activity in CFS/ME patients.ConclusionsImpaired TRPM3 activity was validated in NK cells isolated from CFS/ME patients using different pharmacological tools and whole-cell patch-clamp technique as the gold standard for ion channel research. This investigation further helps to establish TRPM3 channels as a prognostic marker and/ or a potential therapeutic target for CFS/ME.

中文翻译：

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慢性疲劳综合征/肌痛性脑脊髓炎患者自然杀伤细胞中受损的瞬时受体电位美拉司他汀 3 离子通道活性的验证

背景慢性疲劳综合征/肌痛性脑脊髓炎 (CFS/ME) 是一种复杂的多因素疾病，原因不明，具有多系统表现。尽管 CFS/ME 的病因仍然难以捉摸，但免疫功能障碍，尤其是自然杀伤 (NK) 细胞的细胞毒活性降低是最一致的实验室发现。阳离子通道的瞬时受体电位 (TRP) 超家族在免疫疾病的病理生理学中起关键作用，因此是潜在的治疗靶点。我们之前已经确定了 CFS/ME 患者外周 NK 细胞中 TRP 基因的单核苷酸多态性。我们还描述了 CFS/ME 患者 NK 细胞的生化途径变化和钙信号扰动。尤其，我们之前曾报道过，与健康对照相比，在使用膜片钳技术用孕烯醇酮硫酸盐和洋葱头素调制后，从 CFS/ME 患者中分离出的 NK 细胞中 TRP 阳离子通道亚家族褪黑素成员 3 (TRPM3) 功能降低。在本研究中，我们的目标是证实先前的结果，该结果描述了在用可逆 TRPM3 激动剂、硫酸孕烯醇酮和硝苯地平以及有效的 TRPM3 调节后，使用全细胞膜片钳技术在一组新的 CFS/ME 患者中 TRPM3 活性受损的结果。拮抗剂，黄连素。事实上，没有正式的研究评论使用硫酸孕烯醇酮或硝苯地平治疗 CFS/ME 患者，而有证据表明临床医生开出钙通道阻滞剂来改善不同的症状。方法全细胞膜片钳技术用于测量来自 12 个年龄和性别匹配的健康对照和 CFS/ME 患者的分离 NK 细胞中的 TRPM3 活性，在用硫酸孕烯醇酮和硝苯地平激活并用 ononetin 抑制后。结果我们证实显着降低与健康对照相比，来自另一组 CFS/ME 患者的分离 NK 细胞在孕烯醇酮硫酸盐刺激后 TRPM3 电流的振幅。与 CFS/ME 患者相比，从健康对照分离的 NK 细胞中，通过 TRPM3 通道的孕烯醇酮硫酸盐诱发的离子电流再次受到 ononetin 的显着调节。此外，我们使用了另一种可逆 TRPM3 激动剂硝苯地平来支持先前的发现，并发现类似的结果证实了 CFS/ME 患者中 TRPM3 通道活性的显着丧失。结论使用不同的药理学工具和全细胞膜片钳技术作为离子通道研究的金标准，在从 CFS/ME 患者分离的 NK 细胞中验证了受损的 TRPM3 活性。这项研究进一步有助于建立 TRPM3 通道作为 CFS/ME 的预后标志物和/或潜在的治疗靶点。