Efficacy of agonists.

A member of THIK (two pore domain halothane-inhibited K+) channels, THIK-1, was reported as a target of Gi/o-coupled receptors (Gi/o-Rs) in neurons and microglia. We confirmed that in HEK293T cells the THIK-1 channel is activated by Gi/o-Rs and found that Gq-coupled receptors (Gq-Rs) also activates the channel. The effects of Gi/o-Rs and Gq-Rs were inhibited by the Gi/o inhibitor pertussis toxin and phospholipase C (PLC) inhibitor, respectively. The effects of Gi/o-Rs were attenuated when consensus Gβγ binding motif at the C-tail of the THIK-1 channel was mutated, suggesting that Gβγ serves as a THIK-1 channel activator upon the stimulation of Gi/o-Rs. As to the effects of Gq-Rs on the THIK-1 channel, a protein kinase C inhibitor and calcium chelators failed to inhibit the effect of a Gq coupled muscarinic M1R. Neither the hydrolysis of phosphatidyl inositol bisphosphate induced by voltage sensitive phosphatase nor the application of a diacylglycerol analogue, OAG, increased the channel current. The mediator of Gq-dependent activation of the THIK-1 channel remained unsolved. The effects of Gi/o- and Gq-Rs on the THIK-2 channel were also investigated, by using a THIK-2 mutant channel whose N-terminal domain is deleted to improve the surface membrane expression. We observed that Gi/o- and Gq-Rs activate the mutated THIK-2 channel, similarly to the THIK-1 channel. Interestingly, heterodimeric channels of THIK-1 and THIK-2 responded to Gi/o-R and Gq-R stimulation. Taken together, Gi/o- or Gq-Rs activates the THIK-1 and THIK-2 channels in a Gβγ or PLC dependent manner, respectively.

THIK（双孔域卤烷抑制性钾离子通道，two pore domain halothane-inhibited K+ channels）家族成员THIK-1，此前被报道为神经元与小胶质细胞中Gi/o型偶联受体（Gi/o-coupled receptors, Gi/o-Rs）的作用靶点。本研究在HEK293T细胞中验证了THIK-1通道可被Gi/o-Rs激活，同时发现Gq型偶联受体（Gq-coupled receptors, Gq-Rs）同样能够激活该通道。Gi/o-Rs与Gq-Rs的调控效应分别可被Gi/o特异性抑制剂百日咳毒素（pertussis toxin）与磷脂酶C（phospholipase C, PLC）抑制剂阻断。当THIK-1通道C端的保守Gβγ结合基序发生突变时，Gi/o-Rs的激活效应显著减弱，提示Gi/o-Rs刺激后，Gβγ可作为THIK-1通道的激活因子发挥作用。针对Gq-Rs对THIK-1通道的调控机制，蛋白激酶C抑制剂与钙螯合剂均无法阻断Gq偶联的毒蕈碱型M1受体（muscarinic M1R）所介导的通道激活效应。电压敏感磷酸酶诱导的磷脂酰肌醇二磷酸水解，以及二酰甘油类似物OAG的外源施加，均未能提升THIK-1通道的电流水平，因此Gq依赖型THIK-1通道激活的具体介导因子仍未明确。本研究同时针对THIK-2通道开展了相关实验：为提升其细胞膜表面表达量，我们构建了N端结构域缺失的THIK-2突变通道，并以此为模型探究Gi/o-Rs与Gq-Rs对其的调控作用。实验结果显示，Gi/o-Rs与Gq-Rs均可激活该突变型THIK-2通道，这一点与THIK-1通道的调控规律一致。值得注意的是，THIK-1与THIK-2形成的异二聚体通道，同样可响应Gi/o-Rs与Gq-Rs的刺激并发生激活。综上，Gi/o-Rs与Gq-Rs可分别通过Gβγ依赖与PLC依赖的通路，激活THIK-1与THIK-2通道。