Greene Derek L, Kosenko Anastasia, Hoshi Naoto
Department of Pharmacology, University of California, Irvine, Irvine, California.
Department of Physiology and Biophysics, University of California, Irvine, Irvine, California.
Epilepsia. 2018 Oct;59(10):1908-1918. doi: 10.1111/epi.14541. Epub 2018 Aug 26.
The M-current is a low-threshold voltage-gated potassium current generated by Kv7 subunits that regulates neural excitation. It is important to note that M-current suppression, induced by activation of Gq-coupled neurotransmitter receptors, can dynamically regulate the threshold of action-potential firing and firing frequency. Here we sought to directly examine whether M-current suppression is involved in seizures and epileptogenesis.
Kv7.2 knock-in mice lacking the key protein kinase C (PKC) phosphorylation acceptor site for M-current suppression were generated by introducing an alanine substitution at serine residue 559 of mouse Kv7.2, mKv7.2(S559A). Basic electrophysiologic properties of the M-current between wild-type and Kv7.2(S559A) knock-in mice were analyzed in primary cultured neurons. Homozygous Kv7.2(S559A) knock-in mice were used to evaluate the protective effect of mutant Kv7.2 channel against chemoconvulsant-induced seizures. In addition, pilocarpine-induced neuronal damage and spontaneously recurrent seizures were evaluated after equivalent chemoconvulsant-induced status epilepticus was achieved by coadministration of the M-current-specific channel inhibitor, XE991.
Neurons from Kv7.2(S559A) knock-in mice showed normal basal M-currents. Knock-in mice displayed reduced M-current suppression when challenged by a muscarinic agonist, oxotremorine-M. Kv7.2(S559A) mice were resistant to chemoconvulsant-induced seizures with no mortality. Administration of XE991 transiently exacerbated seizures in knock-in mice equivalent to those of wild-type mice. Valproate, which disrupts neurotransmitter-induced M-current suppression, showed no additional anticonvulsant effect in Kv7.2(S559A) mice. After experiencing status epilepticus, Kv7.2(S559A) knock-in mice did not show seizure-induced cell death or spontaneous recurring seizures.
This study provides evidence that neurotransmitter-induced suppression of M-current generated by Kv7.2-containing channels exacerbates behavioral seizures. In addition, prompt recovery of M-current after status epilepticus prevents subsequent neuronal death and the development of spontaneously recurrent seizures. Therefore, prompt restoration of M-current activity may have a therapeutic benefit for epilepsy.
M电流是一种由Kv7亚基产生的低阈值电压门控钾电流，可调节神经兴奋性。需要注意的是，由Gq偶联神经递质受体激活所诱导的M电流抑制，能够动态调节动作电位发放阈值和发放频率。在此，我们试图直接探究M电流抑制是否参与癫痫发作和癫痫发生过程。
通过将小鼠Kv7.2的丝氨酸残基559替换为丙氨酸（mKv7.2(S559A)），构建缺乏M电流抑制关键蛋白激酶C（PKC）磷酸化受体位点的Kv7.2基因敲入小鼠。在原代培养神经元中分析野生型和Kv7.2(S559A)基因敲入小鼠之间M电流的基本电生理特性。使用纯合Kv7.2(S559A)基因敲入小鼠评估突变型Kv7.2通道对化学惊厥剂诱导癫痫发作的保护作用。此外，在联合给予M电流特异性通道抑制剂XE991达到等效化学惊厥剂诱导的癫痫持续状态后，评估毛果芸香碱诱导的神经元损伤和自发复发性癫痫发作情况。
Kv7.2(S559A)基因敲入小鼠的神经元显示出正常的基础M电流。基因敲入小鼠在受到毒蕈碱激动剂氧化震颤素-M刺激时，M电流抑制作用减弱。Kv7.2(S559A)小鼠对化学惊厥剂诱导的癫痫发作具有抗性，且无死亡情况。给予XE991会使基因敲入小鼠的癫痫发作短暂加剧，程度与野生型小鼠相当。丙戊酸可破坏神经递质诱导的M电流抑制，但在Kv7.2(S559A)小鼠中未显示出额外的抗惊厥作用。经历癫痫持续状态后，Kv7.2(S559A)基因敲入小鼠未出现癫痫发作诱导的细胞死亡或自发复发性癫痫发作。
本研究提供了证据表明神经递质诱导的含Kv7.2通道产生的M电流抑制会加剧行为性癫痫发作。此外，癫痫持续状态后M电流的迅速恢复可防止随后的神经元死亡和自发复发性癫痫发作的发生。因此，迅速恢复M电流活性可能对癫痫具有治疗益处。
