Devaux Jérôme, Dhifallah Sandra, De Maria Michela, Stuart-Lopez Geoffrey, Becq Hélène, Milh Mathieu, Molinari Florence, Aniksztejn Laurent
CNRS, CRN2M-UMR7286, Aix-Marseille University, Marseille, France.
INSERM UMR_S901, Mediterranean Neurobiology Institute (INMED), Aix-Marseille University, Marseille, France.
Epilepsia. 2017 Dec;58(12):2073-2084. doi: 10.1111/epi.13927. Epub 2017 Oct 25.
Kv7 channels mediate the voltage-gated M-type potassium current. Reduction of M current due to KCNQ2 mutations causes early onset epileptic encephalopathies (EOEEs). Mutations in STXBP1 encoding the syntaxin binding protein 1 can produce a phenotype similar to that of KCNQ2 mutations, suggesting a possible link between STXBP1 and Kv7 channels. These channels are known to be modulated by syntaxin-1A (Syn-1A) that binds to the C-terminal domain of the Kv7.2 subunit and strongly inhibits M current. Here, we investigated whether STXBP1could prevent this inhibitory effect of Syn-1A and analyzed the consequences of two mutations in STXBP1 associated with EOEEs.
Electrophysiologic analysis of M currents mediated by homomeric Kv7.2 or heteromeric Kv7.2/Kv7.3 channels in Chinese hamster ovary (CHO) cells coexpressing Syn-1A and/or STXBP1 or mutants STXBP1 p.W28* and p.P480L. Expression and interaction of these different proteins have been investigated using biochemical and co-immunoprecipitation experiments.
Syn-1A decreased M currents mediated by Kv7.2 or Kv7.2/Kv7.3 channels. STXBP1 had no direct effects on M current but dampened the inhibition produced by Syn-1A by abrogating Syn-1A binding to Kv7 channels. The mutation p.W28*, but not p.P480L, failed to rescue M current from Syn-1A inhibition. Biochemical analysis showed that unlike the mutation p.W28*, the mutation p.P480L did not affect STXBP1 expression and reduced the interaction of Syn-1A with Kv7 channels.
These data indicate that there is a functional link between STXBP1 and Kv7 channels via Syn-1A, which may be important for regulating M-channel activity and neuronal excitability. They suggest also that a defect in Kv7 channel activity or regulation could be one of the consequences of some STXBP1 mutations associated with EOEEs. Furthermore, our data reveal that STXBP1 mutations associated with the Ohtahara syndrome do not necessarily result in protein haploinsufficiency.
Kv7通道介导电压门控M型钾电流。KCNQ2突变导致的M电流减少会引发早发性癫痫性脑病（EOEEs）。编码 syntaxin结合蛋白1的STXBP1突变可产生与KCNQ2突变相似的表型，提示STXBP1与Kv7通道之间可能存在联系。已知这些通道受 syntaxin-1A（Syn-1A）调节，Syn-1A与Kv7.2亚基的C末端结构域结合并强烈抑制M电流。在此，我们研究了STXBP1是否能阻止Syn-1A的这种抑制作用，并分析了与EOEEs相关的STXBP1中的两个突变的后果。
对在中国仓鼠卵巢（CHO）细胞中共表达Syn-1A和/或STXBP1或突变体STXBP1 p.W28*和p.P480L的同聚体Kv7.2或异聚体Kv7.2/Kv7.3通道介导的M电流进行电生理分析。使用生化和免疫共沉淀实验研究了这些不同蛋白质的表达和相互作用。
Syn-1A降低了由Kv7.2或Kv7.2/Kv7.3通道介导的M电流。STXBP1对M电流没有直接影响，但通过消除Syn-1A与Kv7通道的结合减轻了Syn-1A产生的抑制作用。突变p.W28不能从Syn-1A抑制中挽救M电流，而p.P480L则可以。生化分析表明，与突变p.W28不同，突变p.P480L不影响STXBP1的表达，并减少了Syn-1A与Kv7通道的相互作用。
这些数据表明STXBP1与Kv7通道之间通过Syn-1A存在功能联系，这可能对调节M通道活性和神经元兴奋性很重要。它们还表明，Kv7通道活性或调节缺陷可能是与EOEEs相关的一些STXBP1突变的后果之一。此外，我们的数据表明与大田原综合征相关的STXBP1突变不一定导致蛋白质单倍剂量不足。
