Molecular dynamics of the pathogenic KCNQ2 variant G256W reveals mechanisms of channel dysfunction in epileptic encephalopathy

Brain potassium channels containing the subunit KCNQ2 are essential for regulating electrical signals contributing to sensation, learning, memory and motor control. De novo KCNQ2 variants are among the more common Mendelian causes of early-life epilepsy and neurodevelopmental impairment. Some patients with KCNQ2 variants are affectedby KCNQ2 developmental and epileptic encephalopathy (KCNQ2 DEE) characterized by seizures and developmental delays. Children with KCNQ2 DEE exhibit a range of impairment patterns that appear to be correlated with specific consequences of the variant for protein function. Here, we used all-atom molecular dynamics to analyze KCNQ2 G256W, a pathogenic missense variant located in the pore turret. G256W subunit simulations showed migration of the hydrophobic W256 side chain toward the lipid membrane. This movement affected overall turret structure and mobility prominently involving K255. We identified hydrogen bonding interactions in the wild type KCNQ2 turret region forming a network that extended to the selectivity filter, with N258, H260 and K283 as key residues. Simulations comparing WT and G256W tetrameric channels exhibited more conformationally unstable ion selectivity filters for G256W subunits. We analyzed how different stoichiometries of wild type and G256W subunits, as expected in heterozygous individuals, impacted dynamics and compared the G256W results to three additional turret-selectivity filter network variants. Our results provide support for an integral role of the KCNQ2 turret in selectivity filter stability. The majority of severe KCNQ2 DEE variants are clustered near the selectivity filter. Our study provides insights that may be broadly applicable to this clinically important allele subgroup. A serious childhood illness, called KCNQ2 developmental and epileptic encephalopathy, usually arises from single amino acid substitutions, or missense variants. This paper provides insight into how such a local change can profoundly disrupt the function of a large oligomeric channel protein containing over 3,400 residues. Molecular dynamics simulations of the pathogenic KCNQ2 pore domain variant, G256W, revealed that W256 changed the structure and flexibility of the pore domain turret and altered the ion selectivity filter. These findings shed light on the functional impact of KCNQ2 pathogenic variants and may help illuminate general mechanisms underlying severe KCNQ2 variants that occur commonly near the ion pore. Abbreviations: CaM: calmodulin; KCNQ2 DEE: KCNQ2 developmental and epileptic encephalopathy; MD: molecular dynamics; PGD: pore gating domain; PIP2: phosphatidylinositol 4,5-bisphosphate; POPC: 1-palmitoyl-2-oleoylphosphatidylcholine; RMSD: root mean square deviation; RMSE: root mean square error; RMSF: root mean square fluctuation; SASA: solvent accessible surface area; SD: standard deviation; SF: selectivity filter; SLFNE: self-limited familial neonatal epilepsy; VMD: visual molecular dynamics; VSD: voltage-sensing domain; WT: wild type携带KCNQ2亚基的脑钾离子通道，对于调控与感知、学习、记忆及运动控制相关的电信号至关重要。新发KCNQ2变异是早期癫痫及神经发育障碍较为常见的孟德尔式病因之一。部分携带KCNQ2变异的患者会罹患KCNQ2发育性癫痫性脑病（KCNQ2 developmental and epileptic encephalopathy, KCNQ2 DEE），该疾病以癫痫发作与发育迟缓为特征。罹患KCNQ2 DEE的儿童会表现出多种障碍表型，且这些表型似乎与该变异对蛋白质功能产生的特定影响相关。本研究采用全原子分子动力学（all-atom molecular dynamics）技术，对位于孔道袢（pore turret）区域的致病性错义变异KCNQ2 G256W进行分析。G256W亚基的模拟结果显示，疏水性的W256侧链会向脂质膜发生迁移。该迁移显著影响了孔道袢的整体结构与流动性，其中K255的作用尤为突出。我们在野生型（wild type, WT）KCNQ2孔道袢区域中鉴定出了氢键相互作用网络，该网络可延伸至离子选择性滤器（selectivity filter, SF），其中N258、H260及K283为关键残基。对比野生型与G256W四聚体通道的模拟结果显示，G256W亚基的离子选择性滤器构象稳定性更差。我们分析了野生型与G256W亚基的不同化学计量比——正如杂合个体中所见——对通道动力学的影响，并将G256W的实验结果与另外3种孔道袢-选择性滤器网络变异进行了对比。本研究结果支持了KCNQ2孔道袢在维持选择性滤器稳定性方面的核心作用。绝大多数重症KCNQ2 DEE变异均聚集于选择性滤器附近。本研究的结论可为这一临床意义重大的等位基因亚型提供新的见解。KCNQ2发育性癫痫性脑病是一种严重的儿童疾病，通常由单个氨基酸替换（即错义变异）所引发。本研究阐明了此类局部氨基酸改变，如何对含有超过3400个残基的大型寡聚通道蛋白功能造成严重破坏。对致病性KCNQ2孔道域变异G256W进行的分子动力学（molecular dynamics, MD）模拟显示，W256改变了孔道域袢的结构与柔性，并改变了离子选择性滤器的特性。上述研究结果阐明了KCNQ2致病性变异的功能影响机制，或可为普遍存在于离子孔道附近的重症KCNQ2变异的潜在发病机制提供新的阐释思路。缩写说明：CaM: 钙调蛋白（calmodulin）; KCNQ2 DEE: KCNQ2发育性癫痫性脑病（KCNQ2 developmental and epileptic encephalopathy）; MD: 分子动力学（molecular dynamics）; PGD: 孔道门控域（pore gating domain）; PIP2: 磷脂酰肌醇4,5-二磷酸（phosphatidylinositol 4,5-bisphosphate）; POPC: 1-棕榈酰-2-油酰磷脂酰胆碱（1-palmitoyl-2-oleoylphosphatidylcholine）; RMSD: 均方根偏差（root mean square deviation）; RMSE: 均方根误差（root mean square error）; RMSF: 均方根波动（root mean square fluctuation）; SASA: 溶剂可及表面积（solvent accessible surface area）; SD: 标准差（standard deviation）; SF: 选择性滤器（selectivity filter）; SLFNE: 自限性家族性新生儿癫痫（self-limited familial neonatal epilepsy）; VMD: 可视化分子动力学（visual molecular dynamics）; VSD: 电压感受域（voltage-sensing domain）; WT: 野生型（wild type）