Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA; Neuroscience Program, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Neurobiol Dis. 2018 Oct;118:76-93. doi: 10.1016/j.nbd.2018.07.004. Epub 2018 Jul 6.
Neuronal K7/KCNQ channels are voltage-gated potassium channels composed of K7.2/KCNQ2 and K7.3/KCNQ3 subunits. Enriched at the axonal membrane, they potently suppress neuronal excitability. De novo and inherited dominant mutations in K7.2 cause early onset epileptic encephalopathy characterized by drug resistant seizures and profound psychomotor delay. However, their precise pathogenic mechanisms remain elusive. Here, we investigated selected epileptic encephalopathy causing mutations in calmodulin (CaM)-binding helices A and B of K7.2. We discovered that R333W, K526N, and R532W mutations located peripheral to CaM contact sites decreased axonal surface expression of heteromeric channels although only R333W mutation reduced CaM binding to K7.2. These mutations also altered gating modulation by phosphatidylinositol 4,5-bisphosphate (PIP), revealing novel PIP binding residues. While these mutations disrupted K7 function to suppress excitability, hyperexcitability was observed in neurons expressing K7.2-R532W that displayed severe impairment in voltage-dependent activation. The M518 V mutation at the CaM contact site in helix B caused most defects in K7 channels by severely reducing their CaM binding, K currents, and axonal surface expression. Interestingly, the M518 V mutation induced ubiquitination and accelerated proteasome-dependent degradation of K7.2, whereas the presence of K7.3 blocked this degradation. Furthermore, expression of K7.2-M518V increased neuronal death. Together, our results demonstrate that epileptic encephalopathy mutations in helices A and B of K7.2 cause abnormal K7 expression and function by disrupting K7.2 binding to CaM and/or modulation by PIP. We propose that such multiple K7 channel defects could exert more severe impacts on neuronal excitability and health, and thus serve as pathogenic mechanisms underlying Kcnq2 epileptic encephalopathy.
神经元 K7/KCNQ 通道是由 K7.2/KCNQ2 和 K7.3/KCNQ3 亚基组成的电压门控钾通道。它们在轴突膜上丰富，强烈抑制神经元兴奋性。K7.2 的新生和显性遗传突变导致早发性癫痫性脑病，其特征是耐药性癫痫发作和严重的精神运动发育迟缓。然而，其确切的致病机制仍不清楚。在这里，我们研究了 K7.2 的钙调蛋白（CaM）结合螺旋 A 和 B 中的选定的癫痫性脑病致病突变。我们发现，位于 CaM 接触点外围的 R333W、K526N 和 R532W 突变降低了异源通道的轴突表面表达，尽管只有 R333W 突变减少了 CaM 与 K7.2 的结合。这些突变还改变了磷脂酰肌醇 4,5-二磷酸（PIP）的门控调制，揭示了新的 PIP 结合残基。虽然这些突变破坏了 K7 抑制兴奋性的功能，但在表达 K7.2-R532W 的神经元中观察到了过度兴奋，这些神经元表现出严重的电压依赖性激活受损。位于螺旋 B 中 CaM 接触点的 M518V 突变通过严重降低其 CaM 结合、K 电流和轴突表面表达，导致 K7 通道的大多数缺陷。有趣的是，M518V 突变诱导了 K7.2 的泛素化和加速蛋白酶体依赖性降解，而 K7.3 的存在阻止了这种降解。此外，K7.2-M518V 的表达增加了神经元死亡。总之，我们的结果表明，K7.2 螺旋 A 和 B 中的癫痫性脑病突变通过破坏 K7.2 与 CaM 的结合和/或通过 PIP 调节导致异常的 K7 表达和功能。我们提出，这种多种 K7 通道缺陷可能对神经元兴奋性和健康产生更严重的影响，从而成为 Kcnq2 癫痫性脑病的致病机制。
