KV1.2通道,由KCNA2基因编码,通过在去极化时传导K来调节神经元兴奋性。在一名癫痫患者中发现了一种新的KCNA2错义变异,在KV1.2电压感应域中,在螺旋S4处引起氨基酸取代F302L。免疫细胞化学和流式细胞术显示F302L不损害KCNA2亚基表面运输。分子动力学模拟表明F302L改变了S4残基对膜脂质的暴露。电压钳荧光法显示,KV1.2-F302L通道的电压传感域对去极化更敏感。因此,KV1.2-F302L通道打开更快,负电势更多;然而,它们还表现出增强的失活:也就是说,F302L引起功能增益和丧失效应。KCNA2-WT和-F302L的共表达不能完全挽救这些作用。先证者的症状在KCNA2功能丧失患者中更具特征性。增强的KV1.2失活可导致兴奋性神经元的突触释放增加,使神经元回路转向癫痫。
在患有癫痫的婴儿中进行的基于外显子组的诊断小组显示了KCNA2中先前未报道的从头错义变异,其编码电压门控K通道KV1.2。该变体导致取代F302L,在KV1.2电压传感域(VSD)的螺旋S4中。F302L不影响KCNA2亚基膜运输。然而,它确实改变了通道的功能特性,在更高的超极化膜电位下加速通道开放,表明功能的增益。F302L还通过加速失活发作导致KV1.2功能丧失,减速恢复,并将失活电压依赖性转移到更多的负电位。这些影响,它们不能通过野生型和突变型KCNA2亚基的共表达来完全拯救,可能是由于VSD功能的增强,如光学跟踪VSD去极化诱发的构象重排所证明的。反过来,分子动力学模拟表明VSD暴露于膜脂质改变。与其他具有KCNA2突变的脑病患者相比,先证者表现出轻度的神经损伤,KCNA2功能丧失的患者更具特征性。根据这些信息,我们提出了一种基于增强KV1.2失活导致兴奋性神经元突触释放增加的癫痫发生机制,从而扰乱了神经元回路的兴奋/抑制平衡。
KV1.2 channels, encoded by the KCNA2 gene, regulate neuronal excitability by conducting K+ upon depolarization. A new KCNA2 missense variant was discovered in a patient with epilepsy, causing amino acid substitution F302L at helix S4, in the KV1.2 voltage-sensing domain. Immunocytochemistry and flow cytometry showed that F302L does not impair KCNA2 subunit surface trafficking. Molecular dynamics simulations indicated that F302L alters the exposure of S4 residues to membrane lipids. Voltage clamp fluorometry revealed that the voltage-sensing domain of KV1.2-F302L channels is more sensitive to depolarization. Accordingly, KV1.2-F302L channels opened faster and at more negative potentials; however, they also exhibited enhanced inactivation: that is, F302L causes both gain- and loss-of-function effects. Coexpression of KCNA2-WT and -F302L did not fully rescue these effects. The proband\'s symptoms are more characteristic of patients with loss of KCNA2 function. Enhanced KV1.2 inactivation could lead to increased synaptic release in excitatory neurons, steering neuronal circuits towards epilepsy.
An exome-based diagnostic panel in an infant with epilepsy revealed a previously unreported de novo missense variant in KCNA2, which encodes voltage-gated K+ channel KV1.2. This variant causes substitution F302L, in helix S4 of the KV1.2 voltage-sensing domain (VSD). F302L does not affect KCNA2 subunit membrane trafficking. However, it does alter channel functional properties, accelerating channel opening at more hyperpolarized membrane potentials, indicating gain of function. F302L also caused loss of KV1.2 function via accelerated inactivation onset, decelerated recovery and shifted inactivation voltage dependence to more negative potentials. These effects, which are not fully rescued by coexpression of wild-type and mutant KCNA2 subunits, probably result from the enhancement of VSD function, as demonstrated by optically tracking VSD depolarization-evoked conformational rearrangements. In turn, molecular dynamics simulations suggest altered VSD exposure to membrane lipids. Compared to other encephalopathy patients with KCNA2 mutations, the proband exhibits mild neurological impairment, more characteristic of patients with KCNA2 loss of function. Based on this information, we propose a mechanism of epileptogenesis based on enhanced KV1.2 inactivation leading to increased synaptic release preferentially in excitatory neurons, and hence the perturbation of the excitatory/inhibitory balance of neuronal circuits.