Hyperpolarization-gated, cyclic nucleotide-activated (HCN1-4) channels are inwardly rectifying cation channels that display voltage dependent activation and de-activation. Pathogenic variants in HCN1 are associated with severe developmental and epileptic encephalopathies including the de novo HCN1 M305L variant. M305 is located in the S5 domain that is implicated in coupling voltage sensor domain movement to pore opening. This variant lacks voltage-dependent activation and de-activation and displays normal cation selectivity. To elucidate the impact of the mutation on the channel structure-function relations, molecular dynamics simulations of the wild type and mutant homotetramers were compared and identified a sulphur-aromatic interaction between M305 and F389 that contributes to the coupling of the voltage-sensing domain to the pore domain. To mimic the heterozygous condition as a heterotetrameric channel assembly, Xenopus oocytes were co-injected with various ratios of wild-type and mutant subunit cRNAs and the biophysical properties of channels with different subunit stoichiometries were determined. The results showed that a single mutated subunit was sufficient to significantly disrupt the voltage dependence of activation. The functional data were qualitatively consistent with predictions of a model that assumes independent activation of the voltage sensing domains allosterically controlling the closed to open transition of the pore. Overall, the M305L mutation results in an HCN1 channel that lacks voltage dependence and facilitates excitatory cation flow at membrane potentials that would normally close the channel. Our findings provide molecular insights into HCN1 channels and reveal the structural and biophysical basis of the severe epilepsy phenotype associated with the M305L mutation.

超极化门控环核苷酸激活 (HCN1-4) 通道是向内整流的阳离子通道，显示电压依赖性激活和去激活。HCN1 的致病性变异与严重的发育性和癫痫性脑病相关，包括新发HCN1 M305L 变体。M305 位于 S5 域中，该域涉及将电压传感器域运动与孔隙开口耦合。这种变体缺乏电压依赖性激活和失活，并显示出正常的阳离子选择性。为了阐明突变对通道结构-功能关系的影响，对野生型和突变型同源四聚体的分子动力学模拟进行了比较，并确定了 M305 和 F389 之间的硫芳香相互作用，这有助于电压传感域与孔域。为了将杂合条件模拟为异四聚体通道组件，非洲爪蟾卵母细胞与不同比例的野生型和突变亚基 cRNAs 共同注射，并确定了具有不同亚基化学计量的通道的生物物理特性。结果表明，单个突变亚基足以显着破坏激活的电压依赖性。功能数据在定性上与模型的预测一致，该模型假设电压传感域的独立激活变构控制孔隙的闭合到开放过渡。总体而言，M305L 突变导致 HCN1 通道缺乏电压依赖性，并在通常会关闭通道的膜电位下促进兴奋性阳离子流动。