Loss-of-function variant in the gene encoding the KCNQ4 potassium channel causes autosomal dominant nonsyndromic hearing loss (DFNA2), and no effective pharmacotherapeutics have been developed to reverse channel activity impairment. Phosphatidylinositol 4,5-bisphosphate (PIP₂), an obligatory phospholipid for maintaining KCNQ channel activity, confers differential pharmacological sensitivity of channels to KCNQ openers. Through whole-exome sequencing of DFNA2 families, we identified three novel KCNQ4 variants related to diverse auditory phenotypes in the proximal C-terminus (p.Arg331Gln), the C-terminus of the S6 segment (p.Gly319Asp), and the pore region (p.Ala271_Asp272del). Potassium currents in HEK293T cells expressing each KCNQ4 variant were recorded by patch-clamp, and functional recovery by PIP₂ expression or KCNQ openers was examined. In the homomeric expression setting, the three novel KCNQ4 mutant proteins lost conductance and were unresponsive to KCNQ openers or PIP₂ expression. Loss of p.Arg331Gln conductance was slightly restored by a tandem concatemer channel (WT-p.R331Q), and increased PIP₂ expression further increased the concatemer current to the level of the WT channel. Strikingly, an impaired homomeric p.Gly319Asp channel exhibited hyperactivity when a concatemer (WT-p.G319D), with a negative shift in the voltage dependence of activation. Correspondingly, a KCNQ inhibitor and chelation of PIP₂ effectively downregulated the hyperactive WT-p.G319D concatemer channel. Conversely, the pore-region variant (p.Ala271_Asp272del) was nonrescuable under any condition. Collectively, these novel KCNQ4 variants may constitute therapeutic targets that can be manipulated by the PIP₂ level and KCNQ-regulating drugs under the physiological context of heterozygous expression. Our research contributes to the establishment of a genotype/mechanism-based therapeutic portfolio for DFNA2.

编码 KCNQ4 钾通道的基因功能丧失变异导致常染色体显性遗传非综合征性听力损失 (DFNA2)，并且尚未开发出有效的药物治疗来逆转通道活动障碍。磷脂酰肌醇 4,5-二磷酸 (PIP ₂ ) 是维持 KCNQ 通道活性的必需磷脂，赋予通道对 KCNQ 开放剂的不同药理学敏感性。通过对 DFNA2 家族的全外显子组测序，我们确定了三个新的KCNQ4变异与近端 C 末端 (p.Arg331Gln)、S6 片段的 C 末端 (p.Gly319Asp) 和孔区域 (p.Ala271_Asp272del) 中的不同听觉表型相关。通过膜片钳记录表达每个 KCNQ4 变体的 HEK293T 细胞中的钾电流，并检查 PIP ₂表达或 KCNQ 开放剂的功能恢复。在同聚体表达设置中，三种新型 KCNQ4 突变蛋白失去电导并且对 KCNQ 开放剂或 PIP ₂表达无反应。串联连接通道 (WT-p.R331Q) 略微恢复了 p.Arg331Gln 电导的损失，并增加了 PIP ₂表达进一步将多联体电流增加到WT通道的水平。引人注目的是，受损的同聚体 p.Gly319Asp 通道在多联体 (WT-p.G319D) 时表现出过度活跃，激活的电压依赖性出现负移。相应地，KCNQ 抑制剂和 PIP ₂螯合有效地下调了过度活跃的 WT-p.G319D 多联体通道。相反，孔隙区域变体 (p.Ala271_Asp272del) 在任何条件下都是不可挽救的。总的来说，这些新的 KCNQ4 变体可能构成治疗靶点，可以在杂合表达的生理背景下由 PIP ₂水平和 KCNQ 调节药物操纵。我们的研究有助于为 DFNA2 建立基于基因型/机制的治疗组合。