We identified six novel de novo human variants in children with motor/language delay, intellectual disability (ID), and/or epilepsy by whole exome sequencing. These variants, comprising two nonsense and four missense alterations, were functionally characterized by electrophysiology in HEK293/CHO cells, together with four previously reported missense variants (Lehman A, Thouta S, Mancini GM, Naidu S, van Slegtenhorst M, McWalter K, Person R, Mwenifumbo J, Salvarinova R; CAUSES Study; EPGEN Study; Guella I, McKenzie MB, Datta A, Connolly MB, Kalkhoran SM, Poburko D, Friedman JM, Farrer MJ, Demos M, Desai S, Claydon T. 101: 65-74, 2017). Surprisingly, all eight missense variants resulted in gain of function (GOF) due to hyperpolarized voltage dependence of activation or slowed deactivation kinetics, whereas the two nonsense variants were confirmed to be loss of function (LOF). One severe GOF allele () was tested and found to extend a dominant GOF effect to heteromeric KCNQ5/3 channels. Clinical presentations were associated with altered KCNQ5 channel gating: milder presentations with LOF or smaller GOF shifts in voltage dependence [change in voltage at half-maximal conduction (Δ) = ∼-15 mV] and severe presentations with larger GOF shifts in voltage dependence (Δ = ∼-30 mV). To examine LOF pathogenicity, two LOF mouse lines were created with CRISPR/Cas9. Both lines exhibited handling- and thermal-induced seizures and abnormal cortical EEGs consistent with epileptiform activity. Our study thus provides evidence for in vivo LOF pathogenicity and strengthens the contribution of both LOF and GOF mutations to global pediatric neurological impairment, including ID/epilepsy. Six novel de novo human variants were identified from children with neurodevelopmental delay, intellectual disability, and/or epilepsy. Expression of these variants along with four previously reported variants from a similar cohort revealed GOF potassium channels, negatively shifted in of activation and/or delayed deactivation kinetics. GOF is extended to KCNQ5/3 heteromeric channels, making these the predominant channels affected in heterozygous de novo patients. LOF mice exhibited seizures, consistent with in vivo pathogenicity.