Key points K(V)1.2 channels, encoded by theKCNA2gene, regulate neuronal excitability by conducting K(+)upon depolarization. A newKCNA2missense variant was discovered in a patient with epilepsy, causing amino acid substitution F302L at helix S4, in the K(V)1.2 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 K(V)1.2-F302L channels is more sensitive to depolarization. Accordingly, K(V)1.2-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 probands symptoms are more characteristic of patients with loss ofKCNA2function. Enhanced K(V)1.2 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 unreportedde novomissense variant inKCNA2, which encodes voltage-gated K(+)channel K(V)1.2. This variant causes substitution F302L, in helix S4 of the K(V)1.2 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 K(V)1.2 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 withKCNA2mutations, the proband exhibits mild neurological impairment, more characteristic of patients withKCNA2loss of function. Based on this information, we propose a mechanism of epileptogenesis based on enhanced K(V)1.2 inactivation leading to increased synaptic release preferentially in excitatory neurons, and hence the perturbation of the excitatory/inhibitory balance of neuronal circuits.

Significance: A child with epilepsy has a previously unreported, heterozygous mutation in KCNA2, the gene encoding K_V1.2 proteins. Four K_V1.2 assemble into a potassium-selective channel, a protein complex at the neuronal cell surface regulating electrical signaling. K_V1.2 subunits assemble with other K_V1-family members to form heterotetrameric channels, contributing to neuronal potassium-channel diversity. The most striking consequence of this mutation is preventing K_V1.2-subunit trafficking, i.e., their ability to reach the cell surface. Moreover, the mutation is dominant negative, as mutant subunits can assemble with wild-type K_V1.2 and K_V1.4, trapping them into nontrafficking heterotetramers and decreasing their functional expression. Thus, K_V1-family genes’ ability to form heterotetrameric channels is a double-edged sword, rendering K_V1-family members vulnerable to dominant-negative mutations in a single member gene.

Abstract: We report on a heterozygous KCNA2 variant in a child with epilepsy. KCNA2 encodes K_V1.2 subunits, which form homotetrameric potassium channels and participate in heterotetrameric channel complexes with other K_V1-family subunits, regulating neuronal excitability. The mutation causes substitution F233S at the K_V1.2 charge transfer center of the voltage-sensing domain. Immunocytochemical trafficking assays showed that K_V1.2(F233S) subunits are trafficking deficient and reduce the surface expression of wild-type K_V1.2 and K_V1.4: a dominant-negative phenotype extending beyond KCNA2, likely profoundly perturbing electrical signaling. Yet some K_V1.2(F233S) trafficking was rescued by wild-type K_V1.2 and K_V1.4 subunits, likely in permissible heterotetrameric stoichiometries: electrophysiological studies utilizing applied transcriptomics and concatemer constructs support that up to one or two K_V1.2(F233S) subunits can participate in trafficking-capable heterotetramers with wild-type K_V1.2 or K_V1.4, respectively, and that both early and late events along the biosynthesis and secretion pathway impair trafficking. These studies suggested that F233S causes a depolarizing shift of ∼48 mV on K_V1.2 voltage dependence. Optical tracking of the K_V1.2(F233S) voltage-sensing domain (rescued by wild-type K_V1.2 or K_V1.4) revealed that it operates with modestly perturbed voltage dependence and retains pore coupling, evidenced by off-charge immobilization. The equivalent mutation in the Shaker K⁺ channel (F290S) was reported to modestly affect trafficking and strongly affect function: an ∼80-mV depolarizing shift, disrupted voltage sensor activation and pore coupling. Our work exposes the multigenic, molecular etiology of a variant associated with epilepsy and reveals that charge-transfer-center disruption has different effects in K_V1.2 and Shaker, the archetypes for potassium channel structure and function.