Two Epilepsy-Associated Mutations at Same Site in Kv1.2 (KCNA2) Oppositely Affect Channel Functional Expression

We report a new variant of KCNA2 from a pediatric patient with early-onset epilepsy and intellectual disability. KCNA2 encodes the pore-forming subunit of voltage-gated potassium channel KV1.2 and the heterozygous mutation causes amino-acid substitution H310Y. H310 is well-conserved among KV channels, located subsequent to the last arginine (R6) of helix S4 in the voltage-sensing domain (VSD). Interestingly, another mutation at the same position (H310R) was previously reported (ClinVar variation ID: 975835) in a patient with epileptic encephalopathy. Here, we tested the hypothesis that the two mutations affect human KV1.2 channels in the same manner. We first evaluated surface trafficking. Flow cytometry studies on transiently-transfected COS-7 cells showed that KV1.2(H310Y) channels exhibited increased membrane trafficking compared to KV1.2(WT): H310Y surface-label mean fluorescence intensity (MFI) =3.2±0.04log10 (A.U.); WT: MFI=2.8±0.01log10 (A.U.); p=5.02E-10. In stark contrast, KV1.2(H310R) channels exhibited strongly diminished membrane trafficking, being detected in 15.6±3.4% of cells, compared to KV1.2(WT) transfected cells. We then probed the functional consequences of H310 mutations using cut-open oocyte vaseline gap experiments. H310Y significantly slowed KV1.2 channel deactivation kinetics (WT: τ-80=3.9±0.12 ms; n=4; H310Y: τ-80=17±0.55 ms; n=6; p=8.4E-8). H310Y also shifted the voltage-dependence of channel activation towards negative potentials (ΔV0.5=-5.7 mV, n=6). Conversely, VSD activation, optically tracked using voltage-clamp fluorometry, shifted towards positive potentials (ΔV0.5=7.3 mV, n=7), suggesting that H310Y increases VSD-pore coupling. As anticipated from the flow cytometry experiments, no currents were detected in Xenopus oocytes injected with KV1.2(H310R) cRNA. Our work classifies two KCNA2 variants associated with epileptic encephalopathy; H310Y, first-reported here, produces a striking “double” gain-of-function effect (enhanced trafficking and current), while H310R is a loss-of-function mutation that decreases surface trafficking. This highlights the importance of highly-conserved H310 in regulating diverse aspects of channel functional expression.