An Epilepsy-Associated Mutation at the KV1.2 (KCNA2) Charge Transfer Center Strongly Suppresses Channel Surface Trafficking

Abstract

We report on a new variant of KCNA2, which encodes the subunits of tetrameric, voltage-gated, potassium-selective channels KV1.2. The de novo, heterozygous mutation was discovered in an infant patient with epilepsy and causes amino-acid substitution F233S. F233 is highly-conserved and constitutes the charge transfer center at the KV1.2 voltage-sensing domain. Our immunocytochemistry (confocal microscopy) investigations in transiently-transfected COS-7 cells revealed that KCNA2(F233S) subunits do not traffic to the cell surface. In flow cytometry experiments, surface-labeled KCNA2(F233S) was detected in 1.60±0.08% of cells, relative to cells transfected with KCNA2(WT). In medium-throughput electrophysiological experiments using the OpusXpress TEVC robot, no currents were detectable in Xenopus oocytes injected with KCNA2(F233S) cRNA (p=0.17 compared with sham-injected oocytes; n≥10). In COVG experiments (n=5), no significant KV1.2(F233S) current was detected in oocytes during depolarizing pulses up to 180mV. We then investigated the interaction of KCNA2(WT) and KCNA2(F233S) subunits on the OpusXpress by comparing oocytes injected with KCNA2 cRNA to emulate homozygous WT, haploinsufficiency (half-dose WT) or heterozygous WT/F233S conditions. All oocytes exhibited currents with similar V0.5, indicating that they were produced by wild-type KV1.2 channels. However, heterozygous WT/F233S cells exhibited a mean macroscopic conductance of 11±1μS, 16% that of homozygous WT (70±12μS) and 28% that of the haploinsufficiency condition (40±4μS). KCNA2(F233S)-mediated suppression of KV1.2 channels was dependent on WT:F233S ratio. These results show that F233S has a dominant loss-of-function effect via a severe trafficking defect, which affects subunits from both alleles and potently suppresses KV1.2 expression. Preliminary experiments show that KCNA4 subunits, which can form heterotetrameric channels with KCNA2, are not sequestered by KCNA2(F233S). We are actively studying the interaction of KCNA2(F233S) with KCNA4 and KCNAB2 subunits, both known to facilitate KCNA2 expression.