Multigenic, dominant-negative loss-of-function caused by an epilepsy-associated mutation in Kv1.2 (KCNA2)

We have identified an epilepsy-associated mutation in KCNA2. This gene encodes the subunits of tetrameric voltage-gated potassium channel KV1.2, which controls neuronal excitability. Moreover, K+ conductance in the brain is diversified by the heteromerization of KV1.2 subunits with other members of the KV1 family. The mutation, F233S, is located at the well-conserved charge-transfer centre in helix S2. We previously reported that KV1.2(F233S) subunits have a severe trafficking deficiency. Here, we investigated how the mutation affects i) heteromeric KV1.2(WT/F233S)-channels, as in the heterozygous patient and ii) heteromers with known molecular associate KV1.4. Medium-throughput electrophysiological studies showed that “heterozygous” cells co-injected with KV1.2(WT) and KV1.2(F233S) cRNA had a maximum conductance of 21±4% relative to “homozygous-WT,” indicating a dominant-negative loss-of-function. Indeed, increasing the molar ratio of KV1.2(F233S) resulted in a dose-dependent loss of conductance. A binomial distribution model predicted that only KV1.2(WT) homomers and 3WT:1F233S heteromers are trafficking-capable. Accordingly, a flow-cytometry-based surface trafficking assay showed simultaneous i) sequestration of KV1.2(WT) in the presence of KV1.2(F233S) (p=1.3E-7) and ii) rescue of KV1.2(F233S) in the presence of KV1.2(WT) (p=1.7E-7). KV1.4 subunits are more trafficking-efficient than KV1.2. In fact, cells injected with KV1.4 cRNA and increasing proportions of KV1.2(F233S) exhibited macroscopic conductance consistent with the trafficking of KV1.4 homomers, 3Kv1.4:1F233S and 2Kv1.4:2F233S heteromers, but sequestration of 1Kv1.4:3F233S heteromers. Flow cytometry confirmed a partial rescue of KV1.2(F233S) in the presence of KV1.4 (p=1.2E-4). Crucially, a significant reduction of KV1.4 surface trafficking was observed in the presence of KV1.2(F233S) (p=6.6E-7). Our findings show that F233S causes a multigenic dominant-negative loss-of-function, decreasing the functional expression of its own subunits, KV1.2(WT) and KV1.4 subunits. Thus, the heteromerization of KV1 members promotes functional diversity of K+ conductance, but also confers vulnerability to disease-causing mutations in a single KV1 gene.