Negative charges in the loop between the A and B box of the T1 domain are involved in Kv2.1 and Kv2.1/Kv6.3 channel tetramerization

Abstract

Voltage-gated potassium (Kv) channels are tetramers of four α-subunits. Formation of homo- or heterotetramers is determined by their subfamily specific N-terminal T1 domain. This domain contains two regions designated A and B box, which display a pattern of conservation within and between the Kv subfamilies assumed to determine subfamily specific channel assembly. However, the linker between both boxes is not conserved and its function is uncertain. Here we report that negatively charged residues in this linker are involved in Kv2.1 channel assembly. Mutating these negative charges to arginines caused at least a 15-fold down regulation in current density. To investigate if this was due to a trafficking or a tetramerization deficiency we used FRET analysis to determine the amount of unpaired channel subunits in the plasma membrane versus the ER. Using N-terminal CFP and YFP constructs we observed a 2.5 to >10-fold decline in the FRET efficiency in the ER for these charge reversal substitutions, indicating that there were more unpaired CFP-labeled subunits and that the reduction in current is most likely due to deficient tetramerization. These negative charges are also present in Kv6.3, an electrically silent subunit that fails to form functional homotetramers but selectively co-assembles with the Kv2.x subunits and generates heterotetrameric channels in which Kv6.3 modulates the Kv2.x currents. Mutating this charge cluster in Kv6.3 resulted likewise in a tetramerization defect with Kv2.1 as shown by the reduced FRET efficiencies and the lack of modulating Kv6.3 effects. These results indicate that besides the T1 domain's A and B boxes, the A-B linker is involved in channel tetramerization and could be one of the determinants for the selective co-assembly of Kv6.3 with Kv2.x channels.