Modulation of KV2.1 and KV2.1/KV6.4 Channels by Auxiliary KCNE Subunits

Abstract

Based on sequence homology, eight Shaker-related subfamilies of voltage-gated K+ (Kv) channel α-subunits have been identified: Kv1-Kv6 and Kv8-Kv9. The Kv1-Kv4 subfamilies generate functional channels in both homo- and heterotetrameric configuration while the silent (KvS) channel subunits (Kv5-9) do not form functional homotetramers. These KvS subunits assemble with Kv2 subunits generating functional Kv2/KvS heterotetramers with unique biophysical properties. The diversity within the Kv2 subfamily is further increased by interactions with auxiliary β-subunits (e.g. AMIGO and KCNE1) resulting in altered biophysical properties compared to Kv2.1 alone. While Kv2.1 subunits interact with both the modulatory KvS α-subunits and auxiliary β-subunits, it has not been investigated whether auxiliary β-subunits also interact with Kv2/KvS heterotetramers. We demonstrate here that the transmembrane β-subunit KCNE1 also interacts with Kv2.1/Kv6.4 heterotetramers. Co-expression of KCNE1 with Kv2.1 and Kv6.4 shifts the voltage-dependence of activation approximately 10 mV into hyperpolarizing potentials compared to that of Kv2.1/Kv6.4 alone, without influencing the voltage-dependence of the Kv2.1/Kv6.4 inactivation. This Kv2.1/Kv6.4/KCNE1 interaction was confirmed by Fluorescence Resonance Energy Transfer (FRET) experiments. Co-expression of N-terminal CFP-tagged Kv6.4 with C-terminal YFP-tagged KCNE1 in HEK293 cells did not result in FRET while co-expression of CFP-Kv6.4 with KCNE1-YFP and unlabeled Kv2.1 yielded a FRET efficiency of 6 % which is similar to the FRET efficiency of the positive CFP-Kv2.1 + KCNE1-YFP combination. Furthermore, RT-PCR experiments revealed that Kv6.4 and KCNE1 subunits posses an overlapping expression pattern. These results suggest that a triple complex consisting of Kv2.1, Kv6.4 and KCNE1 subunits can be formed in heterologous cells and might also exist and play a function in vivo.(Supported by FWO fellowship to EB & grant FWO-G.0449.11N to DJS)