Abstract
Ether-a-go-go family (EAG) channels play a major role in many physiological processes in humans, including cardiac repolarization and cell proliferation. Cryo-EM structures of two of them, KV10.1 and human ether-a-go-go-related gene (hERG or KV11.1), have revealed an original nondomain-swapped structure, suggesting that the mechanism of voltage-dependent gating of these two channels is quite different from the classical mechanical-lever model. Molecular aspects of hERG voltage-gating have been extensively studied, indicating that the S4-S5 linker (S4-S5L) acts as a ligand binding to the S6 gate (S6 C-terminal part, S6T) and stabilizes it in a closed state. Moreover, the N-terminal extremity of the channel, called N-Cap, has been suggested to interact with S4-S5L to modulate channel voltage-dependent gating, as N-Cap deletion drastically accelerates hERG channel deactivation. In this study, using COS-7 cells, site-directed mutagenesis, electrophysiological measurements, and immunofluorescence confocal microscopy, we addressed whether these two major mechanisms of voltage-dependent gating are conserved in KV10.2 channels. Using cysteine bridges and S4-S5L-mimicking peptides, we show that the ligand/receptor model is conserved in KV10.2, suggesting that this model is a hallmark of EAG channels. Truncation of the N-Cap domain, Per-Arnt-Sim (PAS) domain, or both in KV10.2 abolished the current and altered channel trafficking to the membrane, unlike for the hERG channel in which N-Cap and PAS domain truncations mainly affected channel deactivation. Our results suggest that EAG channels function via a conserved ligand/receptor model of voltage gating, but that the N-Cap and PAS domains have different roles in these channels.
Highlights
Ether-a-go-go family (EAG) channels play a major role in many physiological processes in humans, including cardiac repolarization and cell proliferation
Using cysteine bridges and S4-S5 linker (S4-S5L)–mimicking peptides, we show that the ligand/receptor model is conserved in KV10.2, suggesting that this model is a hallmark of EAG channels
We could observe that a S4-S5L mimicking peptide inhibits the hERG channel, suggesting a ligand/receptor model, in which S4-S5L, directly under the control of the voltage sensor S4, binds to S6T to lock the channel in a closed state
Summary
The similar structures of hERG and KV10.1 [12, 13] showing a nonswapped arrangement of the pore and voltage sensor domains suggest that voltage-gating of these channels does not follow the classical mechanical-lever model in which S4-S5L constricts the S6T gate [21]. We could observe that a S4-S5L mimicking peptide inhibits the hERG channel, suggesting a ligand/receptor model, in which S4-S5L, directly under the control of the voltage sensor S4, binds to S6T to lock the channel in a closed state (see Fig. 1B, right). Another study has shown that injection of the purified eag domain, corresponding to the first 135 amino acids of hERG, into oocytes expressing eag-truncated hERG, restores the deactivation kinetics to WT-like in more than 24 h [17] Based on these previous observations on hERG, we proposed that co-expression of specific KV10.2 N-Cap mimicking peptides with the N-Cap– truncated KV10.2 channel should recover its expression and activity at the plasma membrane. Combined activities of tetrameric channels containing different ratios of the WT and truncated subunits, it appears that N-terminal deletion of KV10.2 impacts the steady-state activation curve rather than deactivation kinetics
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