Voltage-dependent activation in EAG channels follows a ligand-receptor rather than a mechanical-lever mechanism

Abstract

Edited by Roger J. ColbranEther-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, K V 10.1 and human ether-a-go-go-related gene (hERG or K V 11.1), have revealed an original nondomainswapped structure, suggesting that the mechanism of voltagedependent 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-S5 L ) acts as a ligand binding to the S6 gate (S6 C-terminal part, S6 T ) 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-S5 L 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 K V 10.2 channels. Using cysteine bridges and S4-S5 L -mimicking peptides, we show that the ligand/receptor model is conserved in K V 10.2, suggesting that this model is a hallmark of EAG channels. Truncation of the N-Cap domain, Per-Arnt-Sim (PAS) domain, or both in K V 10.2 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.Voltage-gated potassium (K V ) channels regulate a variety of cellular processes, including membrane polarization (1, 2), apoptosis (3), cell proliferation (4), and cell volume (5). In connection with such a variety of functions of K V channels, mutations in these channels cause a variety of pathological conditions in humans: neurological disorders (6, 7), cardiac arrhythmias (2), multiple sclerosis (8), and pain syndrome (9). It has also been shown that K V channels are associated with the development of malignant tumors cancer (10). K V 10 channels belong to the ether-a-go-go family (EAG), 5 as hERG channels. Two isoforms of K V 10 channels are expressed in mammals: K V 10.1 (eag1) and K V 10.2 (eag2), which show 70% amino acid sequence identity. K V 10.1 has been detected mainly in the brain, whereas K V 10.2 is also expressed in other tissues such as the skeletal muscles, heart, placenta, lungs, and liver (11).Atomic structures of rat K V 10.1 (12) and human hERG (K V 11.1) channels (13) highlighted many structural similarities: nonswapped pore and voltage domains, i.e. facing voltage-sensor and pore domains are from the same subunit, short S4-S5 linkers (S4-S5 L ) and similar N-terminal structures such as Per-Arnt-Sim (PAS) and N-Cap domains. We hypothesi...

The EAG Voltage-Dependent K+ Channel Subfamily: Similarities and Differences in Structural Organization and Gating

“…Finally, it is important to note that the proposed existence of a network of interactions involving the N-terminus, the S4-S5 linker and the final portion of S6, and other C-terminal regions, is also compatible with the named ligand/receptor (allosteric) model of voltage-dependent gating (Malak et al, 2017;Malak et al, 2019), recently shown to be shared by both erg1 and eag2 channels, in which the VSD and PD are weakly coupled via a ligand constituted by the S4-S5 linker and a part of S5, and a receptor, the C-terminal part of the S6 segment (Malak et al, 2017;Malak et al, 2019). The reason why the presence of a substantial part of the S5 helix is necessary for the soluble peptides used as ligands to stabilize the closed channel state (Malak et al, 2017) remains to be established.…”

“…Moreover, the possibility that the amino terminal end of the eag2 channel modulates the S4-S5 linker interaction with S6 and, as a consequence, that a hypothetical ligand/receptor gating mechanism exists, has been considered. Unfortunately, as previously shown with erg1 channels split at the S4-S5 linker, deletions of amino terminal sequences of eag2 obliterated by themselves the functional expression of the channels, complicating the possible interpretation of the data (Malak et al, 2019). In any case, it is tempting to speculate that the existence of allosteric components for modulation of gating, documented in Kv10-12 (EAG) channels, but also in other Kv relatives (Barros et al, 2019), constitutes a way to provide evolutionary functional diversification to a common and particularly successful 6TM1P molecular channel design.…”

Up-regulation of voltage-gated sodium channels by peptides mimicking S4-S5 linkers reveals a variation of the ligand-receptor mechanism

“…In this work, we used a S4-S5 L mimicking peptide approach to test whether voltage-gated sodium channels follow the ligand/receptor model previously proposed for hK V 7.1 25 , hK V 11.1 26 and hK V 10.2 29 channels. We identified one activating S4-S5 L peptide in Na V Sp1 and four in hNa V 1.4, suggesting that Na V channels follow a ligand/ receptor model of voltage-dependent gating (Fig.…”

“…This is consistent with the observation that specific S4-S5 L -mimicking peptides inhibit hK V 7.1 and hK V 11.1 channels, by replacing the endogenous segment in the binding pocket 25,26 . This mechanism was recently extended to hK V 10.2 channels 29 .…”

“…From the interaction motif observed in Na V Ms channel 8,16,17 , we hypothesized that S4-S5 L acts as a ligand binding to S6 T and stabilizing the channel open-state and not the closed state. We used the same peptide approach previously used for hK V 7.1, hK V 11.1 and hK V 10.2 channels 25,26,29 to test whether S4-S5 L peptides lead to a gain of function in both prokaryotic and eukaryotic Na V channels.…”