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...
Styrene-maleic acid (SMA) copolymers are used to extract lipid-encased membrane proteins from lipid bilayers in a detergent-free manner, yielding SMA lipid particles (SMALPs). SMALPs can serve as stable water-soluble nanocontainers for structural and functional studies of membrane proteins. Here, we used SMA copolymers to study full-length pore-forming αsubunits hKCNH5 and hKCNQ1 of human neuronal and cardiac voltage-gated potassium (Kv) channels, as well as the fusion construct comprising of an α-subunit hKCNQ1 and its regulatory transmembrane KCNE1 β-subunit (hKCNE1-hKCNQ1) with added affinity tags, expressed in mammalian COS-1 cells. All these recombinant proteins were shown to be functionally active. Treatment with the SMA copolymer, followed by purification on the affinity column, enabled extraction of all three channels. A DLS experiment demonstrated that Negative stain electron microscopy and single particle image analysis revealed a four-fold symmetry within channelcontaining SMALPs, which indicates that purified hKCNH5 and hKCNQ1 channels, as well as the hKCNE1-hKCNQ1 fusion construct, retained their structural integrity as tetramers.
N-terminally substituted lysine derivatives of gramicidin A (gA), [Lys1]gA and [Lys3]gA, but not glutamate- or aspartate-substituted peptides have been previously shown to cause the leakage of carboxyfluorescein from liposomes. Here, the leakage induction was also observed for [Arg1]gA and [Arg3]gA, while [His1]gA and [His3]gA were inactive at neutral pH. The Lys3-containing analogue with all tryptophans replaced by isoleucines did not induce liposome leakage, similar to gA. This suggests that the presence of both tryptophans and N-terminal cationic residues is critical for pore formation. Remarkably, the addition of gA blocked the leakage induced by [Lys3]gA. By examining with fluorescence correlation spectroscopy the peptide-induced leakage of fluorescent markers from liposomes, we estimated the diameter of pores responsible for the leakage to be about 1.6 nm. Transmission electron cryo-microscopy imaging of liposomes with [Lys3]gA showed that the liposomal membranes contained high electron density particles with a size of about 40 Å, suggesting the formation of peptide clusters. No such clusterization was observed in liposomes incorporating gA or a mixture of gA with [Lys3]gA. Three-dimensional reconstruction of the clusters was compatible with their pentameric arrangement. Based on experimental data and computational modeling, we suggest that the large pore formed by [Lys3]gA represents a barrel-stave oligomeric cluster formed by antiparallel double-stranded helical dimers (DH). In a tentative model, the pentamer of dimers may be stabilized by aromatic Trp-Trp and cation-π Trp-Lys interactions between the neighboring DHs. The inhibiting effect of gA on the [Lys3]gA-induced leakage can be attributed to breaking of cation-π interactions, which prevents peptide clusterization and pore formation.
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