Voltage-gated Ion Channel Superfamily Research Articles

Structure of the human K2P13.1(THIK-1) channel reveals a novel hydrophilic pore restriction and lipid cofactor site.

The halothane-inhibited K2P leak potassium channel K2P13.1 (THIK-1)1-3 is found in diverse cells1,4 including neurons1,5 and microglia6-8 where it affects surveillance6, synaptic pruning7, phagocytosis7, and inflammasome-mediated interleukin-1β release6,8,9. As with many K2Ps1,5,10-14 and other voltage-gated ion channel (VGIC) superfamily members3,15,16, polyunsaturated fatty acid (PUFA) lipids modulate K2P13.1 (THIK-1)1,5,14,17 via a poorly understood mechanism. Here, we present cryo-electronmicroscopy (cryo-EM) structures of human K2P13.1 (THIK-1) and mutants in lipid nanodiscs and detergent. These reveal that, unlike other K2Ps13,18-24, K2P13.1 (THIK-1) has a two-chamber aqueous inner cavity obstructed by a M4 transmembrane helix tyrosine (Tyr273, the flow restrictor). This hydrophilic barrier can be opened by an activatory mutation, S136P25, at natural break in the M2 transmembrane helix and by intrinsic channel dynamics. The structures also reveal a buried lipid in the P1/M4 intersubunit interface at a location, the PUFA site, that coincides with the TREK subfamily K2P modulator pocket for small molecule agonists18,26,27. This overlap, together with the effects of mutation on K2P13.1 (THIK-1) PUFA responses, indicates that the PUFA site lipids are K2P13.1 (THIK-1) cofactors. Comparison with the PUFA-responsive VGIC Kv7.1 (KCNQ1)28-31 reveals a shared role for the equivalent pore domain intersubunit interface in lipid modulation, providing a framework for dissecting the effects of PUFAs on the VGIC superfamily. Our findings reveal the unique architecture underlying K2P13.1 (THIK-1) function, highlight the importance of the P1/M4 interface in control of K2Ps by both natural and synthetic agents, and should aid development of THIK subfamily modulators for diseases such as neuroinflammation6,32 and autism6.

The Pentameric Ligand-Gated Ion Channel Family: A New Member of the Voltage Gated Ion Channel Superfamily?

In this report we present seven lines of bioinformatic evidence supporting the conclusion that the Pentameric Ligand-gated Ion Channel (pLIC) Family is a member of the Voltage-gated Ion Channel (VIC) Superfamily. In our approach, we used the Transporter Classification Database (TCDB) as a reference and applied a series of bioinformatic methods to search for similarities between the pLIC family and members of the VIC superfamily. These include: (1) sequence similarity, (2) compatibility of topology and hydropathy profiles, (3) shared domains, (4) conserved motifs, (5) similarity of Hidden Markov Model profiles between families, (6) common 3D structural folds, and (7) clustering analysis of all families. Furthermore, sequence and structural comparisons as well as the identification of a 3-TMS repeat unit in the VIC superfamily suggests that the sixth transmembrane segment evolved into a re-entrant loop. This evidence suggests that the voltage-sensor domain and the channel domain have a common origin. The classification of the pLIC family within the VIC superfamily sheds light onto the topological origins of this family and its evolution, which will facilitate experimental verification and further research into this superfamily by the scientific community.

Deletion of Dictyostelium tpc2 gene forms multi-tipped structures, regulates autophagy and cell-type patterning.

Two pore channels (TPCs) are voltage-gated ion channel superfamily members that release Ca2+ from acidic intracellular stores and are ubiquitously present in both animals and plants. Starvation initiates multicellular development in Dictyostelium discoideum. Increased intracellular calcium levels bias Dictyostelium cells towards the stalk pathway and thus we decided to analyze the role of TPC2 in development, differentiation, and autophagy. We showed TPC2 protein localizes in lysosome-like acidic vesicles and the in situ data showed stalk cell biasness. Deletion of tpc2 showed defective and delayed development with formation of multi-tipped structures attached to a common base, while tpc2OE cells showed faster development with numerous small-sized aggregates and wiry fruiting bodies. The tpc2OE cells showed higher intracellular cAMP levels as compared to the tpc2- cells while pinocytosis was found to be higher in the tpc2- cells. Also, TPC2 regulates cell-substrate adhesion and cellular morphology. Under nutrient starvation, deletion of tpc2 reduced autophagic flux as compared to Ax2. During chimera formation, tpc2- cells showed a bias towards the prestalk/stalk region while tpc2OE cells showed a bias towards the prespore/spore region. tpc2 deficient strain exhibits aberrant cell-type patterning and loss of distinct boundary between the prestalk/prespore regions. TPC2 is required for effective development and differentiation in Dictyostelium and supports autophagic cell death and cell-type patterning. Decreased calcium due to deletion of tpc2 inhibit autophagic flux.

Voltage-Gated Proton Channels in the Tree of Life.

With a single gene encoding HV1 channel, proton channel diversity is particularly low in mammals compared to other members of the superfamily of voltage-gated ion channels. Nonetheless, mammalian HV1 channels are expressed in many different tissues and cell types where they exert various functions. In the first part of this review, we regard novel aspects of the functional expression of HV1 channels in mammals by differentially comparing their involvement in (1) close conjunction with the NADPH oxidase complex responsible for the respiratory burst of phagocytes, and (2) in respiratory burst independent functions such as pH homeostasis or acid extrusion. In the second part, we dissect expression of HV channels within the eukaryotic tree of life, revealing the immense diversity of the channel in other phylae, such as mollusks or dinoflagellates, where several genes encoding HV channels can be found within a single species. In the last part, a comprehensive overview of the biophysical properties of a set of twenty different HV channels characterized electrophysiologically, from Mammalia to unicellular protists, is given.

Interplay between VSD, pore and membrane lipids in electromechanical coupling in HCN channels.

Hyperpolarized-activated and Cyclic Nucleotide-gated (HCN) channels are the only members of the voltage-gated ion channel superfamily in mammals that open upon hyperpolarization, conferring them pacemaker properties that are instrumental for rhythmic firing of cardiac and neuronal cells. Activation of their voltage-sensor domains (VSD) upon hyperpolarization occurs through a downward movement of the S4 helix bearing the gating charges, which triggers a break in the alpha-helical hydrogen bonding pattern at the level of a conserved Serine residue. Previous structural and molecular simulation studies had however failed to capture pore opening that should be triggered by VSD activation, presumably because of a low VSD/pore electromechanical coupling efficiency and the limited timescales accessible to such techniques. Here, we have used advanced modeling strategies, including enhanced sampling molecular dynamics simulations exploiting comparisons between non-domain swapped voltage-gated ion channel structures trapped in closed and open states to trigger pore gating and characterize electromechanical coupling in HCN1. We propose that the coupling mechanism involves the reorganization of the interfaces between the VSD helices, in particular S4, and the pore-forming helices S5 and S6, subtly shifting the balance between hydrophobic and hydrophilic interactions in a 'domino effect' during activation and gating in this region. Remarkably, our simulations reveal state-dependent occupancy of lipid molecules at this emergent coupling interface, suggesting a key role of lipids in hyperpolarization-dependent gating. Our model provides a rationale for previous observations and a possible mechanism for regulation of HCN channels by the lipidic components of the membrane.

Arachidonic acid reverses cholesterol and zinc inhibition of human voltage-gated proton channels

Unlike other members of the voltage-gated ion channel superfamily, voltage-gated proton (Hv) channels are solely composed of voltage sensor domains without separate ion-conducting pores. Due to their unique dependence on both voltage and transmembrane pH gradients, Hv channels normally open to mediate proton efflux. Multiple cellular ligands were also found to regulate the function of Hv channels, including Zn2+, cholesterol, polyunsaturated arachidonic acid, and albumin. Our previous work showed that Zn2+ and cholesterol inhibit the human voltage-gated proton channel hHv1 by stabilizing its S4 segment at resting state conformations. Released from phospholipids by phospholipase A2 in cells upon infection or injury, arachidonic acid regulates the function of many ion channels, including hHv1. In the present work, we examined the effects of arachidonic acid on purified hHv1 channels using liposome flux assays and revealed underlying structural mechanisms using single-molecule Fluorescence Resonance Energy Transfer (smFRET). Our data indicated that arachidonic acid strongly activates hHv1 channels by promoting transitions of the S4 segment towards opening or 'pre-opening' conformations. Moreover, we found that arachidonic acid even activates hHv1 channels inhibited by Zn2+ and cholesterol, providing a biophysical mechanism to activate hHv1 channels in non-excitable cells upon infection or injury.

Functional characterization of gating polarity in CNBD channels across phylogenetically diverse species.

The cyclic nucleotide binding domain (CNBD) family of the voltage-gated ion channel superfamily (VGIC) is involved in essential physiological processes, e.g. pacemaking, neuronal signaling, olfaction, and photoreception. CNBD channels are found in species throughout the tree of life, and for those with confirmed structures, show striking similarity in structure despite differences in gating polarity. Functional characterization from extant species closely related to ancestral species of each CNBD subfamily would provide more information regarding how functional divergence emerged while maintaining structural conservation. Here, we generated a phylogenetic tree of the CNBD family and functionally characterized the voltage sensitivity of extant species that are closest to each subclade's ancestral node. Our preliminary studies reveal that functional characterization of these extant species match with function of reported species that share the same subclade in our phylogenetic tree. Using this functional data, ancestral protein reconstruction of ancestral nodes of these subfamilies will be characterized and compared in upcoming future work to further elucidate what key residues drive the diversity in gating polarity in the CNBD channel family.

Voltage sensor dynamics of a bacterial voltage-gated sodium channel NavAb reveal three conformational states

High-resolution structures of voltage-gated sodium channels (Nav) were first obtained from a prokaryotic ortholog NavAb, which provided important mechanistic insights into Na+ selectivity and voltage gating. Unlike eukaryotic Navs, the NavAb channel is formed by four identical subunits, but its ion selectivity and pharmacological profiles are very similar to eukaryotic Navs. Recently, the structures of the NavAb voltage sensor at resting and activated states were obtained by cryo-EM, but its intermediate states and transition dynamics remain unclear. In the present work, we used liposome flux assays to show that purified NavAb proteins were functional to conduct both H+ and Na+ and were blocked by the local anesthetic lidocaine. Additionally, we examined the real-time conformational dynamics of the NavAb voltage sensor using single-molecule FRET. Our single-molecule FRET measurements on the tandem NavAb channel labeled with Cy3/5 FRET fluorophore pair revealed spontaneous transitions of the NavAb S4 segment among three conformational states, which fitted well with the kinetic model developed for the S4 segment of the human voltage-gated proton channel hHv1. Interestingly, even under strong activating voltage, the NavAb S4 segment seems to adopt a conformational distribution similar to that of the hHv1 S4 segment at a deep resting state. The conformational behaviors of the NavAb voltage sensor under different voltages need to be further examined to understand the mechanisms of voltage sensing and gating in the canonical voltage-gated ion channel superfamily.

Charge-voltage curves of Shaker potassium channel are not hysteretic at steady state.

Charge-voltage curves of many voltage-gated ion channels exhibit hysteresis but such curves are also a direct measure of free energy of channel gating and, hence, should be path-independent. Here, we identify conditions to measure steady-state charge-voltage curves and show that these are curves are not hysteretic. Charged residues in transmembrane segments of voltage-gated ion channels (VGICs) sense and respond to changes in the electric field. The movement of these gating charges underpins voltage-dependent activation and is also a direct metric of the net free-energy of channel activation. However, for most voltage-gated ion channels, the charge-voltage (Q-V) curves appear to be dependent on initial conditions. For instance, Q-V curves of Shaker potassium channel obtained by hyperpolarizing from 0 mV is left-shifted compared to those obtained by depolarizing from a holding potential of -80 mV. This hysteresis in Q-V curves is a common feature of channels in the VGIC superfamily and raises profound questions about channel energetics because the net free-energy of channel gating is a state function and should be path independent. Due to technical limitations, conventional gating current protocols are limited to test pulse durations of <500 ms, which raises the possibility that the dependence of Q-V on initial conditions reflects a lack of equilibration. Others have suggested that the hysteresis is fundamental thermodynamic property of voltage-gated ion channels and reflects energy dissipation due to measurements under non-equilibrium conditions inherent to rapid voltage jumps (Villalba-Galea. 2017. Channels. https://doi.org/10.1080/19336950.2016.1243190). Using an improved gating current and voltage-clamp fluorometry protocols, we show that the gating hysteresis arising from different initial conditions in Shaker potassium channel is eliminated with ultra-long (18-25 s) test pulses. Our study identifies a modified gating current recording protocol to obtain steady-state Q-V curves of a voltage-gated ion channel. Above all, these findings demonstrate that the gating hysteresis in Shaker channel is a kinetic phenomenon rather than a true thermodynamic property of the channel and the charge-voltage curve is a true measure of the net-free energy of channel gating.

Quaternary structure independent folding of voltage-gated ion channel pore domain subunits.

Every voltage-gated ion channel (VGIC) has a pore domain (PD) made from four subunits, each comprising an antiparallel transmembrane helix pair bridged by a loop. The extent to which PD subunit structure requires quaternary interactions is unclear. Here, we present crystal structures of a set of bacterial voltage-gated sodium channel (BacNaV) 'pore only' proteins that reveal a surprising collection of non-canonical quaternary arrangements in which the PD tertiary structure is maintained. This context-independent structural robustness, supported by molecular dynamics simulations, indicates that VGIC-PD tertiary structure is independent of quaternary interactions. This fold occurs throughout the VGIC superfamily and in diverse transmembrane and soluble proteins. Strikingly, characterization of PD subunit-binding Fabs indicates that non-canonical quaternary PD conformations can occur in full-length VGICs. Together, our data demonstrate that the VGIC-PD is an autonomously folded unit. This property has implications for VGIC biogenesis, understanding functional states, de novo channel design, and VGIC structural origins.

Concurrent sodium channel myotonia and amyotrophic lateral sclerosis supports shared pathogenesis

Amyotrophic lateral sclerosis (ALS) is an invariably fatal adult-onset neurodegenerative disorder; 10% of ALS is autosomal dominant but even sporadic ALS exhibits significant heritability. Dysfunctional voltage- gated sodium channels (NaVs) have been implicated in both sporadic and familial forms of the disease, but it is unclear whether these are an upstream cause or a downstream consequence. Rare missense mutations in the SCN4A gene, encoding NaV1.4, disrupt membrane excitability and cause autosomal dominant muscle channelopathies such as myotonia and hypo/hyperkalaemic periodic paralysis. We report two patients with congenital SCN4A channelopathy due to p.S1159P and p.R672H mutations who developed ALS in later life. Based on our finding we hypothesised that ALS may be linked to genetic mutations in other voltage gated ion channels. Rare-variant burden testing using whole genome sequenc- ing data from 4,495 ALS patients and 1,925 controls within the superfamily of voltage-gated ion channels (Accession: ssf81324) identified one gene, SCN7A, which passed multiple testing correction (p=0.0002, Firth logistic regression) consistent with an enrichment of ALS-associated mutations. We propose that genetic mutations within NaVs leading to neuromuscular excitotoxicity are an upstream cause of ALS. Early identification of ALS patients carrying NaV mutations might lead to personalised therapy with a sodium channel-blocking agent such as riluzole.j.franklin@doctors.org.uk

Activation of endo-lysosomal two-pore channels by NAADP and PI(3,5)P2. Five things to know.

Two-pore channels are ancient members of the voltage-gated ion channel superfamily that are expressed predominantly on acidic organelles such as endosomes and lysosomes. Here we review recent advances in understanding how TPCs are activated by their ligands and identify five salient features: (1) TPCs are Ca2+-permeable non-selective cation channels gated by NAADP. (2) NAADP activation is indirect through associated NAADP receptors. (3) TPCs are also Na+-selective channels gated by PI(3,5)P2. (4) PI(3,5)P2 activation is direct through a structurally-resolved binding site. (5) TPCs switch their ion selectivity in an agonist-dependent manner.

KCNE1 is an auxiliary subunit of two distinct ion channel superfamilies

Determination of what is the specificity of subunits composing a protein complex is essential when studying gene variants on human pathophysiology. The pore-forming α-subunit KCNQ1, which belongs to the voltage-gated ion channel superfamily, associates to its β-auxiliary subunit KCNE1 to generate the slow cardiac potassium IKs current, whose dysfunction leads to cardiac arrhythmia. Using pharmacology, gene invalidation, and single-molecule fluorescence assays, we found that KCNE1 fulfils all criteria of a bona fide auxiliary subunit of the TMEM16A chloride channel, which belongs to the anoctamin superfamily. Strikingly, assembly with KCNE1 switches TMEM16A from a calcium-dependent to a voltage-dependent ion channel. Importantly, clinically relevant inherited mutations within the TMEM16A-regulating domain of KCNE1 abolish the TMEM16A modulation, suggesting that the TMEM16A-KCNE1 current may contribute to inherited pathologies. Altogether, these findings challenge the dogma of the specificity of auxiliary subunits regarding protein complexes and questions ion channel classification.

Dimer interaction in the Hv1 proton channel

The voltage-gated proton channel Hv1 is a member of the voltage-gated ion channel superfamily, which stands out in design: It is a dimer of two voltage-sensing domains (VSDs), each containing a pore pathway, a voltage sensor (S4), and a gate (S1) and forming its own ion channel. Opening of the two channels in the dimer is cooperative. Part of the cooperativity is due to association between coiled-coil domains that extend intracellularly from the S4s. Interactions between the transmembrane portions of the subunits may also contribute, but the nature of transmembrane packing is unclear. Using functional analysis of a mutagenesis scan, biochemistry, and modeling, we find that the subunits form a dimer interface along the entire length of S1, and also have intersubunit contacts between S1 and S4. These interactions exert a strong effect on gating, in particular on the stability of the open state. Our results suggest that gating in Hv1 is tuned by extensive VSD-VSD interactions between the gates and voltage sensors of the dimeric channel.

Dimer Interaction in the Hv1 Proton Channel

The voltage-gated proton channel Hv1 is a member of the voltage-gated ion channel superfamily, which stands out in design: it is a dimer of two voltage-sensing domains (VSDs), each containing a pore pathway, a voltage sensor (S4) and a gate (S1) and forming its own ion channel. Opening of the two channels in the dimer is cooperative. Part of the cooperativity is due to association between coiled-coil domains that extend intracellularly from the S4s, which are thought to couple S4 activation motions.

A Selectivity Filter Gate Controls Voltage-Gated Calcium Channel Calcium-Dependent Inactivation

Calcium-dependent inactivation (CDI) is a fundamental autoregulatory mechanism in CaV1 and CaV2 voltage-gated calcium channels. Although CDI initiates with the cytoplasmic calcium sensor, how this event causes CDI has been elusive. Here, we show that a conserved selectivity filter (SF) domain II (DII) aspartate is essential for CDI. Mutation of this residue essentially eliminates CDI and leaves key channel biophysical characteristics untouched. DII mutants regain CDI by placing an aspartate at the analogous SF site in DIII or DIV, but not DI, indicating that CaV SF asymmetry is key to CDI. Together, our data establish that the CaV SF is the CDI endpoint. Discovery of this SF CDI gate recasts the CaV inactivation paradigm, placing it squarely in the framework of voltage-gated ion channel (VGIC) superfamily members in which SF-based gating is important. This commonality suggests that SF inactivation is an ancient process arising from the shared VGIC pore architecture.

Voltage vs. Ligand II: Structural insights of the intrinsic flexibility in cyclic nucleotide-gated channels

ABSTRACTIn the preceding article, we present a flexibility analysis of the voltage-gated ion channel (VGIC) superfamily. In this study, we describe in detail the flexibility profile of the voltage-sensor domain (VSD) and the pore domain (PD) concerning the evolution of 6TM ion channels. In particular, we highlight the role of flexibility in the emergence of CNG channels and describe a significant level of sequence similarity between the archetypical VSD and the TolQ proteins. A highly flexible S4-like segment exhibiting Lys instead Arg for these membrane proteins is reported. Sequence analysis indicates that, in addition to this S4-like segment, TolQ proteins also show similarity with specific motifs in S2 and S3 from typical V-sensors. Notably, S3 flexibility profiles from typical VSDs and S3-like in TolQ proteins are also similar. Interestingly, TolQ from early divergent prokaryotes are comparatively more flexible than those in modern counterparts or true V-sensors. Regarding the PD, we also found that 2TM K+-channels in early prokaryotes are considerably more flexible than the ones in modern microbes, and such flexibility is comparable to the one present in CNG channels. Voltage dependence is mainly exhibited in prokaryotic CNG channels whose VSD is rigid whereas the eukaryotic CNG channels are considerably more flexible and poorly V-dependent. The implication of the flexibility present in CNG channels, their sensitivity to cyclic nucleotides and the cation selectivity are discussed. Finally, we generated a structural model of the putative cyclic nucleotide-modulated ion channel, which we coined here as AqK, from the thermophilic bacteria Aquifex aeolicus, one of the earliest diverging prokaryotes known. Overall, our analysis suggests that V-sensors in CNG-like channels were essentially rigid in early prokaryotes but raises the possibility that this module was probably part of a very flexible stator protein of the bacterial flagellum motor complex.

Bipolar switching by HCN voltage sensor underlies hyperpolarization activation

Despite sharing a common architecture with archetypal voltage-gated ion channels (VGICs), hyperpolarization- and cAMP-activated ion (HCN) channels open upon hyperpolarization rather than depolarization. The basic motions of the voltage sensor and pore gates are conserved, implying that these domains are inversely coupled in HCN channels. Using structure-guided protein engineering, we systematically assembled an array of mosaic channels that display the full complement of voltage-activation phenotypes observed in the VGIC superfamily. Our studies reveal that the voltage sensor of the HCN channel has an intrinsic ability to drive pore opening in either direction and that the extra length of the HCN S4 is not the primary determinant for hyperpolarization activation. Tight interactions at the HCN voltage sensor-pore interface drive the channel into an hERG-like inactivated state, thereby obscuring its opening upon depolarization. This structural element in synergy with the HCN cyclic nucleotide-binding domain and specific interactions near the pore gate biases the channel toward hyperpolarization-dependent opening. Our findings reveal an unexpected common principle underpinning voltage gating in the VGIC superfamily and identify the essential determinants of gating polarity.

Thermodynamics of voltage-gated ion channels

Ion channels are essential for cellular signaling. Voltage-gated ion channels (VGICs) are the largest and most extensively studied superfamily of ion channels. They possess modular structural features such as voltage-sensing domains that encircle and form mechanical connections with the pore-forming domains. Such features are intimately related to their function in sensing and responding to changes in the membrane potential. In the present work, we discuss the thermodynamic mechanisms of the VGIC superfamily, including the two-state gating mechanism, sliding-rocking mechanism of the voltage sensor, subunit cooperation, lipid-infiltration mechanism of inactivation, and the relationship with their structural features.

Gating interaction maps reveal a noncanonical electromechanical coupling mode in the Shaker K+ channel

Membrane potential regulates the activity of voltage-dependent ion channels via specialized voltage-sensing modules but the mechanisms involved in coupling voltage-sensor movement to pore opening remain unclear due to lack of resting state structures and robust methods to identify allosteric pathways. Here, using a newly developed interaction energy analysis, we probe the interfaces of the voltage-sensing and pore modules in the drosophila Shaker K+ channel. Our measurements reveal unexpectedly strong equilibrium gating interactions between contacts at the S4 and S5 helices in addition to those between S6 and S4–S5 linker. Network analysis of MD trajectories shows that the voltage-sensor and pore motions are linked by two distinct pathways- canonical one through the S4–S5 linker and a hitherto unknown pathway akin to rack and pinion coupling involving S4 and S5 helices. Our findings highlight the central role of the S5 helix in electromechanical transduction in the VGIC superfamily.