Voltage-gated Ion Channel Superfamily Research Articles

Structural insights into the voltage and phospholipid activation of the mammalian TPC1 channel

Organellar two-pore channels (TPCs) function as a homodimer with each subunit containing two homologous Shaker-like 6-TM repeats1. They belong to the voltage-gated ion channel superfamily2 and are ubiquitously expressed in animals and plants3,4. Mammalian TPC1 and TPC2 are localized to the endolysosomal membrane and play critical roles in regulating the physiological functions of these acidic organelles5–7. Here we present the cryo-EM structures of mouse TPC1 (MmTPC1), a voltage-dependent, phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) activated Na+ selective channel, in both the apo closed and ligand-bound open states which, combined with functional analysis, provide comprehensive structural insights into the selectivity and gating mechanisms of mammalian TPC channels. The channel has a coin slot-shaped ion pathway in the filter that defines the selectivity of mammalian TPCs. Only the voltage sensing domain from the second 6-TM domain confers voltage dependence to MmTPC1. Endolysosome-specific PtdIns(3,5)P2 binds to the first 6-TM domain and activates the channel under depolarizing membrane potential. Structural comparison between the apo and PtdIns(3,5)P2-bound structures elucidates the interplay between voltage and ligand in channel activation. In light of the emerging importance of phosphoinositide regulation of ion channels, the MmTPC1 structures exemplify the lipid binding and regulation in a 6-TM voltage-gated channel.

Induction of divalent cation permeability by heterologous expression of a voltage sensor domain

The voltage sensor domain (VSD) is a protein domain that confers sensitivity to membrane potential in voltage-gated ion channels as well as the voltage-sensing phosphatase. Although VSDs have long been considered to function as regulatory units acting on adjacent effectors, recent studies have revealed the existence of direct ion permeation paths in some mutated VSDs and in the voltage-gated proton channel. In this study, we show that calcium currents are evoked upon membrane hyperpolarization in cells expressing a VSD derived from an ascidian voltage-gated ion channel superfamily. Unlike the previously reported omega-pore in the Shaker K+ channel and rNav1.4, mutations are not required. From electrophysiological experiments in heterologous expression systems, we found that the conductance is directly mediated by the VSD itself and is carried by both monovalent and divalent cations. This is the first report of divalent cation permeation through a VSD-like structure.

NAADP-evoked Ca2+ signals through two-pore channel-1 require arginine residues in the first S4-S5 linker

Two-pore channels (TPCs) are two-domain members of the voltage-gated ion channel superfamily that localize to acidic organelles. Their mechanism of activation (ligands such as NAADP/PI(3,5)P2 versus voltage) and ion selectivity (Ca2+ versus Na+) is debated. Here we report that a cluster of arginine residues in the first domain required for selective voltage-gating of TPC1 map not to the voltage-sensing fourth transmembrane region (S4) but to a cytosolic downstream region (S4-S5 linker). These residues are conserved between TPC isoforms suggesting a generic role in TPC activation. Accordingly, mutation of residues in TPC1 but not the analogous region in the second domain prevents Ca2+ release by NAADP in intact cells. Our data affirm the role of TPCs in NAADP-mediated Ca2+ signalling and unite differing models of channel activation through identification of common domain-specific residues.

K2P2.1 (TREK-1)-activator complexes reveal a cryptic selectivity filter binding site.

Polymodal K2P (KCNK) thermo- and mechanosensitive TREK1 potassium channels, generate ‘leak’ currents that regulate neuronal excitability, respond to lipids, temperature, and mechanical stretch, and influence pain, temperature perception, and anesthetic responses1–3. These dimeric voltage-gated ion channel (VGIC) superfamily members have a unique topology comprising two pore forming regions per subunit4–6. Contrasting other potassium channels, K2Ps use a selectivity filter ‘C-type’ gate7–10 as the principal gating site. Despite recent advances3,11,12, K2Ps suffer from a poor pharmacologic profile limiting mechanistic and biological studies. Here, we describe a new small molecule TREK activator class that directly stimulates the C-type gate by acting as molecular wedges that restrict interdomain interface movement behind the selectivity filter. Structures of K2P2.1(TREK-1) alone with two selective K2P2.1(TREK-1) and K2P10.1(TREK-2) activators, an N-aryl-sulfonamide, ML335, and a thiophene-carboxamide, ML402, define a cryptic binding pocket unlike other ion channel small molecule binding sites and, together with functional studies, identify a cation-π interaction that controls selectivity. Together, our data unveil a previously unknown, druggable K2P site that stabilizes the C-type gate ‘leak mode’ and provide direct evidence for K2P selectivity filter gating.

Structure of a eukaryotic cyclic-nucleotide-gated channel

Cyclic-nucleotide-gated channels are essential for vision and olfaction. They belong to the voltage-gated ion channel superfamily but their activities are controlled by intracellular cyclic nucleotides instead of transmembrane voltage. Here we report a 3.5-Å-resolution single-particle electron cryo-microscopy structure of a cyclic-nucleotide-gated channel from Caenorhabditis elegans in the cyclic guanosine monophosphate (cGMP)-bound open state. The channel has an unusual voltage-sensor-like domain, accounting for its deficient voltage dependence. A carboxy-terminal linker connecting S6 and the cyclic-nucleotide-binding domain interacts directly with both the voltage-sensor-like domain and the pore domain, forming a gating ring that couples conformational changes triggered by cyclic nucleotide binding to the gate. The selectivity filter is lined by the carboxylate side chains of a functionally important glutamate and three rings of backbone carbonyls. This structure provides a new framework for understanding mechanisms of ion permeation, gating and channelopathy of cyclic-nucleotide-gated channels and cyclic nucleotide modulation of related channels.

Structural Heterogeneity of CNGA1 Channels Revealed by Electrophysiology and Single-Molecule Force Spectroscopy.

The determination at atomic resolution of the three-dimensional molecular structure of membrane proteins such as receptors and several ion channels has been a major breakthrough in structural biology. The molecular structure of several members of the superfamily of voltage-gated ionic channels such as K+ and Na+ is now available. However, despite several attempts, the molecular structure at atomic resolution of the full cyclic nucleotide-gated (CNG) ion channel, although a member of the same superfamily of voltage-gated ion channels, has not been obtained yet, neither by X-ray crystallography nor by electron cryomicroscopy (cryo-EM). It is possible that CNG channels have a high structural heterogeneity, making difficult crystallization and single-particle analysis. To address this issue, we have combined single-molecule force spectroscopy (SMFS) and electrophysiological experiments to characterize the structural heterogeneity of CNGA1 channels expressed in Xenopus laevis oocytes. The unfolding of the cytoplasmic domain had force peaks, occurring with a probability from 0.2 to 0.96. Force peaks during the unfolding of the transmembrane domain had a probability close to 1, but the distribution of the increase in contour length between two successive force peaks had multiple maxima differing by tens of nanometers. Concomitant electrophysiological experiments showed that the rundown in mutant channels S399C is highly variable and that the effect of thiol reagents when specific residues were mutated was consistent with a dynamic structural heterogeneity. These results show that CNGA1 channels have a wide spectrum of native conformations that are difficult to detect with X-ray crystallography and cryo-EM.

Two-pore channels at the intersection of endolysosomal membrane traffic.

Two-pore channels (TPCs) are ancient members of the voltage-gated ion channel superfamily that localize to acidic organelles such as lysosomes. The TPC complex is the proposed target of the Ca2+-mobilizing messenger NAADP, which releases Ca2+ from these acidic Ca2+ stores. Whereas details of TPC activation and native ion permeation remain unclear, a consensus has emerged around their function in regulating endolysosomal trafficking. This role is supported by recent proteomic data showing that TPCs interact with proteins controlling membrane organization and dynamics, including Rab GTPases and components of the fusion apparatus. Regulation of TPCs by PtdIns(3,5)P2 and/or NAADP (nicotinic acid adenine dinucleotide phosphate) together with their functional and physical association with Rab proteins provides a mechanism for coupling phosphoinositide and trafficking protein cues to local ion fluxes. Therefore, TPCs work at the regulatory cross-roads of (patho)physiological cues to co-ordinate and potentially deregulate traffic flow through the endolysosomal network. This review focuses on the native role of TPCs in trafficking and their emerging contributions to endolysosomal trafficking dysfunction.

Poring over two-pore channel pore mutants.

Two-pore channels are members of the voltage-gated ion channel superfamily. They localise to the endolysosomal system and are likely targets for the Ca2+ mobilising messenger NAADP. In this brief review, we relate mutagenesis of the TPC pore to a recently published homology model and discuss how pore mutants are informing us of TPC function. Molecular physiology of these ubiquitous proteins is thus emerging.

Molecular Determinants of Temperature Dependent Gating of Ion Channels

Physiological sensation of heat or cold in higher organisms is mediated by specialized ion channels whose opening and closing is exquisitely regulated by ambient temperatures. Members of TRP channel family, a branch of the much larger voltage-gated ion channel superfamily, serve as the primary physiological thermo-sensors. However, the physicochemical underpinnings of high temperature-sensitivity of channel gating remain poorly understood. Here, using a heuristic protein design approach, we have transmuted a temperature-insensitive potassium channel into a heat or a cold-sensitive channel. By varying amino acid polarities at sites undergoing state-dependent changes in solvation, we were able to systematically confer temperature-sensing phenotype to a prototypical voltage-dependent potassium channel. We also demonstrate that magnitude of voltage-sensing charges inversely modulate temperature-sensitivity consistent with predictions of thermodynamic coupling. These emerging molecular principles provide a template to understand varied temperature-dependent gating phenotype in channels with conserved transmembrane architecture.

Interplay between R513 methylation and S516 phosphorylation of the cardiac voltage-gated sodium channel.

Arginine methylation is a novel post-translational modification within the voltage-gated ion channel superfamily, including the cardiac sodium channel, NaV1.5. We show that NaV1.5 R513 methylation decreases S516 phosphorylation rate by 4 orders of magnitude, the first evidence of protein kinase A inhibition by arginine methylation. Reciprocally, S516 phosphorylation blocks R513 methylation. NaV1.5 p.G514C, associated to cardiac conduction disease, abrogates R513 methylation, while leaving S516 phosphorylation rate unchanged. This is the first report of methylation-phosphorylation cross-talk of a cardiac ion channel.

Structure of the rabbit ryanodine receptor RyR1 at near-atomic resolution.

The ryanodine receptors (RyRs) are high-conductance intracellular Ca2+ channels that play a pivotal role in the excitation-contraction coupling of skeletal and cardiac muscles. RyRs are the largest known ion channels, with a homotetrameric organization and approximately 5000 residues in each protomer. Here we report the structure of the rabbit RyR1 in complex with its modulator FKBP12 at an overall resolution of 3.8 Å, determined by single-particle electron cryo-microscopy. Three previously uncharacterized domains, named Central, Handle, and Helical domains, display the armadillo repeat fold. These domains, together with the amino-terminal domain, constitute a network of superhelical scaffold for binding and propagation of conformational changes. The channel domain exhibits the voltage-gated ion channel superfamily fold with distinct features. A negative charge-enriched hairpin loop connecting S5 and the pore helix is positioned above the entrance to the selectivity filter vestibule. The four elongated S6 segments form a right-handed helical bundle that closes the pore at the cytoplasmic border of the membrane. Allosteric regulation of the pore by the cytoplasmic domains is mediated through extensive interactions between the Central domains and the channel domain. These structural features explain high ion conductance by RyRs and the long-range allosteric regulation of channel activities.

Identification of N-terminal protein acetylation and arginine methylation of the voltage-gated sodium channel in end-stage heart failure human heart

The α subunit of the cardiac voltage-gated sodium channel, NaV1.5, provides the rapid sodium inward current that initiates cardiomyocyte action potentials. Here, we analyzed for the first time the post-translational modifications of NaV1.5 purified from end-stage heart failure human cardiac tissue. We identified R526 methylation as the major post-translational modification of any NaV1.5 arginine or lysine residue. Unexpectedly, we found that the N terminus of NaV1.5 was: 1) devoid of the initiation methionine, and 2) acetylated at the resulting initial alanine residue. This is the first evidence for N-terminal acetylation in any member of the voltage-gated ion channel superfamily. Our results open the door to explore NaV1.5 N-terminal acetylation and arginine methylation levels as drivers or markers of end-stage heart failure.

Cortical HCN channels: function, trafficking and plasticity.

The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels belong to the superfamily of voltage-gated potassium ion channels. They are, however, activated by hyperpolarizing potentials and are permeable to cations. Four HCN subunits have been cloned, of which HCN1 and HCN2 subunits are predominantly expressed in the cortex. These subunits are principally located in pyramidal cell dendrites, although they are also found at lower concentrations in the somata of pyramidal neurons as well as other neuron subtypes. HCN channels are actively trafficked to dendrites by binding to the chaperone protein TRIP8b. Somato-dendritic HCN channels in pyramidal neurons modulate spike firing and synaptic potential integration by influencing the membrane resistance and resting membrane potential. Intriguingly, HCN channels are present in certain cortical axons and synaptic terminals too. Here, they regulate synaptic transmission but the underlying mechanisms appear to vary considerably amongst different synaptic terminals. In conclusion, HCN channels are expressed in multiple neuronal subcellular compartments in the cortex, where they have a diverse and complex effect on neuronal excitability.

General flexible nature of the cytosolic regions of fungal transient receptor potential (TRP) channels, revealed by expression screening using GFP-fusion techniques.

Transient receptor potential (TRP) channels are members of the voltage gated ion channel superfamily and display the unique characteristic of activation by diverse stimuli. We performed an expression analysis of fungal TRP channels, which possess relatively simple structures yet share the common functional characteristics with the other members, using a green fluorescent protein-based screening methodology. The analysis revealed that all the tested fungal TRP channels were severely digested in their cytosolic regions during expression, implying the common flexibility of this region, as observed in the recent structural analyses of the fungal member, TRPGz. These characteristics are likely to be important for their diverse functions.

Evolutionarily conserved intracellular gate of voltage-dependent sodium channels

Members of the voltage-gated ion channel superfamily (VGIC) regulate ion flux and generate electrical signals in excitable cells by opening and closing pore gates. The location of the gate in voltage-gated sodium channels, a founding member of this superfamily, remains unresolved. Here we explore the chemical modification rates of introduced cysteines along the S6 helix of domain IV in an inactivation-removed background. We find that state-dependent accessibility is demarcated by an S6 hydrophobic residue; substituted cysteines above this site are not modified by charged thiol reagents when the channel is closed. These accessibilities are consistent with those inferred from open- and closed-state structures of prokaryotic sodium channels. Our findings suggest that an intracellular gate composed of a ring of hydrophobic residues is not only responsible for regulating access to the pore of sodium channels, but is also a conserved feature within canonical members of the VGIC superfamily.

Thermodynamic Analysis of Voltage-Sensing Mechanisms

Voltage-gated ion channels (VGIC) form a large superfamily of ion channels and the activation of these channels underlie electrical and chemical signaling in a variety of cell types. Structure-function studies are widely used to deduce the energetic effects of a mutation by measuring macroscopic currents and fitting their voltage-dependence to a Boltzmann function. However, in absence of detailed kinetic models, this approach can introduce serious errors in free-energy estimates because of the inherent assumption that the channel activation is a two-state process. We recently developed analytical tools that allows us to calculate the free energies required for activation of voltage-dependent processes without any prior knowledge of the underlying gating scheme. Our method involves measurement of conjugate displacement associated with the force that drives the activation of these channels. In the case of voltage-gated ion channels, gating charge movement is the conjugate displacement and the force is voltage across the membrane. We show that by measuring the median voltage of charge transfer, VM, and the total gating charge per channel, we can calculate the chemical free energy difference between the resting and activated state of the channels. These free-energy measurements can be extended to other members of the VGIC superfamily to obtain a measure of interaction energies between voltage- and ligand-dependent pathways. Development of well-defined model-independent metrics of interaction energies will be crucial for delineating molecular interaction pathways and understand the mechanisms of voltage-transduction.

Phylogeny Unites Animal Sodium Leak Channels with Fungal Calcium Channels in an Ancient, Voltage-Insensitive Clade

Proteins in the superfamily of voltage-gated ion channels mediate behavior across the tree of life. These proteins regulate the movement of ions across cell membranes by opening and closing a central pore that controls ion flow. The best-known members of this superfamily are the voltage-gated potassium, calcium (Ca(v)), and sodium (Na(v)) channels, which underlie impulse conduction in nerve and muscle. Not all members of this family are opened by changes in voltage, however. NALCN (NA(+) leak channel nonselective) channels, which encode a voltage-insensitive "sodium leak" channel, have garnered a growing interest. This study examines the phylogenetic relationship among Na(v)/Ca(v) voltage-gated and voltage-insensitive channels in the eukaryotic group Opisthokonta, which includes animals, fungi, and their unicellular relatives. We show that NALCN channels diverged from voltage-gated channels before the divergence of fungi and animals and that the closest relatives of NALCN channels are fungal calcium channels, which they functionally resemble.

Voltage-Gated Sodium Channel (NaV) Pore-Only Proteins

The general topology repeating in the Voltage-Gated Ion Channel (VGIC) superfamily members includes six-transmembrane α-helical segments in which the first four form a voltage-sensing domain and the last two form the pore domain (PD). Studies of potassium channels from the VGIC superfamily together with identification of voltage-sensor only proteins have suggested that the VSD and the PD can fold independently. Whether such transmembrane modularity is common to other VGIC superfamily members has remained untested. Here we show, using protein dissection, that the Silicibacter pomeroyi voltage-gated sodium channel (NaVSp1) PD forms a stand-alone, ion selective pore (NaVSp1p) that is tetrameric, α-helical, and forms functional, sodium-selective channels when reconstituted into lipid bilayers. Mutation of the NaVSp1p selectivity filter from LESWSM to LDDWSD, creates a calcium-selective pore-only channel, CaVSp1p. We further show that production of PDs can be generalized by making pore-only proteins from two other extremophile NaVs: one from the hydrocarbon degrader Alcanivorax borkumensis (NaVAb1p), and one from the arsenite oxidizer Alkalilimnicola ehrlichei (NaVAe1p). Together, our data establish a family of active pore-only ion channels that should be excellent model systems for study of the factors that govern both sodium and calcium selectivity and permeability. Further, our findings suggest that similar dissection approaches may be applicable to a wide range of VGICs and, thus, serve as a means to simplify and accelerate biophysical, structural, and drug development efforts.

Characterization of Cyclic Nucleotide Gated Channels using Atomic Force Microscope

Cyclic nucleotide-gated (CNG) channels have a key role in the conversion of sensory information, such as light and scent, into primary electrical signals. Structurally, these channels belong to the superfamily of voltage-gated ion channels and share a significant sequence identity with K+ channels suggesting a common ancestral three-dimensional structure. However, unlike K+ channels, to date no crystal structure of CNG channel has been solved. Therefore, we decided to analyze the structure of CNG channels with Force Spectroscopy using Atomic Force Microscopy (AFM), capable of giving structural information about proteins hard to crystallize in their native environment. We expressed the GFP-CNG channel in Xenopus laevis oocytes and isolated Xenopus plasma membrane containing channels' high density (∼500/μm2). Combining AFM with total internal reflection fluorescence (TIRF) microscopy, we were able to detect CNG single-molecule fluorescence attached to membrane patches. We have then proceeded to characterize CNG channels by measuring their mechanical properties by Force Spectroscopy. These measurements allow to extract a signature of their mechanical properties and to estimate their content of α-helices, β-sheet, hydrophobic and hydrophilic domains. These measurements do not require the purification or the crystallization of the CNG protein but provide important information on conformational changes during channel gating of CNG channels.

The Cardiac Sodium Channel Is Post-Translationally Modified by Arginine Methylation

The α subunit of the cardiac sodium channel (Na(v)1.5) is an essential protein in the initial depolarization phase of the cardiomyocyte action potential. Post-translational modifications such as phosphorylation are known to regulate Na(v)1.5 function. Here, we used a proteomic approach for the study of the post-translational modifications of Na(v)1.5 using tsA201 cells as a model system. We generated a stable cell line expressing Na(v)1.5, purified the sodium channel, and analyzed Na(v)1.5 by MALDI-TOF and LC-MS/MS. We report the identification of arginine methylation as a novel post-translational modification of Na(v)1.5. R513, R526, and R680, located in the linker between domains I and II in Na(v)1.5, were found in mono- or dimethylated states. The functional relevance of arginine methylation in Na(v)1.5 is underscored by the fact that R526H and R680H are known Na(v)1.5 mutations causing Brugada and long QT type 3 syndromes, respectively. Our work describes for the first time arginine methylation in the voltage-gated ion channel superfamily.