Voltage Sensor Movements during Hyperpolarization in the HCN Channel.

The hyperpolarization-activated cyclic nucleotide-gated (HCN) channel is a voltage-gated cation channel that mediates neuronal and cardiac pacemaker activity. The HCN channel exhibits reversed voltage dependence, meaning it closes with depolarization and opens with hyperpolarization. Different from Na+, Ca2+, and Kv1-Kv7 channels, the HCN channel does not have domain-swapped voltage sensors. We introduced a reversible, metal-mediated cross bridge into the voltage sensors to create the chemical equivalent of a hyperpolarized conformation and determined the structure using cryoelectron microscopy (cryo-EM). Unlike the depolarized HCN channel, the S4 helix is displaced toward the cytoplasm by two helical turns. Near the cytoplasm, the S4 helix breaks into two helices, one running parallel to the membrane surface, analogous to the S4-S5 linker of domain-swapped voltage-gated channels. These findings suggest a basis for allosteric communication between voltage sensors and the gate in this kind of channel. They also imply that voltage sensor movements are not the same in all voltage-gated channels.

Cholesterol inhibits human voltage-gated proton channel hHv1.

Although human sperm is morphologically mature in the epididymis, it cannot fertilize eggs before capacitation. Cholesterol efflux from the sperm plasma membrane is a key molecular event essential for cytoplasmic alkalinization and hyperactivation, but the underlying mechanism remains unclear. The human voltage-gated proton (hHv1) channel functions as an acid extruder to regulate intracellular pHs of many cell types, including sperm. Aside from voltage and pH, Hv channels are also regulated by distinct ligands, such as Zn2+ and albumin. In the present work, we identified cholesterol as an inhibitory ligand of the hHv1 channel and further investigated the underlying mechanism using the single-molecule fluorescence resonance energy transfer (smFRET) approach. Our results indicated that cholesterol inhibits the hHv1 channel by stabilizing the voltage-sensing S4 segment at resting conformations, a similar mechanism also utilized by Zn2+. Our results suggested that the S4 segment is the central gating machinery in the hHv1 channel, on which voltage and distinct ligands are converged to regulate channel function. Identification of membrane cholesterol as an inhibitory ligand provides a mechanism by which the hHv1 channel regulates fertilization by linking the cholesterol efflux with cytoplasmic alkalinization, a change that triggers calcium influx through the CatSper channel. These events finally lead to hyperactivation, a remarkable change in the mobility pattern indicating fertilization competence of human sperm.

The complex regulation of Slo1 potassium channels from a structural perspective.

Fast and regulated potassium efflux by Slo1 channels is crucial in many tissues in animals including neurons, the kidney and smooth muscle. During the last decade, structures have revealed many details about the gating mechanism and regulation of these large and complex molecular machines. This review summarizes these findings and the current knowledge about the intricate regulation of these important channels. Slo1 integrates sensing of the membrane potential via a voltage-sensor domain that undergoes subtle but significant structural rearrangements with a calcium-induced expansion of parts of the intracellular gating ring. Together, these two signals synergistically lead to changes in the conformation and chemical nature of the pore domain, allowing potassium ions to be translocated. In many native tissues, Slo1 channels are assembled with at least three classes of auxiliary subunits that change the gating kinetics or allow the channel to open also in absence of one of the two signals. Finally, Slo1 is inhibited, activated or deregulated by natural toxins and synthetic compounds, underlining the importance of the channel for the organism and as a potential target for drugs and other molecules.

Voltage-gating and cytosolic Ca2+ activation mechanisms of Arabidopsis two-pore channel AtTPC1.

Arabidopsis thaliana two-pore channel AtTPC1 is a voltage-gated, Ca2+-modulated, nonselective cation channel that is localized in the vacuolar membrane and responsible for generating slow vacuolar (SV) current. Under depolarizing membrane potential, cytosolic Ca2+ activates AtTPC1 by binding at the EF-hand domain, whereas luminal Ca2+ inhibits the channel by stabilizing the voltage-sensing domain II (VSDII) in the resting state. Here, we present 2.8 to 3.3 Å cryoelectron microscopy (cryo-EM) structures of AtTPC1 in two conformations, one in closed conformation with unbound EF-hand domain and resting VSDII and the other in a partially open conformation with Ca2+-bound EF-hand domain and activated VSDII. Structural comparison between the two different conformations allows us to elucidate the structural mechanisms of voltage gating, cytosolic Ca2+ activation, and their coupling in AtTPC1. This study also provides structural insight into the general voltage-gating mechanism among voltage-gated ion channels.

Loose Coupling between the Voltage Sensor and the Activation Gate in Mammalian HCN Channels Suggests a Gating Mechanism.

Voltage-gated potassium (Kv) channels and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels share similar structures but have opposite gating polarity. Kv channels have a strong coupling (>109) between the voltage sensor (S4) and the activation gate: when S4s are activated, the gate is open to >80% but, when S4s are deactivated, the gate is open <10-9 of the time. Using noise analysis, we show that the coupling between S4 and the gate is <200 in HCN channels. In addition, using voltage clamp fluorometry, locking the gate open in a Kv channel drastically altered the energetics of S4 movement. In contrast, locking the gate open or decreasing the coupling between S4 and the gate in HCN channels had only minor effects on the energetics of S4 movement, consistent with a weak coupling between S4 and the gate. We propose that this loose coupling is a prerequisite for the reversed voltage gating in HCN channels.

Gap Junction Channel Regulation: A Tale of Two Gates-Voltage Sensitivity of the Chemical Gate and Chemical Sensitivity of the Fast Voltage Gate.

Gap junction channels are regulated by gates sensitive to cytosolic acidification and trans-junctional voltage (Vj). We propose that the chemical gate is a calmodulin (CaM) lobe. The fast-Vj gate is made primarily by the connexin's NH2-terminus domain (NT). The chemical gate closes the channel slowly and completely, while the fast-Vj gate closes the channel rapidly but incompletely. The chemical gate closes with increased cytosolic calcium concentration [Ca2+]i and with Vj gradients at Vj's negative side. In contrast, the fast-Vj gate closes at the positive or negative side of Vj depending on the connexin (Cx) type. Cxs with positively charged NT close at Vj's negative side, while those with negatively charged NT close at Vj's positive side. Cytosolic acidification alters in opposite ways the sensitivity of the fast-Vj gate: it increases the Vj sensitivity of negative gaters and decreases that of positive gaters. While the fast-Vj gate closes and opens instantaneously, the chemical gate often shows fluctuations, likely to reflect the shifting of the gate (CaM's N-lobe) in and out of the channel's pore.

Voltage-gated potassium channels KCNQs: Structures, mechanisms, and modulations.

KCNQ (Kv7) channels are voltage-gated, phosphatidylinositol 4,5-bisphosphate- (PIP2-) modulated potassium channels that play essential roles in regulating the activity of neurons and cardiac myocytes. Hundreds of mutations in KCNQ channels are closely related to various cardiac and neurological disorders, such as long QT syndrome, epilepsy, and deafness, which makes KCNQ channels important drug targets. During the past several years, the application of single-particle cryo-electron microscopy (cryo-EM) technique in the structure determination of KCNQ channels has greatly advanced our understanding of their molecular mechanisms. In this review, we summarize the currently available structures of KCNQ channels, analyze their special voltage gating mechanism, and discuss their activation mechanisms by both the endogenous membrane lipid and the exogenous synthetic ligands. These structural studies of KCNQ channels will guide the development of drugs targeting KCNQ channels.

Modulation of Voltage-Gating and Hysteresis of Lysenin Channels by Cu2+ Ions.

The intricate voltage regulation presented by lysenin channels reconstituted in artificial lipid membranes leads to a strong hysteresis in conductance, bistability, and memory. Prior investigations on lysenin channels indicate that the hysteresis is modulated by multivalent cations which are also capable of eliciting single-step conformational changes and transitions to stable closed or sub-conducting states. However, the influence on voltage regulation of Cu2+ ions, capable of completely closing the lysenin channels in a two-step process, was not sufficiently addressed. In this respect, we employed electrophysiology approaches to investigate the response of lysenin channels to variable voltage stimuli in the presence of small concentrations of Cu2+ ions. Our experimental results showed that the hysteretic behavior, recorded in response to variable voltage ramps, is accentuated in the presence of Cu2+ ions. Using simultaneous AC/DC stimulation, we were able to determine that Cu2+ prevents the reopening of channels previously closed by depolarizing potentials and the channels remain in the closed state even in the absence of a transmembrane voltage. In addition, we showed that Cu2+ addition reinstates the voltage gating and hysteretic behavior of lysenin channels reconstituted in neutral lipid membranes in which lysenin channels lose their voltage-regulating properties. In the presence of Cu2+ ions, lysenin not only regained the voltage gating but also behaved like a long-term molecular memory controlled by electrical potentials.

Hydrophobic gasket mutation produces gating pore currents in closed human voltage-gated proton channels.

The hydrophobic gasket (HG), a ring of hydrophobic amino acids in the voltage-sensing domain of most voltage-gated ion channels, forms a constriction between internal and external aqueous vestibules. Cationic Arg or Lys side chains lining the S4 helix move through this "gating pore" when the channel opens. S4 movement may occur during gating of the human voltage-gated proton channel, hHV1, but proton current flows through the same pore in open channels. Here, we replaced putative HG residues with less hydrophobic residues or acidic Asp. Substitution of individuals, pairs, or all 3 HG positions did not impair proton selectivity. Evidently, the HG does not act as a secondary selectivity filter. However, 2 unexpected functions of the HG in HV1 were discovered. Mutating HG residues independently accelerated channel opening and compromised the closed state. Mutants exhibited open-closed gating, but strikingly, at negative voltages where "normal" gating produces a nonconducting closed state, the channel leaked protons. Closed-channel proton current was smaller than open-channel current and was inhibited by 10 µM Zn2+ Extreme hyperpolarization produced a deeper closed state through a weakly voltage-dependent transition. We functionally identify the HG as Val109, Phe150, Val177, and Val178, which play a critical and exclusive role in preventing H+ influx through closed channels. Molecular dynamics simulations revealed enhanced mobility of Arg208 in mutants exhibiting H+ leak. Mutation of HG residues produces gating pore currents reminiscent of several channelopathies.

Evolutionary selection of a 19-stranded mitochondrial ß-barrel scaffold bears structural and functional significance.

Transmembrane ß-barrels of eukaryotic outer mitochondrial membranes (OMMs) are major channels of communication between the cytosol and mitochondria and are indispensable for cellular homeostasis. A structurally intriguing exception to all known transmembrane ß-barrels is the unique odd-stranded, i.e. 19-stranded, structures found solely in the OMM. The molecular origins of this 19-stranded structure and its associated functional significance are unclear. In humans, the most abundant OMM transporter is the voltage-dependent anion channel. Here, using the human voltage-dependent anion channel as our template scaffold, we designed and engineered odd- and even-stranded structures of smaller (V216, V217, V218) and larger (V220, V221) barrel diameters. Determination of the structure, dynamics, and energetics of these engineered structures in bilayer membranes reveals that the 19-stranded barrel surprisingly holds modest to low stability in a lipid-dependent manner. However, we demonstrate that this structurally metastable protein possesses superior voltage-gated channel regulation, efficient mitochondrial targeting, and in vivo cell survival, with lipid-modulated stability, all of which supersede the occurrence of a metastable 19-stranded scaffold. We propose that the unique structural adaptation of these transmembrane transporters exclusively in mitochondria bears strong evolutionary basis and is functionally significant for homeostasis.

Molecular mechanism of olesoxime-mediated neuroprotection through targeting α-synuclein interaction with mitochondrial VDAC.

An intrinsically disordered neuronal protein α-synuclein (αSyn) is known to cause mitochondrial dysfunction, contributing to loss of dopaminergic neurons in Parkinson's disease. Through yet poorly defined mechanisms, αSyn crosses mitochondrial outer membrane and targets respiratory complexes leading to bioenergetics defects. Here, using neuronally differentiated human cells overexpressing wild-type αSyn, we show that the major metabolite channel of the outer membrane, the voltage-dependent anion channel (VDAC), is a pathway for αSyn translocation into the mitochondria. Importantly, the neuroprotective cholesterol-like synthetic compound olesoxime inhibits this translocation. By applying complementary electrophysiological and biophysical approaches, we provide mechanistic insights into the interplay between αSyn, VDAC, and olesoxime. Our data suggest that olesoxime interacts with VDAC ß-barrel at the lipid-protein interface thus hindering αSyn translocation through the VDAC pore and affecting VDAC voltage gating. We propose that targeting αSyn translocation through VDAC could represent a key mechanism for the development of new neuroprotective strategies.

Fenestrations control resting-state block of a voltage-gated sodium channel.

Potency of drug action is usually determined by binding to a specific receptor site on target proteins. In contrast to this conventional paradigm, we show here that potency of local anesthetics (LAs) and antiarrhythmic drugs (AADs) that block sodium channels is controlled by fenestrations that allow drug access to the receptor site directly from the membrane phase. Voltage-gated sodium channels initiate action potentials in nerve and cardiac muscle, where their hyperactivity causes pain and cardiac arrhythmia, respectively. LAs and AADs selectively block sodium channels in rapidly firing nerve and muscle cells to relieve these conditions. The structure of the ancestral bacterial sodium channel NaVAb, which is also blocked by LAs and AADs, revealed fenestrations connecting the lipid phase of the membrane to the central cavity of the pore. We cocrystallized lidocaine and flecainide with NavAb, which revealed strong drug-dependent electron density in the central cavity of the pore. Mutation of the contact residue T206 greatly reduced drug potency, confirming this site as the receptor for LAs and AADs. Strikingly, mutations of the fenestration cap residue F203 changed fenestration size and had graded effects on resting-state block by flecainide, lidocaine, and benzocaine, the potencies of which were altered from 51- to 2.6-fold in order of their molecular size. These results show that conserved fenestrations in the pores of sodium channels are crucial pharmacologically and determine the level of resting-state block by widely used drugs. Fine-tuning drug access through fenestrations provides an unexpected avenue for structure-based design of ion-channel-blocking drugs.

Regulation of Mitochondrial Respiration by VDAC Is Enhanced by Membrane-Bound Inhibitors with Disordered Polyanionic C-Terminal Domains.

The voltage-dependent anion channel (VDAC) is the primary regulating pathway of water-soluble metabolites and ions across the mitochondrial outer membrane. When reconstituted into lipid membranes, VDAC responds to sufficiently large transmembrane potentials by transitioning to gated states in which ATP/ADP flux is reduced and calcium flux is increased. Two otherwise unrelated cytosolic proteins, tubulin, and α-synuclein (αSyn), dock with VDAC by a novel mechanism in which the transmembrane potential draws their disordered, polyanionic C-terminal domains into and through the VDAC channel, thus physically blocking the pore. For both tubulin and αSyn, the blocked state is observed at much lower transmembrane potentials than VDAC gated states, such that in the presence of these cytosolic docking proteins, VDAC's sensitivity to transmembrane potential is dramatically increased. Remarkably, the features of the VDAC gated states relevant for bioenergetics-reduced metabolite flux and increased calcium flux-are preserved in the blocked state induced by either docking protein. The ability of tubulin and αSyn to modulate mitochondrial potential and ATP production in vivo is now supported by many studies. The common physical origin of the interactions of both tubulin and αSyn with VDAC leads to a general model of a VDAC inhibitor, facilitates predictions of the effect of post-translational modifications of known inhibitors, and points the way toward the development of novel therapeutics targeting VDAC.

Mapping of voltage sensor positions in resting and inactivated mammalian sodium channels by LRET.

Voltage-gated sodium channels (Navs) play crucial roles in excitable cells. Although vertebrate Nav function has been extensively studied, the detailed structural basis for voltage-dependent gating mechanisms remain obscure. We have assessed the structural changes of the Nav voltage sensor domain using lanthanide-based resonance energy transfer (LRET) between the rat skeletal muscle voltage-gated sodium channel (Nav1.4) and fluorescently labeled Nav1.4-targeting toxins. We generated donor constructs with genetically encoded lanthanide-binding tags (LBTs) inserted at the extracellular end of the S4 segment of each domain (with a single LBT per construct). Three different Bodipy-labeled, Nav1.4-targeting toxins were synthesized as acceptors: ß-scorpion toxin (Ts1)-Bodipy, KIIIA-Bodipy, and GIIIA-Bodipy analogs. Functional Nav-LBT channels expressed in Xenopus oocytes were voltage-clamped, and distinct LRET signals were obtained in the resting and slow inactivated states. Intramolecular distances computed from the LRET signals define a geometrical map of Nav1.4 with the bound toxins, and reveal voltage-dependent structural changes related to channel gating.

Calmodulin-Mediated Regulation of Gap Junction Channels.

Evidence that neighboring cells uncouple from each other as one dies surfaced in the late 19th century, but it took almost a century for scientists to start understanding the uncoupling mechanism (chemical gating). The role of cytosolic free calcium (Ca2+i) in cell-cell channel gating was first reported in the mid-sixties. In these studies, only micromolar [Ca2+]i were believed to affect gating-concentrations reachable only in cell death, which would discard Ca2+i as a fine modulator of cell coupling. More recently, however, numerous researchers, including us, have reported the effectiveness of nanomolar [Ca2+]i. Since connexins do not have high-affinity calcium sites, the effectiveness of nanomolar [Ca2+]i suggests the role of Ca-modulated proteins, with calmodulin (CaM) being most obvious. Indeed, in 1981 we first reported that a CaM-inhibitor prevents chemical gating. Since then, the CaM role in gating has been confirmed by studies that tested it with a variety of approaches such as treatments with CaM-inhibitors, inhibition of CaM expression, expression of CaM mutants, immunofluorescent co-localization of CaM and gap junctions, and binding of CaM to peptides mimicking connexin domains identified as CaM targets. Our gating model envisions Ca2+-CaM to directly gate the channels by acting as a plug ("Cork" gating model), and probably also by affecting connexin conformation.

Cooperativity and Steep Voltage Dependence in a Bacterial Channel.

This paper reports on the discovery of a novel three-membrane channel unit exhibiting very steep voltage dependence and strong cooperative behavior. It was reconstituted into planar phospholipid membranes formed by the monolayer method and studied under voltage-clamp conditions. The behavior of the novel channel-former, isolated from Escherichia coli, is consistent with a linearly organized three-channel unit displaying steep voltage-gating (a minimum of 14 charges in the voltage sensor) that rivals that of channels in mammalian excitable membranes. The channels also display strong cooperativity in that closure of the first channel permits the second to close and closure of the second channel permits closure of the third. All three have virtually the same conductance and selectivity, and yet the first and third close at positive potentials whereas the second closes at negative potentials. Thus, is it likely that the second channel-former is oriented in the membrane in a direction opposite to that of the other two. This novel structure is named "triplin." The extraordinary behavior of triplin indicates that it must have important and as yet undefined physiological roles.

Lipid Headgroup Charge and Acyl Chain Composition Modulate Closure of Bacterial ß-Barrel Channels.

The outer membrane of Gram-negative bacteria contains ß-barrel proteins that form high-conducting ion channels providing a path for hydrophilic molecules, including antibiotics. Traditionally, these proteins have been considered to exist only in an open state so that regulation of outer membrane permeability was accomplished via protein expression. However, electrophysiological recordings show that ß-barrel channels respond to transmembrane voltages by characteristically switching from a high-conducting, open state, to a so-called 'closed' state, with reduced permeability and possibly exclusion of large metabolites. Here, we use the bacterial porin OmpF from E. coli as a model system to gain insight on the control of outer membrane permeability by bacterial porins through the modulation of their open state. Using planar bilayer electrophysiology, we perform an extensive study of the role of membrane lipids in the OmpF channel closure by voltage. We pay attention not only to the effects of charges in the hydrophilic lipid heads but also to the contribution of the hydrophobic tails in the lipid-protein interactions. Our results show that gating kinetics is governed by lipid characteristics so that each stage of a sequential closure is different from the previous one, probably because of intra- or intermonomeric rearrangements.

Sinusoidal voltage protocols for rapid characterisation of ion channel kinetics.

KEY POINTS: Ion current kinetics are commonly represented by current-voltage relationships, time constant-voltage relationships and subsequently mathematical models fitted to these. These experiments take substantial time, which means they are rarely performed in the same cell. Rather than traditional square-wave voltage clamps, we fitted a model to the current evoked by a novel sum-of-sinusoids voltage clamp that was only 8 s long. Short protocols that can be performed multiple times within a single cell will offer many new opportunities to measure how ion current kinetics are affected by changing conditions. The new model predicts the current under traditional square-wave protocols well, with better predictions of underlying currents than literature models. The current under a novel physiologically relevant series of action potential clamps is predicted extremely well. The short sinusoidal protocols allow a model to be fully fitted to individual cells, allowing us to examine cell-cell variability in current kinetics for the first time. ABSTRACT: Understanding the roles of ion currents is crucial to predict the action of pharmaceuticals and mutations in different scenarios, and thereby to guide clinical interventions in the heart, brain and other electrophysiological systems. Our ability to predict how ion currents contribute to cellular electrophysiology is in turn critically dependent on our characterisation of ion channel kinetics - the voltage-dependent rates of transition between open, closed and inactivated channel states. We present a new method for rapidly exploring and characterising ion channel kinetics, applying it to the hERG potassium channel as an example, with the aim of generating a quantitatively predictive representation of the ion current. We fitted a mathematical model to currents evoked by a novel 8 second sinusoidal voltage clamp in CHO cells overexpressing hERG1a. The model was then used to predict over 5 minutes of recordings in the same cell in response to further protocols: a series of traditional square step voltage clamps, and also a novel voltage clamp comprising a collection of physiologically relevant action potentials. We demonstrate that we can make predictive cell-specific models that outperform the use of averaged data from a number of different cells, and thereby examine which changes in gating are responsible for cell-cell variability in current kinetics. Our technique allows rapid collection of consistent and high quality data, from single cells, and produces more predictive mathematical ion channel models than traditional approaches.

Gating of Connexin Channels by transjunctional-voltage: Conformations and models of open and closed states.

Voltage is an important physiologic regulator of channels formed by the connexin gene family. Connexins are unique among ion channels in that both plasma membrane inserted hemichannels (undocked hemichannels) and intercellular channels (aggregates of which form gap junctions) have important physiological roles. The hemichannel is the fundamental unit of gap junction voltage-gating. Each hemichannel displays two distinct voltage-gating mechanisms that are primarily sensitive to a voltage gradient formed along the length of the channel pore (the transjunctional voltage) rather than sensitivity to the absolute membrane potential (Vm or Vi-o). These transjunctional voltage dependent processes have been termed Vj- or fast-gating and loop- or slow-gating. Understanding the mechanism of voltage-gating, defined as the sequence of voltage-driven transitions that connect open and closed states, first and foremost requires atomic resolution models of the end states. Although ion channels formed by connexins were among the first to be characterized structurally by electron microscopy and x-ray diffraction in the early 1980's, subsequent progress has been slow. Much of the current understanding of the structure-function relations of connexin channels is based on two crystal structures of Cx26 gap junction channels. Refinement of crystal structure by all-atom molecular dynamics and incorporation of charge changing protein modifications has resulted in an atomic model of the open state that arguably corresponds to the physiologic open state. Obtaining validated atomic models of voltage-dependent closed states is more challenging, as there are currently no methods to solve protein structure while a stable voltage gradient is applied across the length of an oriented channel. It is widely believed that the best approach to solve the atomic structure of a voltage-gated closed ion channel is to apply different but complementary experimental and computational methods and to use the resulting information to derive a consensus atomic structure that is then subjected to rigorous validation. In this paper, we summarize our efforts to obtain and validate atomic models of the open and voltage-driven closed states of undocked connexin hemichannels. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.

The N-terminus of VDAC: Structure, mutational analysis, and a potential role in regulating barrel shape.

A novel feature of the voltage-dependent anion channel (VDAC, mitochondrial porin), is the barrel, comprising an odd number of ß-strands and closed by parallel strands. Recent research has focused on the N-terminal segment, which in the available structures, resides in the lumen and is not part of the barrel. In this review, the structural data obtained from vertebrate VDAC are integrated with those from VDAC in artificial bilayers, emphasizing the array of native and tagged versions of VDAC used. The data are discussed with respect to a recent gating model (Zachariae et al. (2012) Structure 20:1-10), in which the N-terminus acts not as a gate on a stable barrel, but rather stabilizes the barrel, preventing its shift into a partially collapsed, low-conductance, closed state. Additionally, the role of the N-terminus in VDAC oligomerization, apoptosis through interactions with hexokinase and its interaction with ATP are discussed briefly.