Cryo-EM structures of prokaryotic ligand-gated ion channel GLIC provide insights into gating in a lipid environment.

GLIC, a proton-activated prokaryotic ligand-gated ion channel, served as a model system for understanding the eukaryotic counterparts due to their structural and functional similarities. Despite extensive studies conducted on GLIC, the molecular mechanism of channel gating in the lipid environment requires further investigation. Here, we present the cryo-EM structures of nanodisc-reconstituted GLIC at neutral and acidic pH in the resolution range of 2.6 - 3.4 Å. In our apo state at pH 7.5, the extracellular domain (ECD) displays conformational variations compared to the existing apo structures. At pH 4.0, three distinct conformational states (C1, C2 and O states) are identified. The protonated structures exhibit a compacted and counter-clockwise rotated ECD compared with our apo state. A gradual widening of the pore in the TMD is observed upon reducing the pH, with the widest pore in O state, accompanied by several layers of water pentagons. The pore radius and molecular dynamics (MD) simulations suggest that the O state represents an open conductive state. We also observe state-dependent interactions between several lipids and proteins that may be involved in the regulation of channel gating. Our results provide comprehensive insights into the importance of lipids impact on gating.

Mechanosensitive channel MscL gating transitions coupling with constriction point shift.

The mechanosensitive channel of large conductance (MscL) acts as an "emergency release valve" that protects bacterial cells from acute hypoosmotic stress, and it serves as a paradigm for studying the mechanism underlying the transduction of mechanical forces. MscL gating is proposed to initiate with an expansion without opening, followed by subsequent pore opening via a number of intermediate substates, and ends in a full opening. However, the details of gating process are still largely unknown. Using in vivo viability assay, single channel patch clamp recording, cysteine cross-linking, and tryptophan fluorescence quenching approach, we identified and characterized MscL mutants with different occupancies of constriction region in the pore domain. The results demonstrated the shifts of constriction point along the gating pathway towards cytoplasic side from residue G26, though G22, to L19 upon gating, indicating the closed-expanded transitions coupling of the expansion of tightly packed hydrophobic constriction region to conduct the initial ion permeation in response to the membrane tension. Furthermore, these transitions were regulated by the hydrophobic and lipidic interaction with the constricting "hot spots". Our data reveal a new resolution of the transitions from the closed to the opening substate of MscL, providing insights into the gating mechanisms of MscL.

A binding site for phosphoinositides described by multiscale simulations explains their modulation of voltage-gated sodium channels.

Voltage-gated sodium channels (Naáµ¥) are membrane proteins which open to facilitate the inward flux of sodium ions into excitable cells. In response to stimuli, Naáµ¥ channels transition from the resting, closed state to an open, conductive state, before rapidly inactivating. Dysregulation of this functional cycle due to mutations causes diseases including epilepsy, pain conditions, and cardiac disorders, making Naáµ¥ channels a significant pharmacological target. Phosphoinositides are important lipid cofactors for ion channel function. The phosphoinositide PI(4,5)P2 decreases Naáµ¥1.4 activity by increasing the difficulty of channel opening, accelerating fast inactivation and slowing recovery from fast inactivation. Using multiscale molecular dynamics simulations, we show that PI(4,5)P2 binds stably to inactivated Naáµ¥ at a conserved site within the DIV S4-S5 linker, which couples the voltage-sensing domain (VSD) to the pore. As the Naáµ¥ C-terminal domain is proposed to also bind here during recovery from inactivation, we hypothesize that PI(4,5)P2 prolongs inactivation by competitively binding to this site. In atomistic simulations, PI(4,5)P2 reduces the mobility of both the DIV S4-S5 linker and the DIII-IV linker, responsible for fast inactivation, slowing the conformational changes required for the channel to recover to the resting state. We further show that in a resting state Naáµ¥ model, phosphoinositides bind to VSD gating charges, which may anchor them and impede VSD activation. Our results provide a mechanism by which phosphoinositides alter the voltage dependence of activation and the rate of recovery from inactivation, an important step for the development of novel therapies to treat Naáµ¥-related diseases.

Structural basis for excitatory neuropeptide signaling.

Rapid signaling between neurons is mediated by ligand-gated ion channels, cell-surface proteins with an extracellular ligand-binding domain and a membrane-spanning ion channel domain. The degenerin/epithelial sodium channel (DEG/ENaC) superfamily is diverse in terms of its gating stimuli, with some DEG/ENaCs gated by neuropeptides, and others gated by pH, mechanical force or enzymatic activity. The mechanism by which ligands bind to and activate DEG/ENaCs is poorly understood. Here we dissected the structural basis for neuropeptide-gated activity of a neuropeptide-gated DEG/ENaC, FMRFamide-gated sodium channel 1 (FaNaC1) from the annelid worm Malacoceros fuliginosus, using cryo-electron microscopy. Structures of FaNaC1 in the ligand-free resting state and in several ligand-bound states reveal the ligand-binding site and capture the ligand-induced conformational changes of channel gating, which we verified with complementary mutagenesis experiments. Our results illuminate channel gating in DEG/ENaCs and offer a structural template for experimental dissection of channel pharmacology and ion conduction.

cAMP binding to closed pacemaker ion channels is cooperative.

The cooperative action of the subunits in oligomeric receptors enables fine-tuning of receptor activation, as demonstrated for the regulation of voltage-activated HCN pacemaker ion channels by relating cAMP binding to channel activation in ensemble signals. HCN channels generate electric rhythmicity in specialized brain neurons and cardiomyocytes. There is conflicting evidence on whether binding cooperativity does exist independent of channel activation or not, as recently reported for detergent-solubilized receptors positioned in zero-mode waveguides. Here, we show positive cooperativity in ligand binding to closed HCN2 channels in native cell membranes by following the binding of individual fluorescence-labeled cAMP molecules. Kinetic modeling reveals that the affinity of the still empty binding sites rises with increased degree of occupation and that the transition of the channel to a flip state is promoted accordingly. We conclude that ligand binding to the subunits in closed HCN2 channels not pre-activated by voltage is already cooperative. Hence, cooperativity is not causally linked to channel activation by voltage. Our analysis also shows that single-molecule binding measurements at equilibrium can quantify cooperativity in ligand binding to receptors in native membranes.

A release of local subunit conformational heterogeneity underlies gating in a muscle nicotinic acetylcholine receptor.

Synaptic receptors respond to neurotransmitters by opening an ion channel across the post-synaptic membrane to elicit a cellular response. Here we use recent Torpedo acetylcholine receptor structures and functional measurements to delineate a key feature underlying allosteric communication between the agonist-binding extracellular and channel-gating transmembrane domains. Extensive mutagenesis at this inter-domain interface re-affirms a critical energetically coupled role for the principal α subunit ß1-ß2 and M2-M3 loops, with agonist binding re-positioning a key ß1-ß2 glutamate/valine to facilitate the outward motions of a conserved M2-M3 proline to open the channel gate. Notably, the analogous structures in non-α subunits adopt a locally active-like conformation in the apo state even though each L9' hydrophobic gate residue in each pore-lining M2 α-helix is closed. Agonist binding releases local conformational heterogeneity transitioning all five subunits into a conformationally symmetric open state. A release of conformational heterogeneity provides a framework for understanding allosteric communication in pentameric ligand-gated ion channels.

Trp207 regulation of voltage-dependent activation of human Hv1 proton channel.

In voltage-gated Na+ and K+ channels, the hydrophobicity of noncharged residues in the S4 helix has been shown to regulate the S4 movement underlying the process of voltage-sensing domain (VSD) activation. In voltage-gated proton channel Hv1, there is a bulky noncharged tryptophan residue located at the S4 transmembrane segment. This tryptophan remains entirely conserved across all Hv1 members but is not seen in other voltage-gated ion channels, indicating that the tryptophan contributes different roles in VSD activation. The conserved tryptophan of human voltage-gated proton channel Hv1 is Trp207 (W207). Here, we showed that W207 modifies human Hv1 voltage-dependent activation, and small residues replacement at position 207 strongly perturbs Hv1 channel opening and closing, and the size of the side chain instead of the hydrophobic group of W207 regulates the transition between closed and open states of the channel. We conclude that the large side chain of tryptophan controls the energy barrier during the Hv1 VSD transition.

A high affinity switch for cAMP in the HCN pacemaker channels.

Binding of cAMP to Hyperpolarization activated cyclic nucleotide gated (HCN) channels facilitates pore opening. It is unclear why the isolated cyclic nucleotide binding domain (CNBD) displays in vitro lower affinity for cAMP than the full-length channel in patch experiments. Here we show that HCN are endowed with an affinity switch for cAMP. Alpha helices D and E, downstream of the cyclic nucleotide binding domain (CNBD), bind to and stabilize the holo CNBD in a high affinity state. These helices increase by 30-fold cAMP efficacy and affinity measured in patch clamp and ITC, respectively. We further show that helices D and E regulate affinity by interacting with helix C of the CNBD, similarly to the regulatory protein TRIP8b. Our results uncover an intramolecular mechanism whereby changes in binding affinity, rather than changes in cAMP concentration, can modulate HCN channels, adding another layer to the complex regulation of their activity.

Transcendent Aspects of Proton Channels.

A handful of biological proton-selective ion channels exist. Some open at positive or negative membrane potentials, others open at low or high pH, and some are light activated. This review focuses on common features that result from the unique properties of protons. Proton conduction through water or proteins differs qualitatively from that of all other ions. Extraordinary proton selectivity is needed to ensure that protons permeate and other ions do not. Proton selectivity arises from a proton pathway comprising a hydrogen-bonded chain that typically includes at least one titratable amino acid side chain. The enormously diverse functions of proton channels in disparate regions of the phylogenetic tree can be summarized by considering the chemical and electrical consequences of proton flux across membranes. This review discusses examples of cells in which proton efflux serves to increase pHi, decrease pHo, control the membrane potential, generate action potentials, or compensate transmembrane movement of electrical charge.

Exploration of the Parameter Space of an Ion Channel Kinetic Model by a Markov-Chain-Based Methodology.

In this work, we propose a methodology based on Monte Carlo Markov chains to explore the parameter space of kinetic models for ion channels. The methodology allows the detection of potential parameter sets of a model that are compatible with experimentally obtained whole-cell currents, which could remain hidden when methods focus on obtaining the parameters that provide the best fit. To show its implementation and utility, we considered a four-state kinetic model proposed in the literature to describe the activation of the voltage-gated proton channel (Hv1), Biophysical Journal, 2014, 107, 1564. In that work, a set of values for the rate transitions that describe the channel kinetics at different intracellular H+ concentration (pHi) were obtained by the Simplex method. With our approach, we find that, in fact, there is more than one parameter set for each pHi, which renders the same open probability temporal course within the experimental error margin for all of the considered voltages. The large differences that we obtained for the values of some rate constants among the different solutions show that there is more than one possible interpretation of this channel behavior as a function of pHi. We also simulated a proposed new experimental condition where it is possible to observe that different sets of parameters yield different results. Our study highlights the importance of a comprehensive analysis of parameter space in kinetic models and the utility of the proposed methodology for finding potential solutions.

Eukaryotic Kv channel Shaker inactivates through selectivity filter dilation rather than collapse.

Eukaryotic voltage-gated K+ channels have been extensively studied, but the structural bases for some of their most salient functional features remain to be established. C-type inactivation, for example, is an auto-inhibitory mechanism that confers temporal resolution to their signal-firing activity. In a recent breakthrough, studies of a mutant of Shaker that is prone to inactivate indicated that this process entails a dilation of the selectivity filter, the narrowest part of the ion conduction pathway. Here, we report an atomic-resolution cryo-electron microscopy structure that demonstrates that the wild-type channel can also adopt this dilated state. All-atom simulations corroborate this conformation is congruent with the electrophysiological characteristics of the C-type inactivated state, namely, residual K+ conductance and altered ion specificity, and help rationalize why inactivation is accelerated or impeded by certain mutations. In summary, this study establishes the molecular basis for an important self-regulatory mechanism in eukaryotic K+ channels, laying a solid foundation for further studies.

Beta-Barrel Channel Response to High Electric Fields: Functional Gating or Reversible Denaturation?

Ion channels exhibit gating behavior, fluctuating between open and closed states, with the transmembrane voltage serving as one of the essential regulators of this process. Voltage gating is a fundamental functional aspect underlying the regulation of ion-selective, mostly α-helical, channels primarily found in excitable cell membranes. In contrast, there exists another group of larger, and less selective, ß-barrel channels of a different origin, which are not directly associated with cell excitability. Remarkably, these channels can also undergo closing, or "gating", induced by sufficiently strong electric fields. Once the field is removed, the channels reopen, preserving a memory of the gating process. In this study, we explored the hypothesis that the voltage-induced closure of the ß-barrel channels can be seen as a form of reversible protein denaturation by the high electric fields applied in model membranes experiments-typically exceeding twenty million volts per meter-rather than a manifestation of functional gating. Here, we focused on the bacterial outer membrane channel OmpF reconstituted into planar lipid bilayers and analyzed various characteristics of the closing-opening process that support this idea. Specifically, we considered the nearly symmetric response to voltages of both polarities, the presence of multiple closed states, the stabilization of the open conformation in channel clusters, the long-term gating memory, and the Hofmeister effects in closing kinetics. Furthermore, we contemplate the evolutionary aspect of the phenomenon, proposing that the field-induced denaturation of membrane proteins might have served as a starting point for their development into amazing molecular machines such as voltage-gated channels of nerve and muscle cells.

Exploring Flexibility and Folding Patterns Throughout Time in Voltage Sensors.

The voltage-sensing domain (VSD) is a module capable of responding to changes in the membrane potential through conformational changes and facilitating electromechanical coupling to open a pore gate, activate proton permeation pathways, or promote enzymatic activity in some membrane-anchored phosphatases. To carry out these functions, this module acts cooperatively through conformational changes. The VSD is formed by four transmembrane segments (S1-S4) but the S4 segment is critical since it carries positively charged residues, mainly Arg or Lys, which require an aqueous environment for its proper function. The discovery of this module in voltage-gated ion channels (VGICs), proton channels (Hv1), and voltage sensor-containing phosphatases (VSPs) has expanded our understanding of the principle of modularity in the voltage-sensing mechanism of these proteins. Here, by sequence comparison and the evaluation of the relationship between sequence composition, intrinsic flexibility, and structural analysis in 14 selected representatives of these three major protein groups, we report five interesting differences in the folding patterns of the VSD both in prokaryotes and eukaryotes. Our main findings indicate that this module is highly conserved throughout the evolutionary scale, however: (1) segments S1 to S3 in eukaryotes are significantly more hydrophobic than those present in prokaryotes; (2) the S4 segment has retained its hydrophilic character; (3) in eukaryotes the extramembranous linkers are significantly larger and more flexible in comparison with those present in prokaryotes; (4) the sensors present in the kHv1 proton channel and the ciVSP phosphatase, both of eukaryotic origin, exhibit relationships of flexibility and folding patterns very close to the typical ones found in prokaryotic voltage sensors; and (5) archaeal channels KvAP and MVP have flexibility profiles which are clearly contrasting in the S3-S4 region, which could explain their divergent activation mechanisms. Finally, to elucidate the obscure origins of this module, we show further evidence for a possible connection between voltage sensors and TolQ proteins.

Dual allosteric modulation of voltage and calcium sensitivities of the Slo1-LRRC channel complex.

Modulation of large conductance intracellular ligand-activated potassium (BK) channel family (Slo1-3) by auxiliary subunits allows diverse physiological functions in excitable and non-excitable cells. Cryoelectron microscopy (cryo-EM) structures of voltage-gated potassium (Kv) channel complexes have provided insights into how voltage sensitivity is modulated by auxiliary subunits. However, the modulation mechanisms of BK channels, particularly as ligand-activated ion channels, remain unknown. Slo1 is a Ca2+-activated and voltage-gated BK channel and is expressed in neurons, muscle cells, and epithelial cells. Using cryo-EM and electrophysiology, we show that the LRRC26-γ1 subunit modulates not only voltage but also Ca2+ sensitivity of Homo sapiens Slo1. LRRC26 stabilizes the active conformation of voltage-senor domains of Slo1 by an extracellularly S4-locking mechanism. Furthermore, it also stabilizes the active conformation of Ca2+-sensor domains of Slo1 intracellularly, which is functionally equivalent to intracellular Ca2+ in the activation of Slo1. Such a dual allosteric modulatory mechanism may be general in regulating the intracellular ligand-activated BK channel complexes.

Recent Developments in Ion Channel and Ion-Related Signaling.

Ion channels play an important role in the cellular functions of various organ systems, such as the nervous, cardiovascular, immune, and endocrine systems, and are potential therapeutic targets for treatments of their dysfunctions, via 'channelopathy' [...].

Hydrophobic gating in bundle-crossing ion channels: a case study of TRPV4.

Transmembrane ion channels frequently regulate ion permeation by forming bundle crossing of the pore-lining helices when deactivated. The resulting physical constriction is believed to serve as the de facto gate that imposes the major free energy barrier to ion permeation. Intriguingly, many ion channels also contain highly hydrophobic inner pores enclosed by bundle crossing, which can undergo spontaneous dewetting and give rise to a "vapor barrier" to block ion flow even in the absence of physical constriction. Using atomistic simulations, we show that hydrophobic gating and bundle-crossing mechanisms co-exist and complement one and another in the human TRPV4 channel. In particular, a single hydrophilic mutation in the lower pore can increase pore hydration and reduce the ion permeation free energy barrier by about half without affecting the bundle crossing. We believe that hydrophobic gating may play a key role in other bundle-crossing ion channels with hydrophobic inner pores.

Direct regulation of the voltage sensor of HCN channels by membrane lipid compartmentalization.

Ion channels function within a membrane environment characterized by dynamic lipid compartmentalization. Limited knowledge exists regarding the response of voltage-gated ion channels to transmembrane potential within distinct membrane compartments. By leveraging fluorescence lifetime imaging microscopy (FLIM) and Förster resonance energy transfer (FRET), we visualized the localization of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in membrane domains. HCN4 exhibits a greater propensity for incorporation into ordered lipid domains compared to HCN1. To investigate the conformational changes of the S4 helix voltage sensor of HCN channels, we used dual stop-codon suppression to incorporate different noncanonical amino acids, orthogonal click chemistry for site-specific fluorescence labeling, and transition metal FLIM-FRET. Remarkably, altered FRET levels were observed between VSD sites within HCN channels upon disruption of membrane domains. We propose that the voltage-sensor rearrangements, directly influenced by membrane lipid domains, can explain the heightened activity of pacemaker HCN channels when localized in cholesterol-poor, disordered lipid domains, leading to membrane hyperexcitability and diseases.

Ion currents through the voltage sensor domain of distinct families of proteins.

The membrane potential of a cell (Vm) regulates several physiological processes. The voltage sensor domain (VSD) is a region that confers voltage sensitivity to different types of transmembrane proteins such as the following: voltage-gated ion channels, the voltage-sensing phosphatase (Ci-VSP), and the sperm-specific Na+/H+ exchanger (sNHE). VSDs contain four transmembrane segments (S1-S4) and several positively charged amino acids in S4, which are essential for the voltage sensitivity of the protein. Generally, in response to changes of the Vm, the positive residues of S4 displace along the plasma membrane without generating ionic currents through this domain. However, some native (e.g., Hv1 channel) and mutants of VSDs produce ionic currents. These gating pore currents are usually observed in VSDs that lack one or more of the conserved positively charged amino acids in S4. The gating pore currents can also be induced by the isolation of a VSD from the rest of the protein domains. In this review, we summarize gating pore currents from all families of proteins with VSDs with classification into three cases: (1) pathological, (2) physiological, and (3) artificial currents. We reinforce the model in which the position of S4 that lacks the positively charged amino acid determines the voltage dependency of the gating pore current of all VSDs independent of protein families.

Asymmetric gating of a human hetero-pentameric glycine receptor.

Hetero-pentameric Cys-loop receptors constitute a major type of neurotransmitter receptors that enable signal transmission and processing in the nervous system. Despite intense investigations into their working mechanism and pharmaceutical potentials, how neurotransmitters activate these receptors remains unclear due to the lack of high-resolution structural information in the activated open state. Here we report near-atomic resolution structures resolved in digitonin consistent with all principle functional states of the human α1ß GlyR, which is a major Cys-loop receptor that mediates inhibitory neurotransmission in the central nervous system of adults. Glycine binding induces cooperative and symmetric structural rearrangements in the neurotransmitter-binding extracellular domain but asymmetrical pore dilation in the transmembrane domain. Symmetric response in the extracellular domain is consistent with electrophysiological data showing cooperative glycine activation and contribution from both α1 and ß subunits. A set of functionally essential but differentially charged amino acid residues in the transmembrane domain of the α1 and ß subunits explains asymmetric activation. These findings provide a foundation for understanding how the gating of the Cys-loop receptor family members diverges to accommodate specific physiological environments.

ATP modulates the activity of the voltage-gated proton channel through direct binding interaction.

ATP is an important molecule implicated in diverse biochemical processes, including the modulation of ion channel and transporter activity. The voltage-gated proton channel (Hv1) controls proton flow through the transmembrane pathway in response to membrane potential, and various molecules regulate its activity. Although it is believed that ATP is not essential for Hv1 activity, a report has indicated that cytosolic ATP may modulate Hv1. However, the detailed molecular mechanism underlying the effect of ATP on Hv1 is unknown, and whether ATP is involved in the physiological regulation of Hv1 activity remains unclear. Here, we report that cytosolic ATP is required to maintain Hv1 activity. To gain insight into the underlying mechanism, we analysed the effects of ATP on the mouse Hv1 channel (mHv1) using electrophysiological and microscale thermophoresis (MST) methods. Intracellular ATP accelerated the activation kinetics of mHv1, thereby increasing the amplitude of the proton current within the physiological concentration range. The increase in proton current was reproduced with a non-hydrolysable ATP analogue, indicating that ATP directly influences Hv1 activity without an enzymatic reaction. The direct molecular interaction between the purified mHv1 protein and ATP was analysed and demonstrated through MST. In addition, ATP facilitation was observed for the endogenous proton current flowing through Hv1 in the physiological concentration range of ATP. These results suggest that ATP influences Hv1 activity via direct molecular interactions and is required for the physiological function of Hv1. KEY POINTS: We found that ATP is required to maintain the activity of voltage-gated proton channels (Hv1) and investigated the underlying molecular mechanism. Application of intracellular ATP increased the amplitude of the proton current flowing through Hv1, accompanied by an acceleration of activation kinetics. The direct interaction between purified Hv1 protein and ATP was quantitatively analysed using microscale thermophoresis. ATP enhanced endogenous proton currents in breast cancer cell lines. These results suggest that ATP influences Hv1 activity via direct molecular interactions and that its functional characteristics are required for the physiological activity of Hv1.