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Ion channels are generally allosteric proteins, involving specialized stimulus sensor domains conformationally linked to the gate to drive channel opening. Temperature receptors are a group of ion channels from the transient receptor potential family. They exhibit an unprecedentedly strong temperature dependence and are responsible for temperature sensing in mammals. Despite intensive studies, however, the nature of the temperature sensor domain in these channels remains elusive. By direct calorimetry of TRPV1 proteins, we have recently provided a proof of principle that temperature sensing by ion channels may diverge from the conventional allosterity theory; rather it is intimately linked to inherent thermal instability of channel proteins. Here, we tackle the generality of the hypothesis and provide key molecular pieces of evidence on the coupling of thermal transitions in the channels. We show that while wild-type channels possess a single concerted thermal transition peak, the chimera, in which strong temperature dependence becomes disrupted, results in multitransition peaks, and the activation enthalpies are accordingly reduced. The data show that the coupling with protein unfolding drives up the energy barrier of activation, leading to a strong temperature dependence of opening. Furthermore, we pinpoint the proximal N-terminus of the channels as a linchpin in coalescing different parts of the channels into concerted activation. Thus, we suggest that coupled interaction networks in proteins underlie the strong temperature dependence of temperature receptors., Competing Interests: Competing interests statement:The authors declare no competing interest.

JGP study (Liu and Bezanilla. https://doi.org/10.1085/jgp.202413667) reveals that a sodium channel mutant blocks fast inactivation downstream of inactivation particle binding, diverting the channel into an alternative open state., (© 2024 Short.)

NMDA receptors (NMDAR) convert the major excitatory neurotransmitter glutamate into a synaptic signal. A key question is how efficiently the ion channel opens in response to the rapid exposure to presynaptic glutamate release. Here, we applied glutamate to single channel outside-out patches and measured the successes of channel openings and the latency to first opening to assay the activation efficiency of NMDARs under different physiological conditions and with different human subunit compositions. For GluN1/GluN2A receptors, we find that various factors, including intracellular ATP and GTP, can enhance the efficiency of activation presumably via the intracellular C-terminal domain. Notably, an energy-based internal solution or increasing the time between applications to increase recovery time improved efficiency. However, even under these optimized conditions and with a 1-s glutamate application, there remained around 10-15% inefficiency. Channel activation became more inefficient with brief synaptic-like pulses of glutamate at 2 ms. Of the different NMDAR subunit compositions, GluN2B-containing NMDARs showed the lowest success rate and longest latency to first openings, highlighting that they display the most distinct activation mechanism. In contrast, putative triheteromeric GluN1/GluN2A/GluN2B receptors showed high activation efficiency. Despite the low open probability, NMDARs containing either GluN2C or GluN2D subunits displayed high activation efficiency, nearly comparable with that for GluN2A-containing receptors. These results highlight that activation efficiency in NMDARs can be regulated by environmental surroundings and varies across different subunits., (© 2024 He and Wollmuth.)

Pannexin 1 (Panx1) constitutes a large pore channel responsible for the release of adenosine triphosphate (ATP) from apoptotic cells. Strong evidence indicates that caspase-mediated cleavage of the C-terminus promotes the opening of the Panx1 channel by unplugging the pore. However, this simple pore-plugging mechanism alone cannot account for the observation that a Panx1 construct ending before the caspase cleavage site remains closed. Here, we show that a helical region located immediately before the caspase cleavage site, referred to as the "C-terminal activating domain (CAD)", plays a pivotal role in facilitating Panx1 activation. Electrophysiology and mutagenesis studies uncovered that two conserved leucine residues within the CAD play a pivotal role. Cryoelectron microscopy (Cryo-EM) analysis of the construct ending before reaching the CAD demonstrated that the N terminus extends into an intracellular pocket. In contrast, the construct including the CAD revealed that this domain occupies the intracellular pocket, causing the N terminus to flip upward within the pore. Analysis of electrostatic free energy landscape in the closed conformation indicated that the intracellular side of the ion permeation pore may be occupied by anions like ATP, creating an electrostatic barrier for anions attempting to permeate the pore. When the N terminus flips up, it diminishes the positively charged surface, thereby reducing the drive to accumulate anions inside the pore. This dynamic change in the electrostatic landscape likely contributes to the selection of permeant ions. Collectively, these experiments put forth a mechanism in which C-terminal cleavage liberates the CAD, causing the repositioning of the N terminus to promote Panx1 channel opening., Competing Interests: Competing interests statement:The authors declare no competing interest.

K V 1.2 is a prominent ion channel in the CNS, where it regulates neuronal excitability. K V 1.2 structure and function are well understood, but there is less consensus on mechanisms of regulation of K V 1.2 and other potassium channels by auxiliary proteins. We previously identified novel regulators of K V 1.2 by a mass spectrometry approach. The neutral amino acid transporter Slc7a5 causes a dramatic hyperpolarizing shift of channel activation. In contrast, the transmembrane lectin LMAN2 is a recently identified candidate regulator that has the opposite effect on gating: large depolarizing voltages are required to activate K V 1.2 channels co-expressed with LMAN2. In this study, we characterized the functional interaction between LMAN2 and Slc7a5 on K V 1.2 gating properties and identified key structural elements that underlie sensitivity to each regulator. When LMAN2 and Slc7a5 are expressed together, K V 1.2 activation exhibits a bi-modal voltage-dependence, suggesting two distinct populations of channels regulated either by LMAN2 or Slc7a5, but not both. Using a K V 1.2:1.5 chimeric approach, we identified specific regions between the S1 to S3 segments of the voltage sensing domain (VSD) that are distinct for either Slc7a5 or LMAN2 sensitivity. By replacing either segment with sequence from K V 1.5, modulation by the corresponding regulator was selectively abolished. These results suggest that Slc7a5 and LMAN2 compete for interaction with the K V 1.2 voltage sensor, leading to complex voltage-dependence of channel activity when both regulators are present in the cell., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

The Drosophila neuromuscular junction (NMJ) is a powerful genetic system that has revealed numerous conserved mechanisms for synapse development and homeostasis. The fly NMJ uses glutamate as the excitatory neurotransmitter and relies on kainate-type glutamate receptors and their auxiliary protein Neto for synapse assembly and function. However, despite decades of study, the reconstitution of NMJ glutamate receptors using heterologous systems has been achieved only recently, and there are no reports on the gating properties for the recombinant receptors. Here, using outside-out, patch clamp recordings and fast ligand application, we examine for the first time the biophysical properties of native type-A and type-B NMJ receptors in complexes with either Neto-α or Neto-β and compare them with recombinant receptors expressed in HEK293T cells. We found that type-A and type-B receptors have strikingly different gating properties that are further modulated by Neto-α and Neto-β. We captured single-channel events and revealed major differences between type-A and type-B receptors and also between Neto splice variants. Surprisingly, we found that deactivation is extremely fast and that the decay of synaptic currents resembles the rate of ionotropic glutamate receptor (iGluR) desensitization. The functional analyses of recombinant iGluRs that we report here should greatly facilitate the interpretation of compound in vivo phenotypes of mutant animals. KEY POINTS: We report the reconstitution of Drosophila neuromuscular junction ionotropic glutamate receptors (iGluRs) with Neto splice forms. Using outside-out patches and fast ligand application, we examine the deactivation and desensitization of the four iGluR/Neto complexes found in vivo. Expression of functional channels is absolutely dependent on Neto. Single-channel recordings revealed different lifetimes for different receptor complexes. The decay of synaptic currents is controlled by desensitization., (© 2024 The Author(s). The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Voltage-gated sodium channels (VGSCs) in the peripheral nervous system shape action potentials (APs) and thereby support the detection of sensory stimuli. Most of the nine mammalian VGSC subtypes are expressed in nociceptors, but predominantly, three are linked to several human pain syndromes: while Nav1.7 is suggested to be a (sub-)threshold channel, Nav1.8 is thought to support the fast AP upstroke. Nav1.9, as it produces large persistent currents, is attributed a role in determining the resting membrane potential. We characterized the gating of Nav1.1-Nav1.3 and Nav1.5-Nav1.9 in manual patch clamp with a focus on the AP subthreshold depolarization phase. Nav1.9 exhibited the most hyperpolarized activation, while its fast inactivation resembled the depolarized inactivation of Nav1.8. For some VGSCs (e.g., Nav1.1 and Nav1.2), a positive correlation between ramp current and window current was detected. Using a modified Hodgkin-Huxley model that accounts for the time needed for inactivation to occur, we used the acquired data to simulate two nociceptive nerve fiber types (an Aδ- and a mechano-insensitive C-nociceptor) containing VGSC conductances according to published human RNAseq data. Our simulations suggest that Nav1.9 is supporting both the AP upstroke and its shoulder. A reduced threshold for AP generation was induced by enhancing Nav1.7 conductivity or shifting its activation to more hyperpolarized potentials, as observed in Nav1.7-related pain disorders. Here, we provide a comprehensive, comparative functional characterization of VGSCs relevant in nociception and describe their gating with Hodgkin-Huxley-like models, which can serve as a tool to study their specific contributions to AP shape and sodium channel-related diseases., (© 2025 Köster et al.)

Voltage-gated ion channels (VGICs) are central to cellular excitation, orchestrating skeletal and cardiac muscle contractions and enabling neural signal transduction. Among these, voltage-gated potassium (Kv) channels are particularly significant in cardiac electrophysiology, especially during the repolarization phase of the cardiac action potential. In cardiac myocytes, Kv channels are integral to a multitude of sophisticated functions, including electrical conduction. Despite their importance, research on Kv channels in the context of cardiovascular diseases is limited. This review offers a comprehensive summary of the structural complexities of Kv channels, delineating the regulatory mechanisms involved in channel gating, expression, and membrane localization. Additionally, we examine the role of different Kv α-subunits in modulating Kv channels and their impact on cardiac remodeling, and assess the potential of targeting Kv channels for the development of anti-arrhythmic therapies.

ω-Grammotoxin-SIA (GrTX-SIA) was originally isolated from the venom of the Chilean rose tarantula and demonstrated to function as a gating modifier of voltage-gated Ca2+ (CaV) channels. Later experiments revealed that GrTX-SIA could also inhibit voltage-gated K+ (KV) channel currents via a similar mechanism of action that involved binding to a conserved S3-S4 region in the voltage-sensing domains (VSDs). Since voltage-gated Na+ (NaV) channels contain homologous structural motifs, we hypothesized that GrTX-SIA could inhibit members of this ion channel family as well. Here, we show that GrTX-SIA can indeed impede the gating process of multiple NaV channel subtypes with NaV1.6 being the most susceptible target. Moreover, molecular docking of GrTX-SIA onto NaV1.6, supported by a p.E1607K mutation, revealed the voltage sensor in domain IV (VSDIV) as being a primary site of action. The biphasic manner in which current inhibition appeared to occur suggested a second, possibly lower-sensitivity binding locus, which was identified as VSDII by using KV2.1/NaV1.6 chimeric voltage-sensor constructs. Subsequently, the NaV1.6p.E782K/p.E838K (VSDII), NaV1.6p.E1607K (VSDIV), and particularly the combined VSDII/VSDIV mutant lost virtually all susceptibility to GrTX-SIA. Together with existing literature, our data suggest that GrTX-SIA recognizes modules in NaV channel VSDs that are conserved among ion channel families, thereby allowing it to act as a comprehensive ion channel gating modifier peptide., (© 2024 COLLAÇO et al.)

The voltage-dependent potassium channels (Kv channels) show several different types of inactivation. N-type inactivation is a fast inactivating mechanism, which is essentially an open pore blockade by the amino-terminal structure of the channel itself or the auxiliary subunit. There are several functionally discriminatable slow inactivation (C-type, P-type, U-type), the mechanism of which is supposed to include rearrangement of the pore region. In some Kv1 channels, the actual inactivation is brought about by coupling of N-type and C-type inactivation (N-C coupling). In the present study, we focused on the N-C coupling of the Aplysia Kv1 channel (AKv1). AKv1 shows a robust N-type inactivation, but its recovery is almost thoroughly from C-type inactivated state owing to the efficient N-C coupling. In the I8Q mutant of AKv1, we found that the inactivation as well as its recovery showed two kinetic components apparently correspond to N-type and C-type inactivation. Also, the cumulative inactivation which depends on N-type mechanism in AKv1 was hindered in I8Q, suggesting that N-type inactivation of I8Q is less stable. We also found that Zn 2 + specifically accelerates C-type inactivation of AKv1 and that H382 in the pore turret is involved in the Zn 2 + binding. Because the region around Ile 8 (I8) in AKv1 has been suggested to be involved in the pre-block binding of the amino-terminal structure, our results strengthen a hypothesis that the stability of the pre-block state is important for stable N-type inactivation as well as the N-C coupling in the Kv1 channel inactivation., (© 2024. The Author(s).)

GABA B receptors (GBRs) are G protein-coupled receptors for GABA, the main inhibitory neurotransmitter in the brain. GBRs regulate fast synaptic transmission by gating Ca 2+ and K + channels via the Gβγ subunits of the activated G protein. It has been demonstrated that auxiliary GBR subunits, the KCTD proteins, shorten onset and rise time and increase desensitization of receptor-induced K + currents. KCTD proteins increase desensitization of K + currents by scavenging Gβγ from the channel, yet the mechanism responsible for the rapid activation of K + currents has remained elusive. In this study, we demonstrate that KCTD proteins preassemble Gβγ at GBRs. The preassembly obviates the need for diffusion-limited G protein recruitment to the receptor, thereby accelerating G protein activation and, as a result, K + channel activation. Preassembly of Gβγ at the receptor relies on the interaction of KCTD proteins with a loop protruding from the seven-bladed propeller of Gβ subunits. The binding site is shared between Gβ1 and Gβ2, limiting the interaction of KCTD proteins to these particular Gβ isoforms. Substituting residues in the KCTD binding site of Gβ1 with those from Gβ3 hinders the preassembly of Gβγ with GBRs, delays onset and prolongs rise time of receptor-activated K + currents. The KCTD-Gβ interface, therefore, represents a target for pharmacological modulation of channel gating by GBRs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The KCNH family of potassium channels serves relevant physiological functions in both excitable and non-excitable cells, reflected in the massive consequences of mutations or pharmacological manipulation of their function. This group of channels shares structural homology with other voltage-gated K + channels, but the mechanisms of gating in this family show significant differences with respect to the canonical electromechanical coupling in these molecules. In particular, the large intracellular domains of KCNH channels play a crucial role in gating that is still only partly understood. Using KCNH1 (K V 10.1) as a model, we have characterized the behavior of a series of modified channels that could not be explained by the current models. With electrophysiological and biochemical methods combined with mathematical modeling, we show that the uncovering of an open state can explain the behavior of the mutants. This open state, which is not detectable in wild-type channels, appears to lack the rapid flicker block of the conventional open state. Because it is accessed from deep closed states, it elucidates intermediate gating events well ahead of channel opening in the wild type. This allowed us to study gating steps prior to opening, which, for example, explain the mechanism of gating inhibition by Ca 2+ -Calmodulin and generate a model that describes the characteristic features of KCNH channels gating., Competing Interests: RA, AT, AN, LP No competing interests declared, (© 2023, Abdelaziz et al.)

K+ channels exist in all living systems. They allow a selective transition to the K+ ion, which enables the activity of various vital tissues such as muscle cells, neurons, and even bacteria and plants. Despite the mechanism variation in the gating process of K+ channels in different tissues, the selectivity for the K+ ion is preserved and the electrochemical cascade is maintained in these tissues. The electrochemical gradient of the K+ ion is very close to the diffusion rate of K+ ions in bulk water. On the molecular level, how does a K+ ion move across the ion conduction pathway? There are many molecular models that describe and answer this question, however, this is rarely described on the macro level. Here, a physical model can serve as a very good basis for enabling a deeper understanding of the K+ ion for ion transport. Classical physical energy and linear and angular momentum laws can provide a good explanation as to how and what happens to K+ ions when they pass through an ion conduction pathway. This model describes the passage of the ion even before it enters the ion conduction path until the last ion at the end exits. The simulation described here is fascinating and depicts the state of the ion at the farthest end released at almost the same speed as the first ion initially, while all the other ions remain almost at rest. How does this occur? What happens if we change the size or mass of the ion? In this work, I describe this principle and the related problems that could be studied., Competing Interests: Declaration of competing interest The author declare that he does not have conflict of interest., (Copyright © 2024. Published by Elsevier Ltd.)

Two-pore channels are pathophysiologically important Na + - and Ca 2+ -permeable channels expressed in lysosomes and other acidic organelles. Unlike most other ion channels, their permeability is malleable and ligand-tuned such that when gated by the signaling lipid PI(3,5)P 2 , they are more Na + -selective than when gated by the Ca 2+ mobilizing messenger nicotinic acid adenine dinucleotide phosphate. However, the structural basis that underlies such plasticity and single-channel behavior more generally remains poorly understood. A recent Cryo-electron microscopy (cryo-EM) structure of TPC2 bound to PI(3,5)P 2 in a proposed open-channel conformation provided an opportunity to address this via molecular dynamics (MD) simulation. To our surprise, simulations designed to compute conductance through this structure revealed almost no Na + permeation events even at very high transmembrane voltages. However further MD simulations identified a spontaneous transition to a dramatically different conformation of the selectivity filter that involved expansion and a flip in the orientation of two core asparagine residues. This alternative filter conformation was remarkably stable and allowed Na + to flow through the channel leading to a conductance estimate that was in very good agreement with direct single-channel measurements. Furthermore, this conformation was more permeable for Na + over Ca 2+ . Our results have important ramifications not just for understanding the control of ion selectivity in TPC2 channels but also more broadly in terms of how ion channels discriminate ions., Competing Interests: Competing interests statement:The authors declare no competing interest.

We used voltage clamp fluorometry to probe the movement of the S4 helix in the voltage-sensing domain of the sea urchin HCN channel (spHCN) expressed in Xenopus oocytes. We obtained markedly different fluorescence responses with either ALEXA-488 or MTS-TAMRA covalently linked to N-terminal Cys332 of the S4 helix. With hyperpolarizing steps, ALEXA-488 fluorescence increased rapidly, consistent with it reporting the initial inward movement of S4, as previously described. In contrast, MTS-TAMRA fluorescence increased more slowly and its early phase correlated with that of channel opening. Additionally, a slow fluorescence component that tracked the development of the mode shift, or channel hysteresis, could be resolved with both labels. We quantitated this component as an increased deactivation tail current delay with concomitantly longer activation periods and found it to depend strongly on the presence of K+ ions in the pore. Using collisional quenching experiments and structural predictions, we established that ALEXA-488 was more exposed to solvent than MTS-TAMRA. We propose that components of S4 movement during channel activation can be kinetically resolved using different fluorescent probes to reveal distinct biophysical properties. Our findings underscore the need to apply caution when interpreting voltage clamp fluorometry data and demonstrate the potential utility of different labels to interrogate distinct biophysical properties of voltage-gated membrane proteins., (© 2024 Wojciechowski et al.)

Phosphorylation enables rapid modulation of voltage-gated calcium channels (VGCC) in physiological and pathophysiological conditions. How phosphorylation modulates human Ca V 1.3 VGCC, however, is largely unexplored. We characterized modulation of Ca V 1.3 gating via S1475, the human equivalent of a phosphorylation site identified in the rat. S1475 is highly conserved in Ca V 1.3 but absent from all other high-voltage activating calcium channel types co-expressed with Ca V 1.3 in similar tissues. Further, it is located in the C-terminal EF-hand motif, which binds calmodulin (CaM). This is involved in calcium-dependent channel inactivation (CDI). We used amino acid exchanges that mimic either sustained phosphorylation (S1475D) or phosphorylation resistance (S1475A). Whole-cell and single-channel recordings of phosphorylation state imitating Ca V 1.3 variants in transiently transfected HEK-293 cells revealed functional relevance of S1475 in human Ca V 1.3. We obtained three main findings: (1) Ca V 1.3_S1475D, imitating sustained phosphorylation, displayed decreased current density, reduced CDI and (in-) activation kinetics shifted to more depolarized voltages compared with both wildtype Ca V 1.3 and the phosphorylation-resistant Ca V 1.3_S1475A variant. Corresponding to the decreased current density, we find a reduced open probability of Ca V 1.3_S1475D at the single-channel level. (2) Using CaM overexpression or depletion, we find that CaM is necessary for modulating Ca V 1.3 through S1475. (3) CaMKII activation led to Ca V 1.3_WT-current properties similar to those of Ca V 1.3_S1475D, but did not affect Ca V 1.3_S1475A, confirming that CaMKII modulates human Ca V 1.3 via S1475. Given the physiological and pathophysiological importance of Ca V 1.3, our findings on the S1475-mediated interplay of phosphorylation, CaM interaction and CDI provide hints for approaches on specific Ca V 1.3 modulation under physiological and pathophysiological conditions. KEY POINTS: Phosphorylation modulates activity of voltage-gated L-type calcium channels for specific cellular needs but is largely unexplored for human Ca V 1.3 channels. Here we report that S1475, a CaMKII phosphorylation site identified in rats, is functionally relevant in human Ca V 1.3. Imitating phosphorylation states at S1475 alters current density and inactivation in a calmodulin-dependent manner. In wildtype Ca V 1.3 but not in the phosphorylation-resistant variant S1475A, CaMKII activation elicits effects similar to constitutively mimicking phosphorylation at S1475. Our findings provide novel insights on the interplay of modulatory mechanisms of human Ca V 1.3 channels, and present a possible target for Ca V 1.3-specific gating modulation in physiological and pathophysiological conditions., (© 2024 The Authors. The Journal of Physiology © 2024 The Physiological Society.)

The transient receptor potential melastatin (TRPM) tetrameric cation channels are involved in a wide range of biological functions, from temperature sensing and taste transduction to regulation of cardiac function, inflammatory pain, and insulin secretion. The structurally conserved TRPM cytoplasmic domains make up >70 % of the total protein. To investigate the mechanism by which the TRPM cytoplasmic domains contribute to gating, we employed electrophysiology and cryo-EM to study TRPM5-a channel that primarily relies on activation via intracellular Ca 2+ . Here, we show that activation of mammalian TRPM5 channels is strongly altered by Ca 2+ -dependent desensitization. Structures of rat TRPM5 identify a series of conformational transitions triggered by Ca 2+ binding, whereby formation and dissolution of cytoplasmic interprotomer interfaces appear to control activation and desensitization of the channel. This study shows the importance of the cytoplasmic assembly in TRPM5 channel function and sets the stage for future investigations of other members of the TRPM family., Competing Interests: Competing interests statement:The authors declare no competing interest.

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., (© 2024. The Author(s).)

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., (© 2024 The Protein Society.)

Mechanically activated Piezo1 channels undergo transitions from closed to open-state in response to pressure and other mechanical stimuli. However, the molecular details of these mechanosensitive gating transitions are unknown. Here, we used cell-attached pressure-clamp recordings to acquire single channel data at steady-state conditions (where inactivation has settled down), at various pressures and voltages. Importantly, we identify and analyze subconductance states of the channel which were not reported before. Pressure-dependent activation of Piezo1 increases the occupancy of open and subconductance state at the expense of decreased occupancy of shut-states. No significant change in the mean open time of subconductance states was observed with increasing negative pipette pressure or with varying voltages (ranging from -40 to -100 mV). Using Markov-chain modeling, we identified a minimal four-states kinetic scheme, which recapitulates essential characteristics of the single channel data, including that of the subconductance level. This study advances our understanding of Piezo1-gating mechanism in response to discrete stimuli (such as pressure and voltage) and paves the path to develop cellular and tissue level models to predict Piezo1 function in various cell types., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2024. The Author(s).)

Voltage-gated sodium channels (Naᵥ) are membrane proteins which open to facilitate the inward flux of sodium ions into excitable cells. In response to stimuli, Naᵥ 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 Naᵥ channels a significant pharmacological target. Phosphoinositides are important lipid cofactors for ion channel function. The phosphoinositide PI(4,5)P 2 decreases Naᵥ1.4 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)P 2 binds stably to inactivated Naᵥ at a conserved site within the DIV S4-S5 linker, which couples the voltage-sensing domain (VSD) to the pore. As the Naᵥ C-terminal domain is proposed to also bind here during recovery from inactivation, we hypothesize that PI(4,5)P 2 prolongs inactivation by competitively binding to this site. In atomistic simulations, PI(4,5)P 2 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 Naᵥ 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 Naᵥ-related diseases., Competing Interests: YL, ET, JC, BC No competing interests declared, (© 2023, Lin, Tao et al.)

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 H v 1, there is a bulky noncharged tryptophan residue located at the S4 transmembrane segment. This tryptophan remains entirely conserved across all H v 1 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 H v 1 is Trp207 (W207). Here, we showed that W207 modifies human H v 1 voltage-dependent activation, and small residues replacement at position 207 strongly perturbs H v 1 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 H v 1 VSD transition., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Published by Elsevier Inc.)

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., Competing Interests: Competing interests statement:The authors declare no competing interest.

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 β1-β2 and M2-M3 loops, with agonist binding re-positioning a key β1-β2 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., (© 2024. The Author(s).)

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 pH i , decrease pH o , control the membrane potential, generate action potentials, or compensate transmembrane movement of electrical charge.

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., (© 2024. The Author(s).)

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 (pH i ) were obtained by the Simplex method. With our approach, we find that, in fact, there is more than one parameter set for each pH i , 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 pH i . 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.

Patch-clamp technique provides a unique possibility to record the ion channels' activity. This method enables tracking the changes in their functional states at controlled conditions on a real-time scale. Kinetic parameters evaluated for the patch-clamp signals form the fundamentals of electrophysiological characteristics of the channel functioning. Nevertheless, the noisy series of ionic currents flowing through the channel protein(s) seem to be bountiful of information, and the standard data processing techniques likely unravel only its part. Rapid development of artificial intelligence (AI) techniques, especially machine learning (ML), gives new prospects for whole channelology. Here we consider the question of the AI applications in the patch-clamp signal analysis. It turns out that the AI methods may not only enable for automatizing of signal analysis, but also they can be used in finding inherent patterns of channel gating and allow the researchers to uncover the details of gating machinery, which had been never considered before. In this work, we outline the currently known AI methods that turned out to be utilizable and useful in the analysis of patch-clamp signals. This chapter can be considered an introductory guide to the application of AI methods in the analysis of the time series of channel currents (together with its advantages, disadvantages, and limitations), but we also propose new possible directions in this field., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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 Ca 2+ -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 Ca 2+ 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 Ca 2+ -sensor domains of Slo1 intracellularly, which is functionally equivalent to intracellular Ca 2+ in the activation of Slo1. Such a dual allosteric modulatory mechanism may be general in regulating the intracellular ligand-activated BK channel complexes., Competing Interests: Declaration of interests O.N. is a cofounder, board member, and scientific advisor of Curreio. Y.K. is chief researcher of Curreio., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

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., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The membrane potential of a cell (V m ) 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 V m , 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., (© 2023. The Author(s).)

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, β-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 β-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.

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., (© 2023. The Author(s).)

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., (© 2023. Springer Nature Limited.)

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 α1β 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 β subunits. A set of functionally essential but differentially charged amino acid residues in the transmembrane domain of the α1 and β 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., (© 2023. Springer Nature Limited.)

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., (© 2023 The Authors. The Journal of Physiology © 2023 The Physiological Society.)

K+ channel activity can be limited by C-type inactivation, which is likely initiated in part by dissociation of K+ ions from the selectivity filter and modulated by the side chains that surround it. While crystallographic and computational studies have linked inactivation to a "collapsed" selectivity filter conformation in the KcsA channel, the structural basis for selectivity filter gating in other K+ channels is less clear. Here, we combined electrophysiological recordings with molecular dynamics simulations, to study selectivity filter gating in the model potassium channel MthK and its V55E mutant (analogous to KcsA E71) in the pore-helix. We found that MthK V55E has a lower open probability than the WT channel, due to decreased stability of the open state, as well as a lower unitary conductance. Simulations account for both of these variables on the atomistic scale, showing that ion permeation in V55E is altered by two distinct orientations of the E55 side chain. In the "vertical" orientation, in which E55 forms a hydrogen bond with D64 (as in KcsA WT channels), the filter displays reduced conductance compared to MthK WT. In contrast, in the "horizontal" orientation, K+ conductance is closer to that of MthK WT; although selectivity filter stability is lowered, resulting in more frequent inactivation. Surprisingly, inactivation in MthK WT and V55E is associated with a widening of the selectivity filter, unlike what is observed for KcsA and reminisces recent structures of inactivated channels, suggesting a conserved inactivation pathway across the potassium channel family., (© 2023 Kopec et al.)

We celebrate this year the 50th anniversary of the first electrophysiological recordings of the gating currents from voltage-dependent ion channels done in 1973. This retrospective tries to illustrate the context knowledge on channel gating and the impact gating-current recording had then, and how it continued to clarify concepts, elaborate new ideas, and steer the scientific debate in these 50 years. The notion of gating particles and gating currents was first put forward by Hodgkin and Huxley in 1952 as a necessary assumption for interpreting the voltage dependence of the Na and K conductances of the action potential. 20 years later, gating currents were actually recorded, and over the following decades have represented the most direct means of tracing the movement of the gating charges and gaining insights into the mechanisms of channel gating. Most work in the early years was focused on the gating currents from the Na and K channels as found in the squid giant axon. With channel cloning and expression on heterologous systems, other channels as well as voltage-dependent enzymes were investigated. Other approaches were also introduced (cysteine mutagenesis and labeling, site-directed fluorometry, cryo-EM crystallography, and molecular dynamics [MD] modeling) to provide an integrated and coherent view of voltage-dependent gating in biological macromolecules. The layout of this retrospective reflects the past 50 years of investigations on gating currents, first addressing studies done on Na and K channels and then on other voltage-gated channels and non-channel structures. The review closes with a brief overview of how the gating-charge/voltage-sensor movements are translated into pore opening and the pathologies associated with mutations targeting the structures involved with the gating currents., (© 2023 Catacuzzeno et al.)

Org 34167 is a small molecule hyperpolarization-activated cyclic nucleotide-gated (HCN) channel modulator that has been trialed in humans for its potential antidepressant activity. The precise action of Org 34167 is not fully understood. Here we use two-electrode voltage clamp recordings and an allosteric model to explore the interaction of Org 34167 with human HCN1 channels. The impact of Org 34167 on channel function included a hyperpolarizing shift in activation voltage dependence and a slowing of activation kinetics. Furthermore, a reduction in the maximum open probability at extreme hyperpolarization argued for an additional voltage-independent mechanism. Org 34167 had a similar impact on a truncated HCN1 channel lacking the C-terminal nucleotide binding domain, thus ruling out an interaction with this domain. Fitting a gating model, derived from a 10-state allosteric scheme, predicted that Org 34167 strongly reduced the equilibrium constant for the voltage-independent pore domain to favor a closed pore, as well as reducing the voltage sensing domain-pore domain coupling and shifting the zero voltage equilibrium constant of the voltage sensing domain to favor the inactive state. SIGNIFICANCE STATEMENT: The brain penetrant small molecule Org 34167 has been reported to have an antidepressant action by targeting HCN channels; however, its mode of action is unknown. We used heterologously expressed human HCN1 channels to show that Org 34167 inhibits channel activity by modulating kinetic parameters associated with the channel pore domain, voltage sensing domain, and interdomain coupling., (Copyright © 2023 by The American Society for Pharmacology and Experimental Therapeutics.)

Recently, three proton channels (H V ) have been identified and characterized in Aplysia californica (AcH V 1-3). Focusing on AcH V 1 and AcH V 2, analysis of Transcriptome Shotgun Assembly and genomic databases of 91 molluscs identified H V homologous channels in other molluscs: channels homologous to AcH V 1 and to AcH V 2 were found in 90 species (56 full-length sequences) and in 33 species (18 full-length sequences), respectively. Here, we report the discovery of a fourth distinct proton channel family, H V 4. This new family has high homology to AcH V 1 and AcH V 2 and was identified only in bivalvian molluscs (13 species, 12 full-length sequences). Typically, these channels possess an extracellular S1-S2 loop of intermediate size (~ 20 amino acids) compared to the shorter loops of molluscan H V 1 channels (~ 13 amino acids) and the much larger loops of molluscan H V 2 channels (> 65 amino acids). The characteristic voltage-sensor motif in S4 possesses only two arginine residues with the common third arginine being replaced by a lysine. Moreover, H V 4 channels are much smaller with only around 200 amino acids in total length. The smallest functional channel found so far in nature (189 amino acids) is expressed in the pacific oyster Crassostrea gigas (CgH V 4) and might be considered an archetypical minimal proton channel. Functional expression and electrophysiological characterization demonstrated that CgH V 4 shares distinctive hallmarks of other investigated proton channels as high proton selectivity, slow activation, and pH- and voltage-regulated gating. This work is the first description of a H V 4 type channel, adding a new member to the recently expanded family of proton channels., (© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Voltage-gated ion channels play essential physiological roles in action potential generation and propagation. Peptidic toxins from animal venoms target ion channels and provide useful scaffolds for the rational design of novel channel modulators with enhanced potency and subtype selectivity. Despite recent progress in obtaining experimental structures of peptide toxin-ion channel complexes, structural determination of peptide toxins bound to ion channels in physiologically important states remains challenging. Here we describe an application of RosettaDock approach to the structural modeling of peptide toxins interactions with ion channels. We tested this approach on 10 structures of peptide toxin-ion channel complexes and demonstrated that it can sample near-native structures in all tested cases. Our approach will be useful for improving the understanding of the molecular mechanism of natural peptide toxin modulation of ion channel gating and for the structural modeling of novel peptide-based ion channel modulators., (© 2023 Wiley Periodicals LLC.)

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., Competing Interests: AE, JC, VB, LW, LZ, BC No competing interests declared, LD Reviewing editor, eLife, (© 2023, Elbahnsi, Cowgill et al.)

The large-conductance, Ca2+-, and voltage-activated K+ (BK) channel consists of the pore-forming α (BKα) subunit and regulatory β and γ subunits. The γ1-3 subunits facilitate BK channel activation by shifting the voltage-dependence of channel activation toward the hyperpolarization direction by about 50-150 mV in the absence of Ca2+. We previously found that the intracellular C-terminal positively charged regions of the γ subunits play important roles in BK channel modulation. In this study, we found that the intracellular C-terminal region of BKα is indispensable in BK channel modulation by the γ1 subunit. Notably, synthetic peptide mimics of the γ1-3 subunits' C-terminal positively charged regions caused 30-50 mV shifts in BKα channel voltage-gating toward the hyperpolarization direction. The cationic cell-penetrating HIV-1 Tat peptide exerted a similar BK channel-activating effect. The BK channel-activating effects of the synthetic peptides were reduced in the presence of Ca2+ and markedly ablated by both charge neutralization of the Ca2+-bowl site and high ionic strength, suggesting the involvement of electrostatic interactions. The efficacy of the γ subunits in BK channel modulation was reduced by charge neutralization of the Ca2+-bowl site. However, BK channel modulation by the γ1 subunit was little affected by high ionic strength and the positively charged peptide remained effective in BK channel modulation in the presence of the γ1 subunit. These findings identify positively charged peptides as BK channel modulators and reveal a role for the Ca2+-bowl site in BK channel modulation by positively charged peptides and the C-terminal positively charged regions of auxiliary γ subunits., (© 2023 Chen et al.)

Voltage-gated sodium (NaV) channels are densely expressed in most excitable cells and activate in response to depolarization, causing a rapid influx of Na+ ions that initiates the action potential. The voltage-dependent activation of NaV channels is followed almost instantaneously by fast inactivation, setting the refractory period of excitable tissues. The gating cycle of NaV channels is subject to tight regulation, with perturbations leading to a range of pathophysiological states. The gating properties of most ion channels are regulated by the membrane phospholipid, phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2). However, it is not known whether PI(4,5)P2 modulates the activity of NaV channels. Here, we utilize optogenetics to activate specific, membrane-associated phosphoinositide (PI)-phosphatases that dephosphorylate PI(4,5)P2 while simultaneously recording NaV1.4 channel currents. We show that dephosphorylating PI(4,5)P2 left-shifts the voltage-dependent gating of NaV1.4 to more hyperpolarized membrane potentials, augments the late current that persists after fast inactivation, and speeds the rate at which channels recover from fast inactivation. These effects are opposed by exogenous diC8PI(4,5)P2. We provide evidence that PI(4,5)P2 is a negative regulator that tunes the gating behavior of NaV1.4 channels., (© 2023 Gada et al.)

Light-controlled availability for phosphorylation reveals dominant roles of select R-domain serines in CFTR channel activation., (© 2023 Csanády.)

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels contribute to the rhythmic firing of pacemaker neurons and cardiomyocytes. Mutations in HCN channels are associated with cardiac arrhythmia and epilepsy. HCN channels belong to the superfamily of voltage-gated K+ channels, most of which are activated by depolarization. HCN channels, however, are activated by hyperpolarization. The mechanism behind this reversed gating polarity of HCN channels is not clear. We here show that sea urchin HCN (spHCN) channels with mutations in the C-terminal part of the voltage sensor use the same voltage-sensor movement to either close or open in response to hyperpolarizations depending on the absence or presence of cAMP. Our results support that non-covalent interactions at the C-terminal end of the voltage sensor are critical for HCN gating polarity. These interactions are also critical for the proper closing of the channels because these mutations exhibit large constitutive currents. Since a similar voltage-sensor movement can cause both depolarization- and hyperpolarization-activation in the same channel, this suggests that the coupling between the voltage sensor and the pore is changed to create channels opened by different polarities. We also show an identical voltage-sensor movement in activated and inactivated spHCN channels and suggest a model for spHCN activation and inactivation. Our results suggest the possibility that channels open by opposite voltage dependence, such as HCN and the related EAG channels, use the same voltage-sensor movement but different coupling mechanisms between the voltage sensor and the gate., (© 2023 Wu et al.)

Calmodulin (CaM) regulates many ion channels to control calcium entry into cells, and mutations that alter this interaction are linked to fatal diseases. The structural basis of CaM regulation remains largely unexplored. In retinal photoreceptors, CaM binds to the CNGB subunit of cyclic nucleotide-gated (CNG) channels and, thereby, adjusts the channel's Cyclic guanosine monophosphate (cGMP) sensitivity in response to changes in ambient light conditions. Here, we provide the structural characterization for CaM regulation of a CNG channel by using a combination of single-particle cryo-electron microscopy and structural proteomics. CaM connects the CNGA and CNGB subunits, resulting in structural changes both in the cytosolic and transmembrane regions of the channel. Cross-linking and limited proteolysis-coupled mass spectrometry mapped the conformational changes induced by CaM in vitro and in the native membrane. We propose that CaM is a constitutive subunit of the rod channel to ensure high sensitivity in dim light. Our mass spectrometry-based approach is generally relevant for studying the effect of CaM on ion channels in tissues of medical interest, where only minute quantities are available.