Pentameric ligand-gated ion channels control synaptic neurotransmission via an allosteric mechanism, whereby agonist binding induces global protein conformational changes that open an ion-conducting pore. For the proton-activated bacterial (Gloeobacter) ligand-gated ion channel (GLIC), high-resolution structures are available in multiple conformational states. We used a library of atomistic molecular dynamics (MD) simulations to study conformational changes and to perform dynamic network analysis to elucidate the communication pathways underlying the gating process. We describe state- and pH-dependent communication between the agonist-binding extracellular domain (ECD) and the ion-conducting transmembrane domain (TMD), revealing variation in pathways associated with conformational changes. We identify five main signal pathway families that connect the TMD and ECD via intra- and intersubunit communication. These pathway families implicate the Cys loop, the β1-β2 loop, and loop F in the ECD, along with the pre-M1 covalent connection and the M2-M3 loop in the TMD, each of which has previously been suggested to be important for gating. The β1-β2 loop and loop F pathway families exhibit stark dependence on the functional state, in contrast to the more constant Cys-loop and pre-M1 pathway families, suggesting that the channel may communicate differently during its transitions between open, closed, and intermediate states of its activity cycle. We interpret the state dependence in terms of the conformational changes that affect ECD-TMD alignment and, in particular, the breakage of the D32-R192 salt bridge, which is crucial in regulating the communication pathway. These communication networks are expected to be conserved within the superfamily of pentameric ligand-gated channels, with potential applications in improved anesthetics, neuromodulatory drugs, antiparasitics, and pesticides.

The fine tuning of biological electrical signaling is mediated by variations in the rates of opening and closing of gates that control ion flux through different ion channels. Human ether-a-go-go related gene (HERG) potassium channels have uniquely rapid inactivation kinetics which are critical to the role they play in regulating cardiac electrical activity. Here, we exploit the K+ sensitivity of HERG inactivation to determine structures of both a conductive and non-conductive selectivity filter structure of HERG. The conductive state has a canonical cylindrical shaped selectivity filter. The non-conductive state is characterized by flipping of the selectivity filter valine backbone carbonyls to point away from the central axis. The side chain of S620 on the pore helix plays a central role in this process, by coordinating distinct sets of interactions in the conductive, non-conductive, and transition states. Our model represents a distinct mechanism by which ion channels fine tune their activity and could explain the uniquely rapid inactivation kinetics of HERG. HERG channel inactivation is critical for normal heart rhythm. Authors determine structures of open and non-conducting states of HERG and identify a key role for S620 on the pore helix in coordinating transitions between open and inactivated states.

Proton-coupled oligopeptide transporters (POTs) are of great pharmaceutical interest owing to their promiscuous substrate binding site that has been linked to improved oral bioavailability of several classes of drugs. Members of the POT family are conserved across all phylogenetic kingdoms and function by coupling peptide uptake to the proton electrochemical gradient. Cryo-EM structures and alphafold models have recently provided new insights into different conformational states of two mammalian POTs, SLC15A1 and SLC15A2. Nevertheless, these studies leave open important questions regarding the mechanism of proton and substrate coupling, while simultaneously providing a unique opportunity to investigate these processes using molecular dynamics (MD) simulations. Here, we employ extensive unbiased and enhanced-sampling MD to map out the full SLC15A2 conformational cycle and its thermodynamic driving forces. By computing conformational free energy landscapes in different protonation states and in the absence or presence of peptide substrate, we identify a likely sequence of intermediate protonation steps that drive inward-directed alternating access. These simulations identify key differences in the extracellular gate between mammalian and bacterial POTs, which we validate experimentally in cell-based transport assays. Our results from constant-PH MD and absolute binding free energy (ABFE) calculations also establish a mechanistic link between proton binding and peptide recognition, revealing key details underpining secondary active transport in POTs. This study provides a vital step forward in understanding proton-coupled peptide and drug transport in mammals and pave the way to integrate knowledge of solute carrier structural biology with enhanced drug design to target tissue and organ bioavailability.

Abstract Over recent years, Induced proximity therapeutics (IPT) and targeted protein degradation (TPD) approaches have become increasingly popular. Whilst significant initial efforts focused upon heterobifunctional degraders, molecular glue degraders (MGDs) represent the next generation proximity-based drugs, but their rational discovery remains challenging. Here, we describe unbiased and broadly applicable approaches for molecular glue hit ID through the development of a suite of orthogonal and highly complementary screening approaches, relying on Homogeneous Time-Resolved Fluorescence (HTRF), Surface Plasmon Resonance (SPR) and High Content Imaging (HCI). To demonstrate the utility of these assays, we have focussed on the therapeutically relevant protein SOS1 as a model target. SOS1 is the major guanine nucleotide exchange factor (GEF) for KRAS, where inhibition of the SOS1/KRAS interaction reduces tumor growth in preclinical settings, and small molecule inhibitors of the SOS1/KRAS interaction are currently undergoing clinical testing for KRAS driven cancers. Our HTRF approach measures the induced interaction between the ubiquitin ligase CRBN/DDB1 and the SOS1 protein. The assay was successfully developed and validated in a miniaturized format utilizing a bifunctional tool compound as a positive control. Complementing our HTRF based approach, we also developed a highly sensitive SPR assay for detecting compounds that generate a ternary complex with CRBN/DDB1. These assay formats are amenable for large scale HTS screening, and as proof-of-concept we have established workflows and screened compound collections from the Sygnature LeadFinder and fragment libraries, in each of these assay platforms. Our combined biochemical/biophysical approach to glue identification using HTRF and SPR demonstrates the successful and scalable use of these methodologies for HTS, with the exciting potential for identification of novel MGD candidates for SOS1. To further complement these strategies for glue identification, we have additionally developed a functional cell-based approach utilizing HCI. HCI offers a medium-high throughput approach to monitor endogenous protein abundance and localization in complex cellular models. One major advantage of imaging assays is that they are amenable to multiplexing, allowing simultaneous detection of target engagement, biomarker analysis and compound toxicity. Here, we showcase HCI as a robust, high throughput method of screening for induced protein degradation at the endogenous level, again focusing on the KRAS pathway. Using this approach we quantified endogenous protein abundance in response to compound treatment, as well as downstream effects in the RAS pathway. Together, these approaches demonstrate multiple, complementary options for identifying molecular glue degraders. Further, the described assays are also applicable for screening and characterization of bifunctional degrader compounds. This platform is poised to identify high quality lead compounds across a range of oncology targets in areas of unmet clinical need. Citation Format: Allan Jordan, Philip Addis, Toby Allen, Tarun Ayra, Sarah Beck, Roslyn Brant, Roland Hjerpe, Benoit Gourdet, Martin Jennings, Rachel Lawrence, Sigrun Campbell-Maurer, Pei Cing Ng, Lyn Parkinson, Eva Rejnowicz, Gonzalo Robles, Elizabeth Rosethorne, Joshua Shaw, Duncan Smith, Denise Swift, Daniel Tait, Stuart Thomson, Chris Tomlinson, Ailsa Townley, Stephanie Ward, Clare Wilson. Building a platform of validated high throughput screening approaches for molecular glue degrader identification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB030.

Understanding membrane charge transport processes, including the actions of ion channels, pumps, carriers, and membrane-active peptides, requires a description of the electrostatics of the lipid bilayer. We have simulated a library of different lipid chemistries to reveal the impact of the headgroup, glycerol backbone, and hydrocarbon chains on the membrane dipole potential. We found a strong dependence of the potential on lipid packing, but this was not caused by the packing of lipid polar components, due to cancellation of their electric fields by electrolyte. In contrast, lipid tail contributions were determined by area per lipid, arising from two countering effects. Increased area per lipid leads to chain tilting that increases methylene dipole projections to strengthen the electric field within the bilayer, while at the same time decreasing the electric field from terminal methyl groups. Moreover, electric fields from some nonterminal groups and the terminal methyl group can extend beyond the bilayer center and be canceled by the opposing leaflet. This interleaflet field annulment explains the experimental reduction in dipole potential for unsaturated and branched lipid bilayers, by as much as ∼200 mV, as well as experiments that substitute chain carbons with sulfur. Replacing ester with ether groups (eliminating two carbonyl groups) causes a significant reduction in potential, also by ∼200 mV, in agreement with experiment. We show that the effect can be largely attributed to the loss of aligned water molecules in the glycerol backbone region, lowering the potential inside the bilayer core. When only one of the two carbonyls is removed (using a hybrid ester-ether lipid or a single-chain lipid), most of this reduction in potential was lost, with the single carbonyl group able to maintain full hydration in the interfacial region. While headgroup chemistry can have a major effect (by as much as ±100 mV relative to phosphatidylcholine), anionic headgroups either decrease or increase the dipole potential, with the variation involving perturbation in hydrogen-bonded water molecules and changes in packing of lipid tails. Overall, these results suggest that membrane electrostatics are dominated by aligned water molecules at the polar-hydrocarbon interface and, surprisingly, by the charge distribution of the nonpolar lipid tails, and not the packing of headgroup and glycerol carbonyl dipoles.

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)P2 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)P2 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 hypothesise 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 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.Voltage-gated sodium channels form pores in the membrane to mediate electrical activity in nerve and muscle cells. They play critical roles throughout the human body and their dysfunction leads to diseases including epilepsy, cardiac arrhythmias and pain disorders. Membrane lipids called phosphoinositides have recently been shown to reduce the activity of a voltage-gated sodium channel, but the molecular basis of this mechanism is not known. Here we use simulations to reveal where these lipids bind to the channels and how they reduce channel activity by making it harder for the pores to open and slower to subsequently recover to the closed resting state.

The cytoplasmic lysine-rich N-termini of the Na+,K+- and H+,K+-ATPases have been designated as R domains because of their possible role in regulation of ion pumping activity. Prior to the publication of any crystal structures of either protein, it was suggested, based on mutagenesis studies, that regulation of the Na+,K+-ATPase could involve the formation and breakage of a salt bridge between Lys30 of the N-terminus and Glu233 in the first M2-M3 cytoplasmic loop of the alpha subunit. In structural studies of the Na+,K+-ATPase, the N-terminus has never been able to be resolved, probably because it is intrinsically disordered. Nevertheless, after theoretically predicting the N-terminus conformation, and adding it to the structure, the distance between residues Lys30 and Glu233 makes it doubtful that a direct long-lived interaction between them is involved in pump regulation. As an alternative hypothesis, we propose that regulation could occur via an interaction of the positively charged lysine residues of the N-terminus with negatively charged lipid headgroups (notably phosphatidylserine) on the cytoplasmic surface of the surrounding membrane, and that such an interaction could potentially be regulated by an electrostatic switch mechanism, whereby serine and tyrosine residues of the N-terminus are phosphorylated by protein kinases. Phosphorylation of these residues would decrease the positive charge of the N-terminus, allowing its release from the membrane. Experimental and theoretical data will be presented which support this hypothesis.

The exquisite fine tuning of biological electrical signalling is mediated by variations in the rates of opening and closing of different ion channels(1). In addition to open and closed conformations, ion channels can exist in an inactivated state, which prevents conduction in the presence of a prolonged activating stimulus(2). Human ether-a-go-go related gene (HERG) K+ channels undergo uniquely rapid and voltage dependent inactivation(3-5), which confers upon them a critical role in protecting against cardiac arrhythmias and sudden death(6). Previous structural studies have captured only the open state of the HERG channel(7,8). Here, we have exploited the K+ sensitivity of HERG inactivation to determine structures of both the conductive state and the elusive inactivated state of HERG. We show that hERG inactivation is facilitated by two competing networks of hydrogen bonds behind the selectivity filter that enable rapid and voltage dependent flipping of the valine carbonyls in the centre of the selectivity filter. Our data also explains how changes in extracellular K+ affects the distribution between conductive and inactivated states(9,10) and thereby explains why hypokalaemia reduces HERG channel activity thereby increasing the risk of cardiac arrhythmias(11).

Quaternary ammonium blockers were previously shown to bind in the pore to block both open and closed conformations of large-conductance calcium-activated potassium (BK and MthK) channels. Because blocker entry was assumed through the intracellular entryway (bundle crossing), closed-pore access suggested that the gate was not at the bundle crossing. Structures of closed MthK, a Methanobacterium thermoautotrophicum homolog of BK channels, revealed a tightly constricted intracellular gate, leading us to investigate the membrane-facing fenestrations as alternative pathways for blocker access directly from the membrane. Atomistic free energy simulations showed that intracellular blockers indeed access the pore through the fenestrations, and a mutant channel with narrower fenestrations displayed no closed-state TPeA block at concentrations that blocked the wild-type channel. Apo BK channels display similar fenestrations, suggesting that blockers may use them as access paths into closed channels. Thus, membrane fenestrations represent a non-canonical pathway for selective targeting of specific channel conformations, opening novel ways to selectively drug BK channels. By solving the cryogenic electron microscopy structures of bacterial calcium-activated potassium channels, Fan et al. report a pathway for blockers to enter the closed pore of the channels through membrane portals rather than through the canonical ion entryway, opening new avenues for drug-targeting this class of channels.

Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract 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. Editor's evaluation In this study the authors aim to describe the electromechanical coupling responsible for activation of a Hyperpolarised-activated and Cyclic Nucleotide-gated (HCN) channel. HCN channels are the only mammalian channels to open under hyperpolarisation, being important for their roles in cardiac and neuronal cells. The authors use enhanced-sampling atomistic simulations to enforce sampling between open and closed states of the channel. The simulations suggest state-dependent interactions involving pore and voltage sensor helices, as well as with lipids, leading the authors to propose a domino-like mechanism of activation. These findings will be of considerable interest to the ion channel community. https://doi.org/10.7554/eLife.80303.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Hyperpolarized-activated and cyclic nucleotide-gated (HCN) channels, also known as pacemaker channels, are non-selective cation channels located at the plasma membrane of pacemaker cells in the heart and brain. These channels are crucial to control the rhythmic activity in many cell types and further regulate cellular excitability in a wide range of electrically excitable cells (Brown et al., 1979; Santoro et al., 1998). The ability to drive rhythmically firing action potentials stems from the unusual and characteristic channel activation under membrane hyperpolarization. Given their ubiquity in excitable organs and unique role in electrical signaling, HCN channels have been implicated in a variety of diseases (e.g. heart arrhythmias, epilepsies, and neuropathic pain) and are considered as promising targets for the development of novel drugs (Benarroch, 2013; Biel et al., 2009; Bleakley et al., 2021; DiFrancesco and DiFrancesco, 2015; DiFrancesco et al., 2019; Oyrer et al., 2019; Tanguay et al., 2019). HCN channels are members of the voltage-gated ion channel (VGIC) superfamily, and as such form tetrameric assemblies around a central pore that opens and closes in response to changes in the transmembrane voltage. Each monomer is composed of six transmembrane helices divided into two domains: the voltage-sensing domain (VSD) is a four-helical bundle formed by the first four helices (S1–S4) (Long et al., 2005) while the pore domain is formed by tetramerization of the final two helices (S5 and S6) around the symmetry axis (Lu et al., 2001). HCN and other VGICs are allosteric systems where movement of gating charges in the VSD upon changes in electric field governs opening and closing of the pore gate located >10 Å away at the C-terminus of the S6 helix in a process known as electromechanical coupling (Blunck and Batulan, 2012; Long et al., 2005). Interestingly, the polarity of the coupling in HCN channels is inverted relative to nearly all other VGICs; HCN channels are opened by the downward movement of the gating charges during hyperpolarization, whereas most other channels are closed (Männikkö et al., 2002). Inverted electromechanical coupling is central to the role of HCN channels in pacemaking, but the molecular mechanisms underlying inverted coupling have remained elusive. The first high-resolution structures of HCN1 revealed that, unlike canonical VGICs, HCN channels adopt a non-domain swapped architecture where the VSD packs against the pore domain of the same subunit (Lee and MacKinnon, 2017). This unusual fold is not unique to HCN channels but rather is characteristic of the subfamily of channels containing a C-terminal cyclic nucleotide-binding domain (CNBD) connected to the pore via the C-linker (James and Zagotta, 2018). Other members of this family include the depolarization-activated KCNH subfamily, such as ether-á-go-go (EAG) and human EAG-related gene (hERG), and voltage-insensitive cyclic nucleotide gated (CNG) channels (Whicher and MacKinnon, 2016; Wang and MacKinnon, 2017; Li et al., 2017). A flood of new structures in addition to the functional and computational studies they fueled have revealed new atomistic details of voltage sensing and pore gating for numerous members of the CNBD family including HCN channels. HCN1 structures were obtained in resting and activated conformations of the VSD but the pore remained closed in both models (Lee and MacKinnon, 2017; Lee and MacKinnon, 2019). The model for VSD activation from these structures agrees with predictions from cysteine accessibility (Bell et al., 2004), patch-clamp fluorometry (Dai et al., 2019), and molecular dynamics (MD) simulations (Kasimova et al., 2019). Furthermore, structures of HCN4 have been resolved in both the closed and open state, but in both cases, the VSD is in the resting conformation (Saponaro et al., 2021). Together, the HCN1 and HCN4 structures represent three out of the four states in a classical allosteric model of channel gating; the resting-closed, activated-closed, and resting-open conformations. The missing activated-open state is central to understanding the electromechanical coupling mechanism in the HCN family. Unfortunately, even with a structure of the activated-open state in hand, the weak nature of the VSD-pore coupling in these channels limits our ability to make inferences about the coupling pathway. The experimentally measured coupling energy of only 3–4.5 kcal/mol indicates that activation of all four voltage sensors only increases the stability of the open pore relative to the closed pore by roughly one hydrogen bond (Ryu and Yellen, 2012). Given this weak coupling, it is important to not only examine the static structures themselves, but also the structural ensembles and dynamics of the interactions comprising each state. In this work, we use a stepwise approach to build an understanding of the electromechanical coupling pathway in HCN1. First, we characterize state-specific interactions by analyzing MD simulations of existing cryoEM structures under equilibrium and activating conditions. We then use a small subset of the interactions likely to stabilize the activated-open channel in enhanced sampling simulations to drive the electromechanical coupling process. Gathering the results, we propose an electromechanical coupling model that relies on a fine-tuned balance between hydrophilic and hydrophobic interactions at the voltage-sensor and pore domain interface. Notably, by characterizing the state-dependent interactions between lipids and the channel domains, we propose an important role for amphiphilic membrane components, rationalizing the effect of changes in membrane composition on the function of this channel. Results State-dependent interactions between VSD and pore domain in HCN1 models In the current model for VSD activation in HCN channels, hyperpolarization triggers a downward motion of S4 coupled to a kinking of the helix at S272. This breaks S4 into two sub-helices with the lower segment becoming almost parallel to the membrane (Figure 1A and C). This non-canonical voltage sensing mechanism is supported by both experimental and computational studies combined with the cryoEM structure of the activated state stabilized by cysteine cross-linking (Lee and MacKinnon, 2017; Lee and MacKinnon, 2019; Vemana et al., 2004; Bell et al., 2004; Dai et al., 2019; Kasimova et al., 2019). An additional structure was resolved for the functionally locked-open Y289D mutant, in which the gating charges are in a resting state and the pore is closed, but the lower S5 helix swivels away from S6 relative to the position in the wild-type channel in a manner similar to the activated state structure (Figure 1B; Lee and MacKinnon, 2019). While the mechanism of VSD activation is well established, the mechanism by which this voltage sensor movement triggers pore opening remains poorly understood as the activated-open state of HCN has not been determined. Figure 1 with 1 supplement see all Download asset Open asset Position and contact network of W281. The three cryoEM structures of the resting (A; PDB ID 5U6P), intermediate (B; PDB ID 6UQG), and activated (C; PDB ID 6UQF) states of hHCN1, shown in blue, yellow, and red, respectively, were investigated by equilibrium molecular dynamics (MD) simulations. The position of W281 is highlighted in ball/stick, and transparent surface and surrounding residues are shown in ball and stick in the resting (D), intermediate (E), and activated (F) conformations. (G) Contact occurrence between W281 and nearby residues and lipid molecules along 1-μs-long MD simulations. The movement of the lower half of the S4 helix upon activation drastically alters the interaction networks between the VSD, N-terminal HCN domain, and pore (Figure 1A–C). To help understand which state-dependent interactions stabilize each of the conformations observed in cryoEM, we carried out 1-µs-MD simulations of the resting, activated, and Y289D mutant conformations under equilibrium conditions (0 mV). A comparison between structures of the resting, activated, and Y289D mutant structures (Figure 1D), preliminary exploratory MD simulations, and a putative important role of the W281-N300 contact in determining the polarity of HCN1 coupling (Ramentol et al., 2020) highlighted W281 in the lower half of S4 as a potentially critical residue in gating, prompting us to further analyze its state-dependent interaction pattern. In the resting state, W281 is facing S1 and nestled in a hydrophobic cavity formed at the interface between S4, S1, and the HCN domain (Figure 1D) and remains stably bound at this position throughout the simulation (Figure 1G, blue bars). This conformation of W281 is stabilized by extensive hydrophobic interactions with S1 and the HCN domain in addition to an electrostatic contact with S1 for >50% of the simulation time (Figure 1G). In the activated state, the intracellular S1-S4-S5 region becomes much less compact and W281 is pulled out of the hydrophobic cavity, disrupting all interactions with S1 (Figure 1F). A new interaction with the pore domain is formed via V296 in S5, but the overall protein contact of W281 is significantly reduced in the activated state. As a result, W281 primarily interacts with lipids (POPC) in the activated state through hydrophobic and electrostatic contacts. The side-chain orientation and contacts formed by W281 in the Y289D mutant retain aspects of both the resting and activated states (Figure 1E). While W281 remains close to S1, the interaction network with S1 is significantly altered relative to the resting state. Furthermore, the strong interaction with the HCN domain enclosing W281 in a hydrophobic cavity in the resting state is disrupted in the mutant. These differences stem from the rotation W281 to an orientation more similar to the activated state of the VSD. Rotation of W281 enables increased contacts with lipids and the pore domain similar to the activated state but interaction with S5 occurs via N300 in addition to the V296 contact observed in the activated state. As a result of the mixed set of W281 contacts shared with the resting and activated states, the structure of the Y289D mutant can be described as an ‘intermediate state’ conformation for VSD activation. The rotation of W281 resembles the activated state, but the interactions with S1 retain the compact nature of the S1-S4-S5 region similar to the resting state. This configuration is quite interesting as the Y289D mutant retains many characteristics of the VSD-pore interactions of the activated state despite the fact that the gating charges are in their resting position and S4 does not adopt the characteristic bend associated with voltage sensor activation. Dynamics of VSD-pore interactions upon voltage sensor activation The state-dependent VSD-pore interactions correlated with rotation of the C-terminus of S4 at W281 outlined above hint at a possible functional role in electromechanical coupling. To gain insight into the evolution of these state-dependent interactions over the course of channel activation, we simulated the resting state under a hyperpolarizing transmembrane potential. We used three new strategies in an attempt to further accelerate VSD activation compared to our previous study in hopes of driving the channel to the activated-open state. First, we started from the cAMP-bound configuration of the CNBD, which has previously been shown to allosterically promote both pore opening and VSD activation (Wu et al., 2011; Kusch et al., 2010). Next, we increased the magnitude of the membrane potential to –1 V (compared to –550 mV previously used). This should reduce the time constant for activation from 23 µs to 3 µs based on our previous kinetic analysis (Kasimova et al., 2019). Finally, we simulated an additional system lacking the HCN-domain (called hereafter HCND-less system), which has previously been suggested to inhibit VSD activation (Saponaro et al., 2021). This is consistent with our results above where the HCN domain acts as a lid enclosing W281 in a hydrophobic cavity in the resting state. In the intact system, full activation occurs in only one VSD as marked by movement of R267 past the charge transfer center (Figure 2A and B and Figure 2—figure supplement 1A). Partial activation as seen by movement of R270 past the charge transfer center occurs in one subunit while the remaining two VSDs show little to no displacement. In the subunits showing full or partial activation, S4 adopts the characteristic bend associated with channel activation observed in our previous simulations and the cryoEM structure of the cadmium-crosslinked activated state (Figure 2—figure supplement 1A). This suggests that the activation mechanism is preserved despite the high magnitude of transmembrane potential used. The incomplete activation of the voltage sensors in this system is not surprising given the length of the simulation (1 µs) compared to the predicted time constant for activation (3 µs). Figure 2 with 3 supplements see all Download asset Open asset HCN1 activation induces an increase in the lower S4/S5-lipids hydrophobic interfaces. (A, D) First and last snapshots of voltage-sensor domains (VSDs) extracted from activating simulations of intact (panel A, subunit D) or HCND-less system (panel D, subunit A). The gating charges and residues W281, Y277 (S4), N300 (S5), and F143 (S1) are highlighted using ball and stick representation. (B, E) Downward displacement of the gating charges with respect to the charge transfer center F186 during the activation of S4 in subunit D (panel B, intact HCN1 model) and subunit A (panel E, HCND-less HCN1 model) during activating simulations. (C, F) Correlation between the S4 bending angle and the rotation of W281 in subunit D (panel C, intact HCN1 model) and subunit A (panel F, HCND-less HCN1 model) during activating simulations. The time evolution is represented by a color gradient, from white/light pink to dark pink. (G) Evolution of the S4/S5 (brown) and S4/S1 (gray) hydrophobic interfaces (contact areas) along activating simulations of HCN1 in the presence (subunit D, top box) or absence (subunit A, bottom box) of the HCN domain. (H) Evolution of the number of lipid carbon atoms, N, in contact with hydrophobic residues from the lower S4/S5 interface along activating simulations of HCN1 in the presence and absence of HCN domain are shown for subunit D in light pink (top box) and for subunit A in dark pink (bottom box), respectively. N count values for the resting and activated states were averaged for the four subunits from the equilibrium simulations and are displayed as blue and red horizontal lines, respectively. (I) Contact area between lipid tails and residues on the lower S4/S5 interface, averaged over the four subunits and over the simulation time, in the resting (blue) and activated (red) states. Error bars are given as standard deviation calculated over the four subunits and over the simulation time. For the HCND-less system, one VSD undergoes full activation while the remaining three undergo partial activation over the course of a 750 ns simulation (Figure 2D and E and Figure 2—figure supplement 1B). This is consistent with an accelerated voltage sensor activation compared to the intact system expected by relieving inhibition of activation provided by the HCN domain. However, the wider distribution of bending angles in S4 for the system lacking the HCN domain indicates that the HCN domain may also contribute to stabilizing the activated state of S4 (Figure 2C and F). During VSD activation, downward movement of the gating charges and bending of S4 is correlated with rotation of the C-terminus of S4, pointing W281 toward the lipid interface (Figure 2—figure supplement 1 and Figure 2C and F). In subunits undergoing complete activation, a more complete rotation of the C-terminus of S4 results in the formation of a hydrogen bond between W281 and N300 that was observed in the Y289D mutant above (Figure 2A and D and Figure 2—figure supplement 2A and D), consistent with equilibrium simulations of the activated state of HCN1 (Figure 1—figure supplement 1B). Interestingly, swapping these two residues inverts the gating polarity in the related spHCN channel (W355N/N370W, spHCN numbering). The proposed basis of this inverted coupling was a similar rotation of the lower S4 helix as we observed, but driven by the introduction of a hydrophobic mismatch of the mutations rather than activation of the voltage sensor (Ramentol et al., 2020). In our simulations of VSD activation, rotation of W281 is accompanied by an increase in the hydrophobic contact area at the lower S4/S5 interface with little change to the electrostatic interactions (Figure 2G and Figure 2—figure supplement 2B and E), reminiscent of the behavior observed in equilibrium simulations of the resting and activated states (Figure 1—figure supplement 1A). On the other hand, analysis of the lower S4-S1 interface showed a decrease in the hydrophobic contacts between S4 and S1 in most (seven out of eight) subunits (Figure 2—figure supplement 2C and F). This suggests that separation of the S1/S4 interface is one of the first steps toward the release of W281 from the S1/S4/HCN domain hydrophobic pocket and enabling rotation of the C-terminus of S4. The combined bending and rotation of the lower S4 accompanies the displacement of S1 away from S4 and exposes the VSD-pore interface to the lipid bilayer. As a result, an increased number of contacts between lipid tails and hydrophobic residues on S4 and S5 accompanies VSD activation (Figure 2I and Figure 2—figure supplement 3). The average number of POPC carbon atoms in contact with the lower S4/S5 interface is ~24 ± 7 in the resting state versus ~40 ± 7 in the activated state equilibrium simulation. This increase in lipidic contact upon activation is preserved whether the HCN domain is present or absent. Thus, lipid tails preferentially contact the VSD/pore interface in the activated state of the voltage sensor, indicating that lipid tails may play a role in electromechanical coupling in HCN1. This may help reconcile the fact that the cryoEM structure of HCN1 with an activated voltage sensor as well as the Y289D mutant in detergent remained closed despite functional evidence suggesting that these channels should remain open at 0 mV (Lee and MacKinnon, 2019). Despite the combination of three strategies to accelerate activation, the pore gate remained shut throughout these simulations. This is unsurprising given the slow intrinsic gating of the pore and the weak electromechanical coupling in the HCN family. Even in the presence of four activated voltage sensors, the closed to open pore transition for HCN1 has a time constant of ~100 ms based on allosteric models of channel gating (Altomare et al., 2001). Consequently, the pore opening process is far outside the current range of unbiased MD simulations for HCN1. We thus set out to further analyze state-dependent interactions in an attempt to pinpoint those that may help favor pore opening, to then be able to use them in enhanced sampling simulations. Displacement of S5 during VSD activation alters interaction between S4-S5 linker and C-linker As a result of the bending and rotation of S4 during VSD activation, the N-terminal end of S5 is displaced relative to the resting state conformation in a movement that was previously observed in MD simulations and cryoEM structures. Interestingly, the N-terminus of S5 is similarly displaced in the Y289D mutant cryoEM structure despite lack of VSD activation in this channel (Figure 3A). These tilted conformations of the S5 helix are also observed in open-channel structures of related CNBD family channels like hERG and TAX-4 (Wang and MacKinnon, 2017; Zheng et al., 2020). Furthermore, this region of S5 directly contacts the S6 helix near the activation gate, thus the tilting movement of S5 following VSD activation was proposed as a critical step in the electromechanical coupling of HCN1 (Lee and MacKinnon, 2019; Kasimova et al., 2019) and experimentally shown in HCN4 (Saponaro et al., 2021). Figure 3 with 1 supplement see all Download asset Open asset Structural comparison of the S4/S5 linker and C-linker regions. (A) Structural comparison of the S4-S6 region of the resting (5U6P; blue), intermediate (6UQG; yellow), and activated (6UQF; red) states of hHCN1. Alignment was performed on the entire structure. (B) Structural comparison of the S4/S5 linker and C-linker regions of the resting, intermediate, and activated states of hHCN1. Residue K412 of the C-linker is shown as grey spheres. (C) D290-K412 distance distribution in simulations of the resting, intermediate, and activated states. Dashed lines represent distances in the starting cryoEM structures of the same color. (D) Peak tail current versus voltage plot for whole-cell recordings from HEK cells expressing HCN1 D290C/K412C with (red) or without (black) 100 uM CdCl2 in the patch pipette. Error bars represent SEM for n = 5 independent cells for each condition. In our simulations of the resting, activated, and intermediate (Y289D) structures above, we noted that tilting of S5 also alters the interaction network between the S4-S5 linker and the C-linker of the channel (Figure 3B). The C-linker directly connects the CNBD to the S6 activation gate and has previously been identified to play a role in electromechanical coupling based on functional studies (Dai et al., 2021); therefore, we suspected these activation-induced interactions may help drive channel opening. The interaction between D290 of the S4-S5 linker and K412 of the C-linker is of particular interest given the clear state-specificity to both the active and intermediate states. The absence of this interaction in the resting state stems in part from the large distance between the positions of the backbones of these residues (Figure 3B). The tilting of S5 caused by activation or the Y289D mutation brings the D290-K412 positions in close proximity and enables stable salt bridge formation. Interestingly, the D290-K412 distance in the original model of the activated VSD-closed pore conformation is close to the distance observed in the resting state structure. However, this distance decreases rapidly in the first few picoseconds of simulations of the activated VSD-closed pore structure and the resulting interaction remains stable for the majority of the trajectory (Figure 3C). We thus hypothesize this interaction is involved in coupling VSD movement to gate opening through the C-linker. Given that this interaction was not observed in the cryoEM structure of the activated VSD, we sought to experimentally validate the D290-K412 interaction in the activated-open state of the channel. Cysteine-crossbridging has been a successful approach to probe state-dependent interactions in HCN channels (Rothberg et al., 2003; Lee and MacKinnon, 2019). In this technique, pairs of residues are mutated to cysteine and then exposed to cadmium, which will form high-affinity bonds between the thiolate groups provided the distance between residues is in the range of 6–8 Å. If the suspected interaction is state-specific, it will ‘lock’ the channel in the corresponding functional state. The HEK cells expressing D290C-K412C double mutant in the absence of cadmium conduct a relatively high basal current and activate at depolarized potentials compared to the HCN1-EM parent constructs (Figure 3—figure supplement 1A). Much of this basal current can be blocked by application of external cesium chloride, indicating that the D290C-K412C mutant does not fully close even in the absence of applied cadmium (Figure 3—figure supplement 1B and C). Disrupting the D290-K412 interaction is expected to destabilize the open state based on our simulations above, in contrast to the observed rightward shift in the activation curve and increase in basal activity we observe in the D290C-K412C mutation. The enhanced apparent stability of the open state in the D290C-K412C mutant may be due to spontaneous disulfide bond formation during the patch experiment or from disruption of closed-state-specific interactions of these residues. In HEK cells patched with 100 uM cadmium chloride in the pipette, the basal conductance increases substantially to 0.77 ± 0.7 compared to 0.44 ± 0.4 in the absence of cadmium (Figure 3D). Furthermore, the voltage dependence of activation shifts by 60 mV to more positive potentials (Figure 3D). This is not observed in the wild-type channel in the presence of cadmium, which only displays a minor rightward shift in the activation curve (Figure 3—figure supplement 1A). The combined increase in the basal current amplitude and rightward shift in the voltage-dependence of activation in the double cysteine mutant in the presence of cadmium strongly indicates that crossbridging these positions preferentially stabilizes the open state, in agreement with our hypothesis that the D290-K412 interaction promotes channel opening. Driving electromechanical coupling in enhanced sampling simulations In all of our simulations described above, the S6 activation gate remained tightly closed and dehydrated for the entirety of the simulation. To gain insights into gating transitions of the HCN1 pore, we turned to enhanced sampling simulations in which pore opening was encouraged by the application of biasing forces. In previous sections, we had identified several interactions that are likely involved in stabilizing the activated voltage sensor and/or open pore. The W281-N300 pair at the S4/S5 interface stabilizes the rotation of the C-terminus of S4 on activation. Interaction between D290-K412 on the S4-S5 linker and C-linker promotes the bending of S5 away from the resting conformation, loosening the packing against the closed state of S6. In addition, a structural comparison of pairs of closed and open structures of CNBD channels revealed that distances between V390 consistently increased upon pore opening in TAX-4; this distance increases from 10.6 to 17.2 Å (PDB IDs 6WEJ-6WEK), in CNGA1 from 9.6 to 16.4 Å (PDB IDs 7LFT-7LFW), in CNGA1/B1 from 9.8 to 15.1 Å (PDB IDs 7RH9-7RHH), in EAG/hERG from 10.7 to 17.3 Å (PDB IDs 5KL7L-5VA1), and in HCN4 from 10.5 to 15.0 Å (PDB IDs 7NP4-7NP3). Using these state-specific distances as collective variables (CVs) in enhanced sampling MD simulations (Figure 4A), we hypothesized that we could drive the electromechanical coupling process in a computationally accessible timescale. Figure 4 with 4 supplements see all Download asset Open asset Pore opening of HCN1. (A) Residues and distances used as collective variables in adiabatic bias molecular dynamics (ABMD) simulations are highlighted by ball and stick and black double arrows, respectively. (B) Inter-subunit distances between opposite V390 residues are represented as

On behalf of the Australian Society for Biophysics (ASB) and the Editors of this Special Issue, I would like to express our appreciation to Editor-in-Chief, Damien Hall, for arranging the publication of this Special Issue. The ASB is about five times smaller than our sister the Biophysical Society for Japan (BSJ) and tenfold smaller than the US Biophysical Society (USBS), but our meetings are notable because of the encouragement the Society gives to emerging biophysicists. It can be a terrifying experience for a PhD student to have to face a roomful of professors and senior academics, but invariably they appreciate the experience. Another feature of the ASB meetings is the inclusion of contributions from the Asian Pacific region. We now have formal ties with our New Zealand colleagues and our meetings with the BSJ contain joint sessions (see below). In 2020, despite the impact of COVID-19 (see Adam Hill's Commentary), there is a joint session with the University of California Davis. This Special Issue comprises 2 Editorials, 3 Commentaries, and 25 reviews.

Despite the progress in modeling complex molecular systems of ever-increasing complexity, a quantitatively accurate computational treatment of ion permeation through narrow membrane channels remains challenging. An important factor to reach this goal is induced electronic polarization, which is likely to impact the permeation rate of small ions through narrow molecular pores. In this work, we extended the recently developed polarizable force field based on the classical Drude oscillators to assess the role of induced polarization effects on the energetics of sodium and potassium ion transport across the gramicidin A (gA) ion channel. The inclusion of induced polarization lowers barriers present in 1D potential of mean force (PMF) for cation permeation by ∼50% compared to those obtained with the additive force field. Conductance properties calculated with 1D PMFs from Drude simulations are in better agreement with experimental results. Polarization of single-file water molecules and protein atoms forming the narrow pore has a direct impact on the free-energy barriers and cation-specific solid-state NMR chemical shifts. Sensitivity analysis indicates that small changes to water-channel interactions can alter the free energy barrier for ion permeation. These results, illustrating polarization effects present in the complex electrostatic environment of the gA channel, have broad implications for revising proposed mechanisms of ion permeation and selectivity in a variety of ion channels.

The human ether á-go-go related gene (hERG) encodes the delayed rectifier potassium channel (Kv11.1), best known for mediation of the repolarising current in the cardiac action potential. Loss of function of hERG can lead to long QT-syndrome which causes cardiac arrhythmias and sudden cardiac arrest. hERG is susceptible to a wide range of drugs which can lead to drug induced long QT-syndrome. Here, the recent cryo-EM structure of hERG in an activated state, together with newly-solved structures that are open or inactivated serve as the basis to investigate the structural determinants of hERG inactivation. We used molecular dynamics flexible fitting to refine and relax these new structures in micelles consisting of detergents, lipids and cholesterol. A combination of long simulations and libraries of short simulations were performed with a variety of ion concentrations to study different conformational states. These simulations highlighted the amino acids important for maintaining each state of the selectivity filter and therefore may be involved in hERG inactivation. Furthermore, we have used replica exchange with solute tempering to enhance sampling of the exchange of ions in the hERG selectivity filter. We observe correlations between channel conformation and ion occupancy that shed light on the coupling of ions to inactivation. Together with mutagenesis experiments, these simulations provide molecular-level understanding of hERG inactivation, with considerable clinical and pharmacological potential.

This Commentary describes a call for submissions for the upcoming Special Issue focused on the research topics presented at the Australian Society of Biophysics (ASB) in 2020 and 2021. Submissions from past and present ASB members who could not attend these meetings are also welcome as contributions to this special issue.

Different mechanisms have been proposed to explain the permeation of charged compounds through lipid membranes. Overall, it is expected that an ion-induced defect permeation mechanism, where substantial membrane deformations accompany ion movement, should be dominant in thin membranes but that a solubility-diffusion mechanism, where ions partition into the membrane core with large associated dehydration energy costs, becomes dominant in thicker membranes. However, while this physical picture is intuitively reasonable, capturing the interconversion between these two permeation mechanisms in molecular dynamics (MD) simulations based on atomic models is challenging. In particular, simulations relying on nonpolarizable force fields are artificially unfavorable to the solubility-diffusion mechanism, as induced polarization of the nonpolar hydrocarbon is ignored, causing overestimated free energy costs for charged molecules to enter into this region of the membrane. In this study, all-atom MD simulations based on nonpolarizable and polarizable force fields are used to quantitatively characterize the permeation process for the arginine side chain analog methyl-guanidinium through bilayer membranes of mono-unsaturated phosphatidylcholine lipids with and without cholesterol, resulting in thicknesses spanning from ∼24 to ∼42 Å. With simulations based on a nonpolarizable force field, ion translocation can take place solely through an ion-induced defect mechanism, with free energy barriers increasing linearly from 14 to 40 kcal/mol, depending on the thickness. However, with simulations based on a polarizable force field, ion translocation is predominantly dominated by an ion-induced defect mechanism in thin membranes, which progressively converts to a solubility-diffusion mechanism as the membranes get thicker. The transition between the two mechanisms occurs at a thickness of ∼29 Å, with lipid tails of 22 or more carbon atoms. This situation appears to represent the upper limit for ion-induced defect permeation within the current polarizable models. Beyond this thickness, it becomes energetically preferable for the ion to dehydrate and partition into the membrane core-a phenomenon that cannot be captured using the nonpolarizable models. Induced electronic polarizability therefore leads not just to a shift in permeation energetics but to an interconversion between two strikingly different physical mechanisms. The result highlights the importance of induced polarizability in modeling lipid membranes.

Quaternary ammonium (QA) compounds have been traditionally used as probes to understand the pore structure and the gate locations in ion channels. Previous reports found that certain QA compounds can bind to the large pore cavity to block not only the open but also the closed states of BK and MthK potassium channels. These results were interpreted to suggest that the intracellular constriction does not constitute an entry barrier to ions in the closed state and that the permeation gates of BK and MthK channels are located at the selectivity filter, unlike those for Shaker-like voltage-dependent potassium channels, whose gates are at the canonical bundle-crossing location. Recently, the structures of MthK in the calcium-free and calcium-bound states, corresponding to closed and open, respectively, were solved using single-particle cryo-EM. These structures indicate that in closed MthK, the intracellular bundle-crossing is constricted enough that ions cannot squeeze through, suggesting that at least one of the gates for permeation is indeed at the intracellular bundle crossing, contrary to previous reports. This left the question of how QA blockers succeed to access their binding sites in the closed pore of MthK. We investigated this issue using structural and molecular dynamics approaches. We found that QA blockers are indeed bound to the closed MthK structure, and we propose that they access their binding site via a pathway different from the canonical bundle-crossing entrance to the pore from the intracellular side.