SnapShot: Channel Gating Mechanisms.

Ion channel families are broadly classified into three types according to their major mechanisms of activation. This SnapShot highlights features of these three classes and conveys general modes of channel regulation. To view this SnapShot, open or download the PDF.

A molecular framework for temperature-dependent gating of ion channels.

Perception of heat or cold in higher organisms is mediated by specialized ion channels whose gating is exquisitely sensitive to temperature. The physicochemical underpinnings of this temperature-sensitive gating have proven difficult to parse. Here, we took a bottom-up protein design approach and rationally engineered ion channels to activate in response to thermal stimuli. By varying amino acid polarities at sites undergoing state-dependent changes in solvation, we were able to systematically confer temperature sensitivity to a canonical voltage-gated ion channel. Our results imply that the specific heat capacity change during channel gating is a major determinant of thermosensitive gating. We also show that reduction of gating charges amplifies temperature sensitivity of designer channels, which accounts for low-voltage sensitivity in all known temperature-gated ion channels. These emerging principles suggest a plausible molecular mechanism for temperature-dependent gating that reconcile how ion channels with an overall conserved transmembrane architecture may exhibit a wide range of temperature-sensing phenotypes. :

cAMP binding to closed pacemaker ion channels is non-cooperative.

Electrical activity in the brain and heart depends on rhythmic generation of action potentials by pacemaker ion channels (HCN) whose activity is regulated by cAMP binding1. Previous work has uncovered evidence for both positive and negative cooperativity in cAMP binding2,3, but such bulk measurements suffer from limited parameter resolution. Efforts to eliminate this ambiguity using single-molecule techniques have been hampered by the inability to directly monitor binding of individual ligand molecules to membrane receptors at physiological concentrations. Here we overcome these challenges using nanophotonic zero-mode waveguides4 to directly resolve binding dynamics of individual ligands to multimeric HCN1 and HCN2 ion channels. We show that cAMP binds independently to all four subunits when the pore is closed, despite a subsequent conformational isomerization to a flip state at each site. The different dynamics in binding and isomerization are likely to underlie physiologically distinct responses of each isoform to cAMP5 and provide direct validation of the ligand-induced flip-state model6-9. This approach for observing stepwise binding in multimeric proteins at physiologically relevant concentrations can directly probe binding allostery at single-molecule resolution in other intact membrane proteins and receptors.

Mapping the contribution of the C-linker domain to gating polarity in CNBD channels.

Ion channels of the cyclic nucleotide-binding domain (CNBD) family play a crucial role in the regulation of key biological processes, such as photoreception and pacemaking activity in the heart. These channels exhibit high sequence and structural similarity but differ greatly in their functional responses to membrane potential. The CNBD family includes hyperpolarization-activated ion channels and depolarization-activated ether-à-go-go channels. Structural and functional studies show that the differences in the coupling interface between these two subfamilies' voltage-sensing domain and pore domain may underlie their differential response to membrane polarity. However, other structural components may also contribute to defining the polarity differences in activation. Here, we focus on the role of the C-terminal domain, which interacts with elements in both the pore and voltage-sensing domains. By generating a series of chimeras involving the C-terminal domain derived from distant members of the CNBD family, we find that the nature of the C-termini profoundly influences the gating polarity of these ion channels. Scanning mutagenesis of the C-linker region, a helix-turn-helix motif connecting the pore helix to the CNBD, reveals that residues at the intersubunit interface between the C-linkers are crucial for hyperpolarization-dependent activation. These findings highlight the unique and unexpected role of the intersubunit interface of the C-linker region in regulating the gating polarity of voltage-gated ion channels.

Dynamics and number of trans-SNARE complexes determine nascent fusion pore properties.

The fusion pore is the first crucial intermediate formed during exocytosis, yet little is known about the mechanisms that determine the size and kinetic properties of these transient structures. Here, we reduced the number of available SNAREs (proteins that mediate vesicle fusion) in neurons and observed changes in transmitter release that are suggestive of alterations in fusion pores. To investigate these changes, we employed reconstituted fusion assays using nanodiscs to trap pores in their initial open state. Optical measurements revealed that increasing the number of SNARE complexes enhanced the rate of release from single pores and enabled the escape of larger cargoes. To determine whether this effect was due to changes in nascent pore size or to changes in stability, we developed an approach that uses nanodiscs and planar lipid bilayer electrophysiology to afford microsecond resolution at the single event level. Both pore size and stability were affected by SNARE copy number. Increasing the number of vesicle (v)-SNAREs per nanodisc from three to five caused a twofold increase in pore size and decreased the rate of pore closure by more than three orders of magnitude. Moreover, pairing of v-SNAREs and target (t)-SNAREs to form trans-SNARE complexes was highly dynamic: flickering nascent pores closed upon addition of a v-SNARE fragment, revealing that the fully assembled, stable SNARE complex does not form at this stage of exocytosis. Finally, a deletion at the base of the SNARE complex, which mimics the action of botulinum neurotoxin A, markedly reduced fusion pore stability. In summary, trans-SNARE complexes are dynamic, and the number of SNAREs recruited to drive fusion determines fundamental properties of individual pores.

Mapping temperature-dependent conformational change in the voltage-sensing domain of an engineered heat-activated K+ channel.

Temperature-dependent regulation of ion channel activity is critical for a variety of physiological processes ranging from immune response to perception of noxious stimuli. Our understanding of the structural mechanisms that underlie temperature sensing remains limited, in part due to the difficulty of combining high-resolution structural analysis with temperature stimulus. Here, we use NMR to compare the temperature-dependent behavior of Shaker potassium channel voltage sensor domain (WT-VSD) to its engineered temperature sensitive (TS-VSD) variant. Further insight into the molecular basis for temperature-dependent behavior is obtained by analyzing the experimental results together with molecular dynamics simulations. Our studies reveal that the overall secondary structure of the engineered TS-VSD is identical to the wild-type channels except for local changes in backbone torsion angles near the site of substitution (V369S and F370S). Remarkably however, these structural differences result in increased hydration of the voltage-sensing arginines and the S4-S5 linker helix in the TS-VSD at higher temperatures, in contrast to the WT-VSD. These findings highlight how subtle differences in the primary structure can result in large-scale changes in solvation and thereby confer increased temperature-dependent activity beyond that predicted by linear summation of solvation energies of individual substituents.

Acylated and alkylated benzo(crown-ethers) form ion-dependent ion channels in biological membranes.

Synthetic ion channels based on benzo(crown-ether) compounds have been previously reported to function as ion-selective channels in planar lipid bilayers, with hydrogen bonding networks implicated in the formation of self-aggregated complexes. Herein, we report the synthesis and characterization of two new families of benzo(crown-ether) compounds, termed monoacylated and monoalkylated benzo(crown-ethers) (MABCE), both of which lack hydrogen bond donors. Depending on the length of alkyl chain substituent and the size of macrocycle, MABCE compounds inhibit bacterial growth and transport ions across biological membranes. Single-channel recordings show that the activity is higher in the presence of K+ as compared with Na+; however, under bionic conditions, open channels do not exhibit any preference between the two ions. These findings reveal that the ionic preference of benzo(crown-ether) compounds is either due to the regulation of assembly of ion-conducting supramolecular complexes or its membrane insertion by cations, as opposed to ion-selective transport through these scaffolds. Furthermore, our data show that the H-bonding network is not needed to form these assemblies in the membrane.

Bipolar switching by HCN voltage sensor underlies hyperpolarization activation.

Despite sharing a common architecture with archetypal voltage-gated ion channels (VGICs), hyperpolarization- and cAMP-activated ion (HCN) channels open upon hyperpolarization rather than depolarization. The basic motions of the voltage sensor and pore gates are conserved, implying that these domains are inversely coupled in HCN channels. Using structure-guided protein engineering, we systematically assembled an array of mosaic channels that display the full complement of voltage-activation phenotypes observed in the VGIC superfamily. Our studies reveal that the voltage sensor of the HCN channel has an intrinsic ability to drive pore opening in either direction and that the extra length of the HCN S4 is not the primary determinant for hyperpolarization activation. Tight interactions at the HCN voltage sensor-pore interface drive the channel into an hERG-like inactivated state, thereby obscuring its opening upon depolarization. This structural element in synergy with the HCN cyclic nucleotide-binding domain and specific interactions near the pore gate biases the channel toward hyperpolarization-dependent opening. Our findings reveal an unexpected common principle underpinning voltage gating in the VGIC superfamily and identify the essential determinants of gating polarity.

NMR Structural Analysis of Isolated Shaker Voltage-Sensing Domain in LPPG Micelles.

The voltage-sensing domain (VSD) is a conserved structural module that regulates the gating of voltage-dependent ion channels in response to a change in membrane potential. Although the structures of many VSD-containing ion channels are now available, our understanding of the structural dynamics associated with gating transitions remains limited. To probe dynamics with site-specific resolution, we utilized NMR spectroscopy to characterize the VSD derived from Shaker potassium channel in 1-palmitoyl-2-hydroxy-sn-glycero-3-phospho-(1'-rac-glycerol) (LPPG) micelles. The backbone dihedral angles predicted based on secondary chemical shifts using torsion angle likeliness obtained from shift (TALOS+) showed that the Shaker-VSD shares many structural features with the homologous Kv1.2/2.1 chimera, including a transition from α-helix to 310 helix in the C-terminal portion of the fourth transmembrane helix. Nevertheless, there are clear differences between the Shaker-VSD and Kv1.2/2.1 chimera in the S2-S3 linker and S3 transmembrane region, where the organization of secondary structure elements in Shaker-VSD appears to more closely resemble the KvAP-VSD. Comparison of microsecond-long molecular dynamics simulations of Kv 1.2-VSD in LPPG micelles and a 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) bilayer showed that LPPG micelles do not induce significant structural distortion in the isolated voltage sensor. To assess the integrity of the tertiary fold, we directly probed the binding of BrMT analog 2-[2-({[3-(2-amino-ethyl)-6-bromo-1H-indol-2-yl]methoxy}k7methyl)-6-bromo-1H-indol-3-yl]ethan-1-amine (BrET), a gating modifier toxin, and identified the location of the putative binding site. Our results suggest that the Shaker-VSD in LPPG micelles is in a native-like fold and is likely to provide valuable insights into the dynamics of voltage-gating and its regulation.

Observing Single-Molecule Dynamics at Millimolar Concentrations.

Single-molecule fluorescence microscopy is a powerful tool for revealing chemical dynamics and molecular association mechanisms, but has been limited to low concentrations of fluorescent species and is only suitable for studying high affinity reactions. Here, we combine nanophotonic zero-mode waveguides (ZMWs) with fluorescence resonance energy transfer (FRET) to resolve single-molecule association dynamics at up to millimolar concentrations of fluorescent species. This approach extends the resolution of molecular dynamics to >100-fold higher concentrations, enabling observations at concentrations relevant to biological and chemical processes, and thus making single-molecule techniques applicable to a tremendous range of previously inaccessible molecular targets. We deploy this approach to show that the binding of cGMP to pacemaking ion channels is weakened by a slower internal conformational change.

Gating transitions in the selectivity filter region of a sodium channel are coupled to the domain IV voltage sensor.

Voltage-dependent ion channels are crucial for generation and propagation of electrical activity in biological systems. The primary mechanism for voltage transduction in these proteins involves the movement of a voltage-sensing domain (D), which opens a gate located on the cytoplasmic side. A distinct conformational change in the selectivity filter near the extracellular side has been implicated in slow inactivation gating, which is important for spike frequency adaptation in neural circuits. However, it remains an open question whether gating transitions in the selectivity filter region are also actuated by voltage sensors. Here, we examine conformational coupling between each of the four voltage sensors and the outer pore of a eukaryotic voltage-dependent sodium channel. The voltage sensors of these sodium channels are not structurally symmetric and exhibit functional specialization. To track the conformational rearrangements of individual voltage-sensing domains, we recorded domain-specific gating pore currents. Our data show that, of the four voltage sensors, only the domain IV voltage sensor is coupled to the conformation of the selectivity filter region of the sodium channel. Trapping the outer pore in a particular conformation with a high-affinity toxin or disulphide crossbridge impedes the return of this voltage sensor to its resting conformation. Our findings directly establish that, in addition to the canonical electromechanical coupling between voltage sensor and inner pore gates of a sodium channel, gating transitions in the selectivity filter region are also coupled to the movement of a voltage sensor. Furthermore, our results also imply that the voltage sensor of domain IV is unique in this linkage and in the ability to initiate slow inactivation in sodium channels.

Probing gating mechanisms of sodium channels using pore blockers.

Several classes of small molecules and peptides bind at the central pore of voltage-gated sodium channels either from the extracellular or intracellular side of the membrane and block ion conduction through the pore. Biophysical studies that shed light on the chemical nature, accessibility, and kinetics of binding of these naturally occurring and synthetic compounds reveal a wealth of information about how these channels gate. Here, we discuss insights into the structural underpinnings of gating of the channel pore and its coupling to the voltage sensors obtained from pore blockers including site 1 neurotoxins and local anesthetics.

Structural basis for hyperpolarization-dependent opening of human HCN1 channel.

Hyperpolarization and cyclic nucleotide (HCN) activated ion channels are critical for the automaticity of action potentials in pacemaking and rhythmic electrical circuits in the human body. Unlike most voltage-gated ion channels, the HCN and related plant ion channels activate upon membrane hyperpolarization. Although functional studies have identified residues in the interface between the voltage-sensing and pore domain as crucial for inverted electromechanical coupling, the structural mechanisms for this unusual voltage-dependence remain unclear. Here, we present cryo-electron microscopy structures of human HCN1 corresponding to Closed, Open, and a putative Intermediate state. Our structures reveal that the downward motion of the gating charges past the charge transfer center is accompanied by concomitant unwinding of the inner end of the S4 and S5 helices, disrupting the tight gating interface observed in the Closed state structure. This helix-coil transition at the intracellular gating interface accompanies a concerted iris-like dilation of the pore helices and underlies the reversed voltage dependence of HCN channels.

WITHDRAWN: Caveolar Compartmentalization is Required for Stable Rhythmicity of Sinus Nodal Cells and is Disrupted in Heart Failure.

The authors have withdrawn their manuscript owing to technical concerns merged during peer review. Therefore, the authors do not wish this work to be cited as a reference. If you have any questions, please contact the corresponding author.

Tethered spectroscopic probes estimate dynamic distances with subnanometer resolution in voltage-dependent potassium channels.

Measurements of inter- and intramolecular distances are important for monitoring structural changes and understanding protein interaction networks. Fluorescence resonance energy transfer and functionalized chemical spacers are the two predominantly used strategies to map short-range distances in living cells. Here, we describe the development of a hybrid approach that combines the key advantages of spectroscopic and chemical methods to estimate dynamic distance information from labeled proteins. Bifunctional spectroscopic probes were designed to make use of adaptable-anchor and length-varied spacers to estimate molecular distances by exploiting short-range collisional electron transfer. The spacers were calibrated using labeled polyproline peptides of defined lengths and validated by molecular simulations. This approach was extended to estimate distance restraints that enable us to evaluate the resting-state model of the Shaker potassium channel.

Deconstructing thermodynamic parameters of a coupled system from site-specific observables.

Cooperative interactions mediate information transfer between structural domains of a protein molecule and are major determinants of protein function and modulation. The prevalent theories to understand the thermodynamic origins of cooperativity have been developed to reproduce the complex behavior of a global thermodynamic observable such as ligand binding or enzyme activity. However, in most cases the measurement of a single global observable cannot uniquely define all the terms that fully describe the energetics of the system. Here we establish a theoretical groundwork for analyzing protein thermodynamics using site-specific information. Our treatment involves extracting a site-specific parameter (defined as χ value) associated with a structural unit. We demonstrate that, under limiting conditions, the χ value is related to the direct interaction terms associated with the structural unit under observation and its intrinsic activation energy. We also introduce a site-specific interaction energy term (χ(diff)) that is a function of the direct interaction energy of that site with every other site in the system. When combined with site-directed mutagenesis and other molecular level perturbations, analyses of χ values of site-specific observables may provide valuable insights into protein thermodynamics and structure.

Charge-voltage curves of Shaker potassium channel are not hysteretic at steady state.

Charge-voltage curves of many voltage-gated ion channels exhibit hysteresis but such curves are also a direct measure of free energy of channel gating and, hence, should be path-independent. Here, we identify conditions to measure steady-state charge-voltage curves and show that these are curves are not hysteretic. Charged residues in transmembrane segments of voltage-gated ion channels (VGICs) sense and respond to changes in the electric field. The movement of these gating charges underpins voltage-dependent activation and is also a direct metric of the net free-energy of channel activation. However, for most voltage-gated ion channels, the charge-voltage (Q-V) curves appear to be dependent on initial conditions. For instance, Q-V curves of Shaker potassium channel obtained by hyperpolarizing from 0 mV is left-shifted compared to those obtained by depolarizing from a holding potential of -80 mV. This hysteresis in Q-V curves is a common feature of channels in the VGIC superfamily and raises profound questions about channel energetics because the net free-energy of channel gating is a state function and should be path independent. Due to technical limitations, conventional gating current protocols are limited to test pulse durations of <500 ms, which raises the possibility that the dependence of Q-V on initial conditions reflects a lack of equilibration. Others have suggested that the hysteresis is fundamental thermodynamic property of voltage-gated ion channels and reflects energy dissipation due to measurements under non-equilibrium conditions inherent to rapid voltage jumps (Villalba-Galea. 2017. Channels. https://doi.org/10.1080/19336950.2016.1243190). Using an improved gating current and voltage-clamp fluorometry protocols, we show that the gating hysteresis arising from different initial conditions in Shaker potassium channel is eliminated with ultra-long (18-25 s) test pulses. Our study identifies a modified gating current recording protocol to obtain steady-state Q-V curves of a voltage-gated ion channel. Above all, these findings demonstrate that the gating hysteresis in Shaker channel is a kinetic phenomenon rather than a true thermodynamic property of the channel and the charge-voltage curve is a true measure of the net-free energy of channel gating.

About hysteresis in Shaker: Response to note by Villalba-Galea.

In this issue, Villalba-Galea (2023. J. Gen. Physiol.https://doi.org/10.1085/jgp.202313371) expresses interest in our recently published work (Cowgill and Chanda. 2023. J. Gen. Physiol.https://doi.org/10.1085/jgp.202112883). Our response points out the deficiencies in the alternative explanation proposed by Villalba-Galea to account for our findings on hysteresis (or lack thereof) in steady state charge-voltage curves of Shaker potassium channel.

Strategies for Overcoming the Single-Molecule Concentration Barrier.

Fluorescence-based single-molecule approaches have helped revolutionize our understanding of chemical and biological mechanisms. Unfortunately, these methods are only suitable at low concentrations of fluorescent molecules so that single fluorescent species of interest can be successfully resolved beyond background signal. The application of these techniques has therefore been limited to high-affinity interactions despite most biological and chemical processes occurring at much higher reactant concentrations. Fortunately, recent methodological advances have demonstrated that this concentration barrier can indeed be broken, with techniques reaching concentrations as high as 1 mM. The goal of this Review is to discuss the challenges in performing single-molecule fluorescence techniques at high-concentration, offer applications in both biology and chemistry, and highlight the major milestones that shatter the concentration barrier. We also hope to inspire the widespread use of these techniques so we can begin exploring the new physical phenomena lying beyond this barrier.