GABAA receptor subunit M2-M3 linkers have asymmetric roles in pore gating and diazepam modulation.

GABAA receptors (GABAARs) are neurotransmitter-gated ion channels critical for inhibitory synaptic transmission as well as the molecular target for benzodiazepines (BZDs), one of the most widely prescribed class of psychotropic drugs today. Despite structural insight into the conformations underlying functional channel states, the detailed molecular interactions involved in conformational transitions and the physical basis for their modulation by BZDs are not fully understood. We previously identified that alanine substitution at the central residue in the α1 subunit M2-M3 linker (V279A) enhances the efficiency of linkage between the BZD site and the pore gate. Here, we expand on this work by investigating the effect of alanine substitutions at the analogous positions in the M2-M3 linkers of ß2 (I275A) and γ2 (V290A) subunits, which together with α1 comprise typical heteromeric α1ß2γ2 synaptic GABAARs. We find that these mutations confer subunit-specific effects on the intrinsic pore closed-open equilibrium and its modulation by the BZD diazepam (DZ). The mutations α1(V279A) or γ2(V290A) bias the channel toward a closed conformation, whereas ß2(I275A) biases the channel toward an open conformation to the extent that the channel becomes leaky and opens spontaneously in the absence of agonist. In contrast, only α1(V279A) enhances the efficiency of DZ-to-pore linkage, whereas mutations in the other two subunits have no effect. These observations show that the central residue in the M2-M3 linkers of distinct subunits in synaptic α1ß2γ2 GABAARs contribute asymmetrically to the intrinsic closed-open equilibrium and its modulation by DZ.

Zinc activation of OTOP proton channels identifies structural elements of the gating apparatus.

Otopetrin proteins (OTOPs) form proton-selective ion channels that are expressed in diverse cell types where they mediate detection of acids or regulation of pH. In vertebrates there are three family members: OTOP1 is required for formation of otoconia in the vestibular system and it forms the receptor for sour taste, while the functions of OTOP2 and OTOP3 are not yet known. Importantly, the gating mechanisms of any of the OTOP channels are not well understood. Here, we show that zinc (Zn2+), as well as other transition metals including copper (Cu2+), potently activates murine OTOP3 (mOTOP3). Zn2+ pre-exposure increases the magnitude of mOTOP3 currents to a subsequent acid stimulus by as much as 10-fold. In contrast, mOTOP2 currents are insensitive to activation by Zn2+. Swapping the extracellular tm 11-12 linker between mOTOP3 and mOTOP2 was sufficient to eliminate Zn2+ activation of mOTOP3 and confer Zn2+ activation on mOTOP2. Mutation to alanine of H531 and E535 within the tm 11-12 linker and H234 and E238 within the 5-6 linker reduced or eliminated activation of mOTOP3 by Zn2+, indicating that these residues likely contribute to the Zn2+ activating site. Kinetic modeling of the data is consistent with Zn2+ stabilizing the opn2+en state of the channel, competing with H+ for activation of the channels. These results establish the tm 11-12 and tm 5-6 linkers as part of the gating apparatus of OTOP channels and a target for drug discovery. Zn2+ is an essential micronutrient and its activation of OTOP channels will undoubtedly have important physiological sequelae.

Benzodiazepine Modulation of GABAA Receptors: A Mechanistic Perspective.

Benzodiazepines (BZDs) are a class of widely prescribed psychotropic drugs that target GABAA receptors (GABAARs) to tune inhibitory synaptic signaling throughout the central nervous system. Despite knowing their molecular target for over 40 years, we still do not fully understand the mechanism of modulation at the level of the channel protein. Nonetheless, functional studies, together with recent cryo-EM structures of GABAA(α1)2(ßX)2(γ2)1 receptors in complex with BZDs, provide a wealth of information to aid in addressing this gap in knowledge. Here, mechanistic interpretations of functional and structural evidence for the action of BZDs at GABAA(α1)2(ßX)2(γ2)1 receptors are reviewed. The goal is not to describe each of the many studies that are relevant to this discussion nor to dissect in detail all the effects of individual mutations or perturbations but rather to highlight general mechanistic principles in the context of recent structural information.

Single-molecule imaging with cell-derived nanovesicles reveals early binding dynamics at a cyclic nucleotide-gated ion channel.

Ligand binding to membrane proteins is critical for many biological signaling processes. However, individual binding events are rarely directly observed, and their asynchronous dynamics are occluded in ensemble-averaged measures. For membrane proteins, single-molecule approaches that resolve these dynamics are challenged by dysfunction in non-native lipid environments, lack of access to intracellular sites, and costly sample preparation. Here, we introduce an approach combining cell-derived nanovesicles, microfluidics, and single-molecule fluorescence colocalization microscopy to track individual binding events at a cyclic nucleotide-gated TAX-4 ion channel critical for sensory transduction. Our observations reveal dynamics of both nucleotide binding and a subsequent conformational change likely preceding pore opening. Kinetic modeling suggests that binding of the second ligand is either independent of the first ligand or exhibits up to ~10-fold positive binding cooperativity. This approach is broadly applicable to studies of binding dynamics for proteins with extracellular or intracellular domains in native cell membrane.

Unsupervised selection of optimal single-molecule time series idealization criterion.

Single-molecule (SM) approaches have provided valuable mechanistic information on many biophysical systems. As technological advances lead to ever-larger data sets, tools for rapid analysis and identification of molecules exhibiting the behavior of interest are increasingly important. In many cases the underlying mechanism is unknown, making unsupervised techniques desirable. The divisive segmentation and clustering (DISC) algorithm is one such unsupervised method that idealizes noisy SM time series much faster than computationally intensive approaches without sacrificing accuracy. However, DISC relies on a user-selected objective criterion (OC) to guide its estimation of the ideal time series. Here, we explore how different OCs affect DISC's performance for data typical of SM fluorescence imaging experiments. We find that OCs differing in their penalty for model complexity each optimize DISC's performance for time series with different properties such as signal/noise and number of sample points. Using a machine learning approach, we generate a decision boundary that allows unsupervised selection of OCs based on the input time series to maximize performance for different types of data. This is particularly relevant for SM fluorescence data sets, which often have signal/noise near the derived decision boundary and include time series of nonuniform length because of stochastic bleaching. Our approach, AutoDISC, allows unsupervised per-molecule optimization of DISC, which will substantially assist in the rapid analysis of high-throughput SM data sets with noisy samples and nonuniform time windows.

A critical residue in the α1M2-M3 linker regulating mammalian GABAA receptor pore gating by diazepam.

Benzodiazepines (BZDs) are a class of widely prescribed psychotropic drugs that modulate activity of GABAA receptors (GABAARs), neurotransmitter-gated ion channels critical for synaptic transmission. However, the physical basis of this modulation is poorly understood. We explore the role of an important gating domain, the α1M2-M3 linker, in linkage between the BZD site and pore gate. To probe energetics of this coupling without complication from bound agonist, we use a gain of function mutant (α1L9'Tß2γ2L) directly activated by BZDs. We identify a specific residue whose mutation (α1V279A) more than doubles the energetic contribution of the BZD positive modulator diazepam (DZ) to pore opening and also enhances DZ potentiation of GABA-evoked currents in a wild-type background. In contrast, other linker mutations have little effect on DZ efficiency, but generally impair unliganded pore opening. Our observations reveal an important residue regulating BZD-pore linkage, thereby shedding new light on the molecular mechanism of these drugs.

Top-down machine learning approach for high-throughput single-molecule analysis.

Single-molecule approaches provide enormous insight into the dynamics of biomolecules, but adequately sampling distributions of states and events often requires extensive sampling. Although emerging experimental techniques can generate such large datasets, existing analysis tools are not suitable to process the large volume of data obtained in high-throughput paradigms. Here, we present a new analysis platform (DISC) that accelerates unsupervised analysis of single-molecule trajectories. By merging model-free statistical learning with the Viterbi algorithm, DISC idealizes single-molecule trajectories up to three orders of magnitude faster with improved accuracy compared to other commonly used algorithms. Further, we demonstrate the utility of DISC algorithm to probe cooperativity between multiple binding events in the cyclic nucleotide binding domains of HCN pacemaker channel. Given the flexible and efficient nature of DISC, we anticipate it will be a powerful tool for unsupervised processing of high-throughput data across a range of single-molecule experiments.

During a chemical or biological process, a molecule may transition through a series of states, many of which are rare or short-lived. Advances in technology have made it easier to detect these states by gathering large amounts of data on individual molecules. However, the increasing size of these datasets has put a strain on the algorithms and software used to identify different molecular states. Now, White et al. have developed a new algorithm called DISC which overcomes this technical limitation. Unlike most other algorithms, DISC requires minimal input from the user and uses a new method to group the data into categories that represent distinct molecular states. Although this new approach produces a similar end-result, it reaches this conclusion much faster than more commonly used algorithms. To test the effectiveness of the algorithm, White et al. studied how individual molecules of a chemical known as cAMP bind to parts of proteins called cyclic nucleotide binding domains (or CNDBs for short). A fluorescent tag was attached to single molecules of cAMP and data were collected on the behavior of each molecule. Previous evidence suggested that when four CNDBs join together to form a so-called tetramer complex, this affects the binding of cAMP. Using the DISC system, White et al. showed that individual cAMP molecules interact with all four domains in a similar way, suggesting that the binding of cAMP is not impacted by the formation of a tetramer complex. Analyzing this data took DISC less than 20 minutes compared to existing algorithms which took anywhere between four hours and two weeks to complete. The enhanced speed of the DISC algorithm could make it easier to analyze much larger datasets from other techniques in addition to fluorescence. This means that a greater number of states can be sampled, providing a deeper insight into the inner workings of biological and chemical processes.

Batrachotoxin acts as a stent to hold open homotetrameric prokaryotic voltage-gated sodium channels.

Batrachotoxin (BTX), an alkaloid from skin secretions of dendrobatid frogs, causes paralysis and death by facilitating activation and inhibiting deactivation of eukaryotic voltage-gated sodium (Nav) channels, which underlie action potentials in nerve, muscle, and heart. A full understanding of the mechanism by which BTX modifies eukaryotic Nav gating awaits determination of high-resolution structures of functional toxin-channel complexes. Here, we investigate the action of BTX on the homotetrameric prokaryotic Nav channels NaChBac and NavSp1. By combining mutational analysis and whole-cell patch clamp with molecular and kinetic modeling, we show that BTX hinders deactivation and facilitates activation in a use-dependent fashion. Our molecular model shows the horseshoe-shaped BTX molecule bound within the open pore, forming hydrophobic H-bonds and cation-π contacts with the pore-lining helices, leaving space for partially dehydrated sodium ions to permeate through the hydrophilic inner surface of the horseshoe. We infer that bulky BTX, bound at the level of the gating-hinge residues, prevents the S6 rearrangements that are necessary for closure of the activation gate. Our results reveal general similarities to, and differences from, BTX actions on eukaryotic Nav channels, whose major subunit is a single polypeptide formed by four concatenated, homologous, nonidentical domains that form a pseudosymmetric pore. Our determination of the mechanism by which BTX activates homotetrameric voltage-gated channels reveals further similarities between eukaryotic and prokaryotic Nav channels and emphasizes the tractability of bacterial Nav channels as models of voltage-dependent ion channel gating. The results contribute toward a deeper, atomic-level understanding of use-dependent natural and synthetic Nav channel agonists and antagonists, despite their overlapping binding motifs on the channel proteins.

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.

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.

The intrinsically liganded cyclic nucleotide-binding homology domain promotes KCNH channel activation.

Channels in the ether-à-go-go or KCNH family of potassium channels are characterized by a conserved, C-terminal domain with homology to cyclic nucleotide-binding homology domains (CNBhDs). Instead of cyclic nucleotides, two amino acid residues, Y699 and L701, occupy the binding pocket, forming an "intrinsic ligand." The role of the CNBhD in KCNH channel gating is still unclear, however, and a detailed characterization of the intrinsic ligand is lacking. In this study, we show that mutating both Y699 and L701 to alanine, serine, aspartate, or glycine impairs human EAG1 channel function. These mutants slow channel activation and shift the conductance-voltage (G-V) relation to more depolarized potentials. The mutations affect activation and the G-V relation progressively, indicating that the gating machinery is sensitive to multiple conformations of the CNBhD. Substitution with glycine at both sites (GG), which eliminates the side chains that interact with the binding pocket, also reduces the ability of voltage prepulses to populate more preactivated states along the activation pathway (i.e., the Cole-Moore effect), as if stabilizing the voltage sensor in deep resting states. Notably, deletion of the entire CNBhD (577-708, ΔCNBhD) phenocopies the GG mutant, suggesting that GG is a loss-of-function mutation and the CNBhD requires an intrinsic ligand to exert its functional effects. We developed a kinetic model for both wild-type and ΔCNBhD mutant channels that describes all our observations on activation kinetics, the Cole-Moore shift, and G-V relations. These findings support a model in which the CNBhD both promotes voltage sensor activation and stabilizes the open pore. The intrinsic ligand is critical for these functional effects.

Structure and dynamics underlying elementary ligand binding events in human pacemaking channels.

Although molecular recognition is crucial for cellular signaling, mechanistic studies have relied primarily on ensemble measures that average over and thereby obscure underlying steps. Single-molecule observations that resolve these steps are lacking due to diffraction-limited resolution of single fluorophores at relevant concentrations. Here, we combined zero-mode waveguides with fluorescence resonance energy transfer (FRET) to directly observe binding at individual cyclic nucleotide-binding domains (CNBDs) from human pacemaker ion channels critical for heart and brain function. Our observations resolve the dynamics of multiple distinct steps underlying cyclic nucleotide regulation: a slow initial binding step that must select a 'receptive' conformation followed by a ligand-induced isomerization of the CNBD. X-ray structure of the apo CNBD and atomistic simulations reveal that the isomerization involves both local and global transitions. Our approach reveals fundamental mechanisms underpinning ligand regulation of pacemaker channels, and is generally applicable to weak-binding interactions governing a broad spectrum of signaling processes.

How to open a proton pore-more than S4?

Pioneering studies in voltage-gated potassium channels have described movement of the voltage-sensing domain (VSD) S4 helix across the membrane electric field in molecular detail, but much less is known regarding opening of the intrinsic proton pore within VSDs of voltage-dependent proton channels. By systematically probing local kinematics, a new study reveals that movements in helix S1 correlate with pore opening and are distinct from voltage-sensing movements of the charged S4 segment.

A nonequilibrium binary elements-based kinetic model for benzodiazepine regulation of GABAA receptors.

Ion channels, like many other proteins, are composed of multiple structural domains. A stimulus that impinges on one domain, such as binding of a ligand to its recognition site, can influence the activity of another domain, such as a transmembrane channel gate, through interdomain interactions. Kinetic schemes that describe the function of interacting domains typically incorporate a minimal number of states and transitions, and do not explicitly model interactions between domains. Here, we develop a kinetic model of the GABAA receptor, a ligand-gated ion channel modulated by numerous compounds including benzodiazepines, a class of drugs used clinically as sedatives and anxiolytics. Our model explicitly treats both the kinetics of distinct functional domains within the receptor and the interactions between these domains. The model describes not only how benzodiazepines that potentiate GABAA receptor activity, such as diazepam, affect peak current dose-response relationships in the presence of desensitization, but also their effect on the detailed kinetics of current activation, desensitization, and deactivation in response to various stimulation protocols. Finally, our model explains positive modulation by benzodiazepines of receptor currents elicited by either full or partial agonists, and can resolve conflicting observations arguing for benzodiazepine modulation of agonist binding versus channel gating.

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.

Evolutionarily conserved intracellular gate of voltage-dependent sodium channels.

Members of the voltage-gated ion channel superfamily (VGIC) regulate ion flux and generate electrical signals in excitable cells by opening and closing pore gates. The location of the gate in voltage-gated sodium channels, a founding member of this superfamily, remains unresolved. Here we explore the chemical modification rates of introduced cysteines along the S6 helix of domain IV in an inactivation-removed background. We find that state-dependent accessibility is demarcated by an S6 hydrophobic residue; substituted cysteines above this site are not modified by charged thiol reagents when the channel is closed. These accessibilities are consistent with those inferred from open- and closed-state structures of prokaryotic sodium channels. Our findings suggest that an intracellular gate composed of a ring of hydrophobic residues is not only responsible for regulating access to the pore of sodium channels, but is also a conserved feature within canonical members of the VGIC superfamily.

Domain IV voltage-sensor movement is both sufficient and rate limiting for fast inactivation in sodium channels.

Voltage-gated sodium channels are critical for the generation and propagation of electrical signals in most excitable cells. Activation of Na(+) channels initiates an action potential, and fast inactivation facilitates repolarization of the membrane by the outward K(+) current. Fast inactivation is also the main determinant of the refractory period between successive electrical impulses. Although the voltage sensor of domain IV (DIV) has been implicated in fast inactivation, it remains unclear whether the activation of DIV alone is sufficient for fast inactivation to occur. Here, we functionally neutralize each specific voltage sensor by mutating several critical arginines in the S4 segment to glutamines. We assess the individual role of each voltage-sensing domain in the voltage dependence and kinetics of fast inactivation upon its specific inhibition. We show that movement of the DIV voltage sensor is the rate-limiting step for both development and recovery from fast inactivation. Our data suggest that activation of the DIV voltage sensor alone is sufficient for fast inactivation to occur, and that activation of DIV before channel opening is the molecular mechanism for closed-state inactivation. We propose a kinetic model of sodium channel gating that can account for our major findings over a wide voltage range by postulating that DIV movement is both necessary and sufficient for fast inactivation.

Multiple pore conformations driven by asynchronous movements of voltage sensors in a eukaryotic sodium channel.

Voltage-dependent Na(+) channels are crucial for electrical signalling in excitable cells. Membrane depolarization initiates asynchronous movements in four non-identical voltage-sensing domains of the Na(+) channel. It remains unclear to what extent this structural asymmetry influences pore gating as compared with outwardly rectifying K(+) channels, where channel opening results from a final concerted transition of symmetric pore gates. Here we combine single channel recordings, cysteine accessibility and voltage clamp fluorimetry to probe the relationships between voltage sensors and pore conformations in an inactivation deficient Nav1.4 channel. We observe three distinct conductance levels such that DI-III voltage sensor activation is kinetically correlated with formation of a fully open pore, whereas DIV voltage sensor movement underlies formation of a distinct subconducting pore conformation preceding inactivation in wild-type channels. Our experiments reveal that pore gating in sodium channels involves multiple transitions driven by asynchronous movements of voltage sensors. These findings shed new light on the mechanism of coupling between activation and fast inactivation in voltage-gated sodium channels.