Structural Basis of Human KCNQ1 Modulation and Gating.

KCNQ1, also known as Kv7.1, is a voltage-dependent K+ channel that regulates gastric acid secretion, salt and glucose homeostasis, and heart rhythm. Its functional properties are regulated in a tissue-specific manner through co-assembly with beta subunits KCNE1-5. In non-excitable cells, KCNQ1 forms a complex with KCNE3, which suppresses channel closure at negative membrane voltages that otherwise would close it. Pore opening is regulated by the signaling lipid PIP2. Using cryoelectron microscopy (cryo-EM), we show that KCNE3 tucks its single-membrane-spanning helix against KCNQ1, at a location that appears to lock the voltage sensor in its depolarized conformation. Without PIP2, the pore remains closed. Upon addition, PIP2 occupies a site on KCNQ1 within the inner membrane leaflet, which triggers a large conformational change that leads to dilation of the pore's gate. It is likely that this mechanism of PIP2 activation is conserved among Kv7 channels.

Activation and Desensitization Mechanism of AMPA Receptor-TARP Complex by Cryo-EM.

AMPA receptors mediate fast excitatory neurotransmission in the mammalian brain and transduce the binding of presynaptically released glutamate to the opening of a transmembrane cation channel. Within the postsynaptic density, however, AMPA receptors coassemble with transmembrane AMPA receptor regulatory proteins (TARPs), yielding a receptor complex with altered gating kinetics, pharmacology, and pore properties. Here, we elucidate structures of the GluA2-TARP γ2 complex in the presence of the partial agonist kainate or the full agonist quisqualate together with a positive allosteric modulator or with quisqualate alone. We show how TARPs sculpt the ligand-binding domain gating ring, enhancing kainate potency and diminishing the ensemble of desensitized states. TARPs encircle the receptor ion channel, stabilizing M2 helices and pore loops, illustrating how TARPs alter receptor pore properties. Structural and computational analysis suggests the full agonist and modulator complex harbors an ion-permeable channel gate, providing the first view of an activated AMPA receptor.

Structural Titration of Slo2.2, a Na+-Dependent K+ Channel.

The stable structural conformations that occur along the complete reaction coordinate for ion channel opening have never been observed. In this study, we describe the equilibrium ensemble of structures of Slo2.2, a neuronal Na+-activated K+ channel, as a function of the Na+ concentration. We find that Slo2.2 exists in multiple closed conformations whose relative occupancies are independent of Na+ concentration. An open conformation emerges from an ensemble of closed conformations in a highly Na+-dependent manner, without evidence of Na+-dependent intermediates. In other words, channel opening is a highly concerted, switch-like process. The midpoint of the structural titration matches that of the functional titration. A maximum open conformation probability approaching 1.0 and maximum functional open probability approaching 0.7 imply that, within the class of open channels, there is a subclass that is not permeable to ions.

A suicidal mechanism for the exquisite temperature sensitivity of TRPV1.

The vanilloid receptor TRPV1 is an exquisite nociceptive sensor of noxious heat, but its temperature-sensing mechanism is yet to define. Thermodynamics dictate that this channel must undergo an unusually energetic allosteric transition. Thus, it is of fundamental importance to measure directly the energetics of this transition in order to properly decipher its temperature-sensing mechanism. Previously, using submillisecond temperature jumps and patch-clamp recording, we estimated that the heat activation for TRPV1 opening incurs an enthalpy change on the order of 100 kcal/mol. Although this energy is on a scale unparalleled by other known biological receptors, the generally imperfect allosteric coupling in proteins implies that the actual amount of heat uptake driving the TRPV1 transition could be much larger. In this paper, we apply differential scanning calorimetry to directly monitor the heat flow in TRPV1 that accompanies its temperature-induced conformational transition. Our measurements show that heat invokes robust, complex thermal transitions in TRPV1 that include both channel opening and a partial protein unfolding transition and that these two processes are inherently coupled. Our findings support that irreversible protein unfolding, which is generally thought to be destructive to physiological function, is essential to TRPV1 thermal transduction and, possibly, to other strongly temperature-dependent processes in biology.

Signal transduction through Cys-loop receptors is mediated by the nonspecific bumping of closely apposed domains.

One of the most fundamental questions in the field of Cys-loop receptors (pentameric ligand-gated ion channels, pLGICs) is how the affinity for neurotransmitters and the conductive/nonconductive state of the transmembrane pore are correlated despite the ∼60-Šdistance between the corresponding domains. Proposed mechanisms differ, but they all converge into the idea that interactions between wild-type side chains across the extracellular-transmembrane-domain (ECD-TMD) interface are crucial for this phenomenon. Indeed, the successful design of fully functional chimeras that combine intact ECD and TMD modules from different wild-type pLGICs has commonly been ascribed to the residual conservation of sequence that exists at the level of the interfacial loops even between evolutionarily distant parent channels. Here, using mutagenesis, patch-clamp electrophysiology, and radiolabeled-ligand binding experiments, we studied the effect of eliminating this residual conservation of sequence on ion-channel function and cell-surface expression. From our results, we conclude that proper state interconversion ("gating") does not require conservation of sequence-or even physicochemical properties-across the ECD-TMD interface. Wild-type ECD and TMD side chains undoubtedly interact with their surroundings, but the interactions between them-straddling the interface-do not seem to be more important for gating than those occurring elsewhere in the protein. We propose that gating of pLGICs requires, instead, that the overall structure of the interfacial loops be conserved, and that their relative orientation and distance be the appropriate ones for changes in one side to result in changes in the other, in a phenomenon akin to the nonspecific "bumping" of closely apposed domains.

The roles of transmembrane family proteins in the regulation of store-operated Ca2+ entry.

Store-operated Ca2+ entry (SOCE) is a major pathway for calcium signaling, which regulates almost every biological process, involving cell proliferation, differentiation, movement and death. Stromal interaction molecule (STIM) and ORAI calcium release-activated calcium modulator (ORAI) are the two major proteins involved in SOCE. With the deepening of studies, more and more proteins are found to be able to regulate SOCE, among which the transmembrane (TMEM) family proteins are worth paying more attention. In addition, the ORAI proteins belong to the TMEM family themselves. As the name suggests, TMEM family is a type of proteins that spans biological membranes including plasma membrane and membrane of organelles. TMEM proteins are in a large family with more than 300 proteins that have been already identified, while the functional knowledge about the proteins is preliminary. In this review, we mainly summarized the TMEM proteins that are involved in SOCE, to better describe a picture of the interaction between STIM and ORAI proteins during SOCE and its downstream signaling pathways, as well as to provide an idea for the study of the TMEM family proteins.

Effects of ethanol on GluN1/GluN2A and GluN1/GluN2B NMDA receptor-ion channel gating kinetics.

BACKGROUND: The N-methyl-D-aspartate receptor (NMDAR) is a major molecular target of alcohol action in the central nervous system, yet many aspects of alcohol's modulation of the activity of this ion channel remain unclear. We and others have shown that ethanol inhibition of NMDAR involves alterations in gating, especially a reduction in mean open time. However, a full description of ethanol's effects on NMDAR kinetics, including fitting them to a kinetic model, has not been reported. METHODS: To determine ethanol's effects on NMDAR kinetics, we used steady-state single-channel recording in outside-out patches from HEK-293 cells transfected with recombinant GluN1/GluN2A or GluN1/GluN2B NMDAR subunits. Very low glutamate concentrations were used to isolate individual activations of the receptor. RESULTS: In both subunit types, ethanol, at approximate whole-cell IC50 values (156 mM, GluN2A; 150 mM, GluN2B), reduced open probability (po ) by approximately 50% and decreased mean open time without changing the frequency of opening. Open and shut time distributions exhibited two and five components, respectively; ethanol selectively decreased the time constant and relative proportion of the longer open time component. In the GluN2A subunit, ethanol increased the time constants of all but the longest shut time components, whereas in the GluN2B subunit, shut times were unchanged by ethanol. Fitting of bursts of openings (representing individual activations of the receptor) to the gating portion of a kinetic model revealed that ethanol altered two rates: the rate associated with activation of the GluN2A or GluN2B subunit, and the rate associated with the closing of the longer of the two open states. CONCLUSIONS: These results demonstrate that ethanol selectively alters individual kinetic rates and thus appears to selectively affect distinct conformational transitions involved in NMDAR gating.

Genetic Variant in Nicotinic Receptor α4-Subunit Causes Sleep-Related Hyperkinetic Epilepsy via Increased Channel Opening.

We describe genetic and molecular-level functional alterations in the α4ß2 neuronal nicotinic acetylcholine receptor (nAChR) from a patient with sleep-related hyperkinetic epilepsy and a family history of epilepsy. Genetic sequencing revealed a heterozygous variant c.851C>G in the CHRNA4 gene encoding the α4 subunit, resulting in the missense mutation p.Ser284Trp. Patch clamp recordings from genetically engineered nAChRs incorporating the α4-Ser284Trp subunit revealed aberrant channel openings in the absence of agonist and markedly prolonged openings in its presence. Measurements of single channel current amplitude distinguished two pentameric stoichiometries of the variant nAChR containing either two or three copies of the α4-Ser284Trp subunit, each exhibiting aberrant spontaneous and prolonged agonist-elicited channel openings. The α4-Ser284 residue is highly conserved and located within the M2 transmembrane α-helix that lines the ion channel. When mapped onto the receptor's three-dimensional structure, the larger Trp substitution sterically clashes with the M2 α-helix from the neighboring subunit, promoting expansion of the pore and stabilizing the open relative to the closed conformation of the channel. Together, the clinical, genetic, functional, and structural observations demonstrate that α4-Ser284Trp enhances channel opening, predicting increased membrane excitability and a pathogenic seizure phenotype.

AMPA receptor structure and auxiliary subunits.

Fast excitatory synaptic transmission in the mammalian brain is largely mediated by AMPA-type ionotropic glutamate receptors (AMPARs), which are activated by the neurotransmitter glutamate. In synapses, the function of AMPARs is tuned by their auxiliary subunits, a diverse set of membrane proteins associated with the core pore-forming subunits of the AMPARs. Each auxiliary subunit provides distinct functional modulation of AMPARs, ranging from regulation of trafficking to shaping ion channel gating kinetics. Understanding the molecular mechanism of the function of these complexes is key to decoding synaptic modulation and their global roles in cognitive activities, such as learning and memory. Here, we review the structural and molecular complexity of AMPAR-auxiliary subunit complexes, as well as their functional diversity in different brain regions. We suggest that the recent structural information provides new insights into the molecular mechanisms underlying synaptic functions of AMPAR-auxiliary subunit complexes.

Thermodynamic and structural basis of temperature-dependent gating in TRP channels.

Living organisms require detecting the environmental thermal clues for survival, allowing them to avoid noxious stimuli or find prey moving in the dark. In mammals, the Transient Receptor Potential ion channels superfamily is constituted by 27 polymodal receptors whose activity is controlled by small ligands, peptide toxins, protons and voltage. The thermoTRP channels subgroup exhibits unparalleled temperature dependence -behaving as heat and cold sensors. Functional studies have dissected their biophysical features in detail, and the advances of single-particle Cryogenic Electron microscopy provided the structural framework required to propose detailed channel gating mechanisms. However, merging structural and functional evidence for temperature-driven gating of thermoTRP channels has been a hard nut to crack, remaining an open question nowadays. Here we revisit the highlights on the study of heat and cold sensing in thermoTRP channels in the light of the structural data that has emerged during recent years.

Molecular basis of allosteric Orai1 channel activation by STIM1.

Store-operated Ca2+ entry through Orai1 channels is a primary mechanism for Ca2+ entry in many cells and mediates numerous cellular effector functions ranging from gene transcription to exocytosis. Orai1 channels are amongst the most Ca2+ -selective channels known and are activated by direct physical interactions with the endoplasmic reticulum Ca2+ sensor stromal interaction molecule 1 (STIM1) in response to store depletion triggered by stimulation of a variety of cell surface G-protein coupled and tyrosine kinase receptors. Work in the last decade has revealed that the Orai1 gating process is highly cooperative and strongly allosteric, likely driven by a wave of interdependent conformational changes throughout the protein originating in the peripheral C-terminal ligand binding site and culminating in pore opening. In this review, we survey the structural and molecular features in Orai1 that contribute to channel gating and consider how they give rise to the unique biophysical fingerprint of Orai1 currents.

Simulating ion channel activation mechanisms using swarms of trajectories.

Atomic-level studies of protein activity represent a significant challenge as a result of the complexity of conformational changes occurring on wide-ranging timescales, often greatly exceeding that of even the longest simulations. A prime example is the elucidation of protein allosteric mechanisms, where localized perturbations transmit throughout a large macromolecule to generate a response signal. For example, the conversion of chemical to electrical signals during synaptic neurotransmission in the brain is achieved by specialized membrane proteins called pentameric ligand-gated ion channels. Here, the binding of a neurotransmitter results in a global conformational change to open an ion-conducting pore across the nerve cell membrane. X-ray crystallography has produced static structures of the open and closed states of the proton-gated GLIC pentameric ligand-gated ion channel protein, allowing for atomistic simulations that can uncover changes related to activation. We discuss a range of enhanced sampling approaches that could be used to explore activation mechanisms. In particular, we describe recent application of an atomistic string method, based on Roux's "swarms of trajectories" approach, to elucidate the sequence and interdependence of conformational changes during activation. We illustrate how this can be combined with transition analysis and Brownian dynamics to extract thermodynamic and kinetic information, leading to understanding of what controls ion channel function. © 2019 Wiley Periodicals, Inc.

Intracellular O-linked glycosylation directly regulates cardiomyocyte L-type Ca2+ channel activity and excitation-contraction coupling.

Cardiomyocyte L-type Ca2+ channels (Cavs) are targets of signaling pathways that modulate channel activity in response to physiologic stimuli. Cav regulation is typically transient and beneficial but chronic stimulation can become pathologic; therefore, gaining a more complete understanding of Cav regulation is of critical importance. Intracellular O-linked glycosylation (O-GlcNAcylation), which is the result of two enzymes that dynamically add and remove single N-acetylglucosamines to and from intracellular serine/threonine residues (OGT and OGA respectively), has proven to be an increasingly important post-translational modification that contributes to the regulation of many physiologic processes. However, there is currently no known role for O-GlcNAcylation in the direct regulation of Cav activity nor is its contribution to cardiac electrical signaling and EC coupling well understood. Here we aimed to delineate the role of O-GlcNAcylation in regulating cardiomyocyte L-type Cav activity and its subsequent effect on EC coupling by utilizing a mouse strain possessing an inducible cardiomyocyte-specific OGT-null-transgene. Ablation of the OGT-gene in adult cardiomyocytes (OGTKO) reduced OGT expression and O-GlcNAcylation by > 90%. Voltage clamp recordings indicated an ~ 40% reduction in OGTKO Cav current (ICa), but with increased efficacy of adrenergic stimulation, and Cav steady-state gating and window current were significantly depolarized. Consistently, OGTKO cardiomyocyte intracellular Ca2+ release and contractility were diminished and demonstrated greater beat-to-beat variability. Additionally, we show that the Cav α1 and ß2 subunits are O-GlcNAcylated while α2δ1 is not. Echocardiographic analyses indicated that the reductions in OGTKO cardiomyocyte Ca2+ handling and contractility were conserved at the whole-heart level as evidenced by significantly reduced left-ventricular contractility in the absence of hypertrophy. The data indicate, for the first time, that O-GlcNAc signaling is a critical and direct regulator of cardiomyocyte ICa achieved through altered Cav expression, gating, and response to adrenergic stimulation; these mechanisms have significant implications for understanding how EC coupling is regulated in health and disease.

String method solution of the gating pathways for a pentameric ligand-gated ion channel.

Pentameric ligand-gated ion channels control synaptic neurotransmission by converting chemical signals into electrical signals. Agonist binding leads to rapid signal transduction via an allosteric mechanism, where global protein conformational changes open a pore across the nerve cell membrane. We use all-atom molecular dynamics with a swarm-based string method to solve for the minimum free-energy gating pathways of the proton-activated bacterial GLIC channel. We describe stable wetted/open and dewetted/closed states, and uncover conformational changes in the agonist-binding extracellular domain, ion-conducting transmembrane domain, and gating interface that control communication between these domains. Transition analysis is used to compute free-energy surfaces that suggest allosteric pathways; stabilization with pH; and intermediates, including states that facilitate channel closing in the presence of an agonist. We describe a switching mechanism that senses proton binding by marked reorganization of subunit interface, altering the packing of ß-sheets to induce changes that lead to asynchronous pore-lining M2 helix movements. These results provide molecular details of GLIC gating and insight into the allosteric mechanisms for the superfamily of pentameric ligand-gated channels.

Phosphatidylinositol 4,5-bisphosphate (PIP2) regulates KCNQ3 K+ channels by interacting with four cytoplasmic channel domains.

Phosphatidylinositol 4,5-bisphosphate (PIP2) in the plasma membrane regulates the function of many ion channels, including M-type (potassium voltage-gated channel subfamily Q member (KCNQ), Kv7) K+ channels; however, the molecular mechanisms involved remain unclear. To this end, we here focused on the KCNQ3 subtype that has the highest apparent affinity for PIP2 and performed extensive mutagenesis in regions suggested to be involved in PIP2 interactions among the KCNQ family. Using perforated patch-clamp recordings of heterologously transfected tissue culture cells, total internal reflection fluorescence microscopy, and the zebrafish (Danio rerio) voltage-sensitive phosphatase to deplete PIP2 as a probe, we found that PIP2 regulates KCNQ3 channels through four different domains: 1) the A-B helix linker that we previously identified as important for both KCNQ2 and KCNQ3, 2) the junction between S6 and the A helix, 3) the S2-S3 linker, and 4) the S4-S5 linker. We also found that the apparent strength of PIP2 interactions within any of these domains was not coupled to the voltage dependence of channel activation. Extensive homology modeling and docking simulations with the WT or mutant KCNQ3 channels and PIP2 were consistent with the experimental data. Our results indicate that PIP2 modulates KCNQ3 channel function by interacting synergistically with a minimum of four cytoplasmic domains.

A Mathematical Model of the Human Cardiac Na+ Channel.

Sodium ion channel is a membrane protein that plays an important role in excitable cells, as it is responsible for the initiation of action potentials. Understanding the electrical characteristics of sodium channels is essential in predicting their behavior under different physiological conditions. We investigated several Markov models for the human cardiac sodium channel NaV1.5 to derive a minimal mathematical model that describes the reported experimental data obtained using major voltage clamp protocols. We obtained simulation results for peak current-voltage relationships, the voltage dependence of normalized ion channel conductance, steady-state inactivation, activation and deactivation kinetics, fast and slow inactivation kinetics, and recovery from inactivation kinetics. Good agreement with the experimental data provides us with the mechanisms of the fast and slow inactivation of the human sodium channel and the coupling of its inactivation states to the closed and open states in the activation pathway.

Positions in the N-methyl-D-aspartate Receptor GluN2C Subunit M3 and M4 Domains Regulate Alcohol Sensitivity and Receptor Kinetics.

BACKGROUND: Alcohol alters synaptic transmission in the brain. The N-methyl-D-aspartate (NMDA) receptor (NMDAR), a subtype of glutamate-gated ion channel, is an important synaptic target of alcohol in the brain. We and others have previously identified 4 alcohol-sensitive positions in the third and fourth membrane-associated (M) domains, designated M31-2 and M41-2 , of the GluN1, GluN2A, and GluN2B NMDAR subunits. In the present study, we tested whether the corresponding positions in the GluN2C subunit also regulate alcohol sensitivity and ion channel gating. METHODS: We performed alanine- and tryptophan-scanning mutagenesis in the GluN2C subunit followed by expression in HEK 293 cells and electrophysiological patch-clamp recording. RESULTS: Alanine substitution at the M31 (F634) and M41-2 (M821 and M823) positions did not alter ethanol (EtOH) sensitivity, whereas substitution of alanine at the M32 position (F635) yielded nonfunctional receptors. Tryptophan substitution at the M31-2 positions did not change EtOH sensitivity, whereas tryptophan substitution at the M41 position increased, and at the M42 position decreased, EtOH sensitivity. The increased EtOH sensitivity of the tryptophan mutant at M41 is in marked contrast to previous results observed in the GluN2A and GluN2B subunits. In addition, this mutant exhibited increased desensitization, but to a much lesser extent compared to the corresponding mutations in GluN2A and GluN2B. A series of mutations at M41 altered EtOH sensitivity, glutamate potency, and desensitization. Seven amino acid substitutions (of 15 tested) at this position yielded nonfunctional receptors. Among the remaining mutants at M41 , EtOH sensitivity was not significantly correlated with hydrophobicity, molecular volume, or polarity of the substituent, or with glutamate EC50 values, but was correlated with maximal steady-state-to-peak current ratio, a measure of desensitization. CONCLUSIONS: The identity and characteristics of alcohol-sensitive positions in the GluN2C subunit differ from those previously reported for GluN2A and GluN2B subunits, despite the high homology among these subunits.

Confined Dynamics of Water in Transmembrane Pore of TRPV1 Ion Channel.

Solvation effects play a key role in chemical and biological processes. The microscopic properties of water near molecular surfaces are radically different from those in the bulk. Furthermore, the behavior of water in confined volumes of a nanometer scale, including transmembrane pores of ion channels, is especially nontrivial. Knowledge at the molecular level of structural and dynamic parameters of water in such systems is necessary to understand the mechanisms of ion channels functioning. In this work, the results of molecular dynamics (MD) simulations of water in the pore and selectivity filter domains of TRPV1 (Transient Receptor Potential Vanilloid type 1) membrane channel are considered. These domains represent nanoscale volumes with strongly amphiphilic walls, where physical behavior of water radically differs from that of free hydration (e.g., at protein interfaces) or in the bulk. Inside the pore and filter domains, water reveals a very heterogeneous spatial distribution and unusual dynamics: It forms compact areas localized near polar groups of particular residues. Residence time of water molecules in such areas is at least 1.5 to 3 times larger than that observed for similar groups at the protein surface. Presumably, these water "blobs" play an important role in the functional activity of TRPV1. In particular, they take part in hydration of the hydrophobic TRPV1 pore by localizing up to six waters near the so-called "lower gate" of the channel and reducing by this way the free energy barrier for ion and water transport. Although the channel is formed by four identical protein subunits, which are symmetrically packed in the initial experimental 3D structure, in the course of MD simulations, hydration of the same amino acid residues of individual subunits may differ significantly. This greatly affects the microscopic picture of the distribution of water in the channel and, potentially, the mechanism of its functioning. Therefore, reconstruction of the full picture of TRPV1 channel solvation requires thorough atomistic simulations and analysis. It is important that the naturally occurring porous volumes, like ion-conducting protein domains, reveal much more sophisticated and fine-tuned regulation of solvation than, e.g., artificially designed carbon nanotubes.

Effects of ion channel noise on neural circuits: an application to the respiratory pattern generator to investigate breathing variability.

Neural activity generally displays irregular firing patterns even in circuits with apparently regular outputs, such as motor pattern generators, in which the output frequency fluctuates randomly around a mean value. This "circuit noise" is inherited from the random firing of single neurons, which emerges from stochastic ion channel gating (channel noise), spontaneous neurotransmitter release, and its diffusion and binding to synaptic receptors. Here we demonstrate how to expand conductance-based network models that are originally deterministic to include realistic, physiological noise, focusing on stochastic ion channel gating. We illustrate this procedure with a well-established conductance-based model of the respiratory pattern generator, which allows us to investigate how channel noise affects neural dynamics at the circuit level and, in particular, to understand the relationship between the respiratory pattern and its breath-to-breath variability. We show that as the channel number increases, the duration of inspiration and expiration varies, and so does the coefficient of variation of the breath-to-breath interval, which attains a minimum when the mean duration of expiration slightly exceeds that of inspiration. For small channel numbers, the variability of the expiratory phase dominates over that of the inspiratory phase, and vice versa for large channel numbers. Among the four different cell types in the respiratory pattern generator, pacemaker cells exhibit the highest sensitivity to channel noise. The model shows that suppressing input from the pons leads to longer inspiratory phases, a reduction in breathing frequency, and larger breath-to-breath variability, whereas enhanced input from the raphe nucleus increases breathing frequency without changing its pattern. NEW & NOTEWORTHY: A major source of noise in neuronal circuits is the "flickering" of ion currents passing through the neurons' membranes (channel noise), which cannot be suppressed experimentally. Computational simulations are therefore the best way to investigate the effects of this physiological noise by manipulating its level at will. We investigate the role of noise in the respiratory pattern generator and show that endogenous, breath-to-breath variability is tightly linked to the respiratory pattern.