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Dynamical diversity of mitochondrial BK channels located in different cell types.
Wawrzkiewicz-Jałowiecka, A, Trybek, P, Machura, Ł, Bednarczyk, P
Bio Systems. 2021;:104310
Abstract
Mitochondrial large-conductance voltage- and Ca2+-activated potassium channels (mitoBK) exhibit substantial similarities in their physiology regardless of the channel's location. Nevertheless, depending on the cell type, composition of membranes can vary, and mitoBK channels can be expressed in different splice variants as well as they can be co-assembled with different types of auxiliary β subunits. These factors can modulate their voltage- and Ca2+-sensitivity, and single-channel current kinetics. It is still an open question to what extent the mentioned factors can affect the complexity of the conformational dynamics of the mitoBK channel gating. In this work the dynamical diversity of mitoBK channels from different cell types was unraveled by the use of nonlinear methods of analysis: multifractal detrended fluctuation analysis (MFDFA) and multiscale entropy (MSE). These techniques were applied to the experimental series of single channel currents. It turns out that the differences in the mitoBK expression systems influence gating machinery by changing the scheme of switching between the stable channel conformations, and affecting the average number of available channel conformations (this effect is visible for mitoBK channels in glioblastoma cells). The obtained results suggest also that a pathological dynamics can be represented by signals of relatively low complexity (low MSE of the mitoBK channel gating in glioblastoma).
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Potency-Enhancing Mutations of Gating Modifier Toxins for the Voltage-Gated Sodium Channel NaV1.7 Can Be Predicted Using Accurate Free-Energy Calculations.
Katz, D, Sindhikara, D, DiMattia, M, Leffler, AE
Toxins. 2021;(3)
Abstract
Gating modifier toxins (GMTs) isolated from venomous organisms such as Protoxin-II (ProTx-II) and Huwentoxin-IV (HwTx-IV) that inhibit the voltage-gated sodium channel NaV1.7 by binding to its voltage-sensing domain II (VSDII) have been extensively investigated as non-opioid analgesics. However, reliably predicting how a mutation to a GMT will affect its potency for NaV1.7 has been challenging. Here, we hypothesize that structure-based computational methods can be used to predict such changes. We employ free-energy perturbation (FEP), a physics-based simulation method for predicting the relative binding free energy (RBFE) between molecules, and the cryo electron microscopy (cryo-EM) structures of ProTx-II and HwTx-IV bound to VSDII of NaV1.7 to re-predict the relative potencies of forty-seven point mutants of these GMTs for NaV1.7. First, FEP predicted these relative potencies with an overall root mean square error (RMSE) of 1.0 ± 0.1 kcal/mol and an R2 value of 0.66, equivalent to experimental uncertainty and an improvement over the widely used molecular-mechanics/generalized born-surface area (MM-GB/SA) RBFE method that had an RMSE of 3.9 ± 0.8 kcal/mol. Second, inclusion of an explicit membrane model was needed for the GMTs to maintain stable binding poses during the FEP simulations. Third, MM-GB/SA and FEP were used to identify fifteen non-standard tryptophan mutants at ProTx-II[W24] predicted in silico to have a at least a 1 kcal/mol gain in potency. These predicted potency gains are likely due to the displacement of high-energy waters as identified by the WaterMap algorithm for calculating the positions and thermodynamic properties of water molecules in protein binding sites. Our results expand the domain of applicability of FEP and set the stage for its prospective use in biologics drug discovery programs involving GMTs and NaV1.7.
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3.
The pore domain in glutamate-gated ion channels: Structure, drug binding and similarity with potassium channels.
Tikhonov, DB, Zhorov, BS
Biochimica et biophysica acta. Biomembranes. 2020;(10):183401
Abstract
Ionotropic glutamate receptors in the CNS excitatory synapses of vertebrates are involved in numerous physiological and pathological processes. Decades of intensive studies greatly advanced our understanding of molecular organization of these transmembrane proteins. Here we focus on the channel pore domain, its selectivity filter and the activation gate, and the pore block by organic ligands. We compare findings from indirect experimental approaches, including site-directed mutagenesis, with recent crystal and cryo-EM structures of different channels in different functional states and complexed with different ligands. We summarize remaining uncertainties and unresolved problems related to the channel structure, function and pharmacology.
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4.
Effects of the KCNQ channel opener ezogabine on functional connectivity of the ventral striatum and clinical symptoms in patients with major depressive disorder.
Tan, A, Costi, S, Morris, LS, Van Dam, NT, Kautz, M, Whitton, AE, Friedman, AK, Collins, KA, Ahle, G, Chadha, N, et al
Molecular psychiatry. 2020;(6):1323-1333
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Abstract
Major depressive disorder (MDD) is a leading cause of disability worldwide, yet current treatment strategies remain limited in their mechanistic diversity. Recent evidence has highlighted a promising novel pharmaceutical target-the KCNQ-type potassium channel-for the treatment of depressive disorders, which may exert a therapeutic effect via functional changes within the brain reward system, including the ventral striatum. The current study assessed the effects of the KCNQ channel opener ezogabine (also known as retigabine) on reward circuitry and clinical symptoms in patients with MDD. Eighteen medication-free individuals with MDD currently in a major depressive episode were enrolled in an open-label study and received ezogabine up to 900 mg/day orally over the course of 10 weeks. Resting-state functional magnetic resonance imaging data were collected at baseline and posttreatment to examine brain reward circuitry. Reward learning was measured using a computerized probabilistic reward task. After treatment with ezogabine, subjects exhibited a significant reduction of depressive symptoms (Montgomery-Asberg Depression Rating Scale score change: -13.7 ± 9.7, p < 0.001, d = 2.08) and anhedonic symptoms (Snaith-Hamilton Pleasure Scale score change: -6.1 ± 5.3, p < 0.001, d = 1.00), which remained significant even after controlling for overall depression severity. Improvement in depression was associated with decreased functional connectivity between the ventral caudate and clusters within the mid-cingulate cortex and posterior cingulate cortex (n = 14, voxel-wise p < 0.005). In addition, a subgroup of patients tested with a probabilistic reward task (n = 9) showed increased reward learning following treatment. These findings highlight the KCNQ-type potassium channel as a promising target for future drug discovery efforts in mood disorders.
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5.
A novel fast-channel myasthenia caused by mutation in β subunit of AChR reveals subunit-specific contribution of the intracellular M1-M2 linker to channel gating.
Shen, XM, Di, L, Shen, S, Zhao, Y, Neumeyer, AM, Selcen, D, Sine, SM, Engel, AG
Experimental neurology. 2020;:113375
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Abstract
Genetic variants causing the fast-channel congenital myasthenic syndrome (CMS) have been identified in the α, δ, and ε but not the β subunit of acetylcholine receptor (AChR). A 16-year-old girl with severe myasthenia had low-amplitude and fast-decaying miniature endplate potentials. Mutation analysis revealed two heteroallelic variants in CHRNB1 encoding the AChR β subunit: a novel c.812C>T (p.P248L) variant in M1-M2 linker (p.P271L in HGVS nomenclature), and a ~430 bp deletion causing loss of exon 8 leading to frame-shift and a premature stop codon (p.G251Dfs*21). P248 is conserved in all β subunits of different species, but not in other AChR subunits. Measurements of radio-labeled α-bungarotoxin binding show that βP248L reduces AChR expression to 60% of wild-type. Patch clamp recordings of ACh-elicited single channel currents demonstrate that βP248L shortens channel opening bursts from 3.3 ms to 1.2 ms, and kinetic analyses predict that the decay of the synaptic response is accelerated 2.4-fold due to reduced probability of channel reopening. Substituting βP248 with threonine, alanine or glycine reduces the burst duration to 2.3, 1.7, and 1.5 ms, respectively. In non-β subunits, substituting leucine for residues corresponding to βP248 prolongs the burst duration to 4.5 ms in the α subunit, shortens it to 2.2 ms in the δ subunit, and has no effect in the ε subunit. Conversely, substituting proline for residues corresponding to βP248 prolongs the burst duration to 8.7 ms in the α subunit, to 4.6 ms in the δ subunit, but has no effect in the ε subunit. Thus, this fast channel CMS is caused by the dual defects of βP248L in reducing expression of the mutant receptor and accelerating the decay of the synaptic response. The results also reveal subunit-specific contributions of the M1-M2 linker to the durations of channel opening bursts.
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6.
A sulfur-aromatic gate latch is essential for opening of the Orai1 channel pore.
Yeung, PS, Ing, CE, Yamashita, M, Pomès, R, Prakriya, M
eLife. 2020
Abstract
Sulfur-aromatic interactions occur in the majority of protein structures, yet little is known about their functional roles in ion channels. Here, we describe a novel molecular motif, the M101 gate latch, which is essential for gating of human Orai1 channels via its sulfur-aromatic interactions with the F99 hydrophobic gate. Molecular dynamics simulations of different Orai variants reveal that the gate latch is mostly engaged in open but not closed channels. In experimental studies, we use metal-ion bridges to show that promoting an M101-F99 bond directly activates Orai1, whereas disrupting this interaction triggers channel closure. Mutational analysis demonstrates that the methionine residue at this position has a unique combination of length, flexibility, and chemistry to act as an effective latch for the phenylalanine gate. Because sulfur-aromatic interactions provide additional stabilization compared to purely hydrophobic interactions, we infer that the six M101-F99 pairs in the hexameric channel provide a substantial energetic contribution to Orai1 activation.
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An Effective Electric Dipole Model for Voltage-induced Gating Mechanism of Lysenin.
Faouri, RA, Krueger, E, Govind Kumar, V, Fologea, D, Straub, D, Alismail, H, Alfaori, Q, Kight, A, Ray, J, Henry, R, et al
Scientific reports. 2019;(1):11440
Abstract
Lysenin is a pore-forming toxin, which self-inserts open channels into sphingomyelin containing membranes and is known to be voltage regulated. The mechanistic details of its voltage gating mechanism, however, remains elusive despite much recent efforts. Here, we have employed a novel combination of experimental and computational techniques to examine a model for voltage gating, that is based on the existence of an "effective electric dipole" inspired by recent reported structures of lysenin. We support this mechanism by the observations that (i) the charge-reversal and neutralization substitutions in lysenin result in changing its electrical gating properties by modifying the strength of the dipole, and (ii) an increase in the viscosity of the solvent increases the drag force and slows down the gating. In addition, our molecular dynamics (MD) simulations of membrane-embedded lysenin provide a mechanistic picture for lysenin conformational changes, which reveals, for the first time, the existence of a lipid-dependent bulge region in the pore-forming module of lysenin, which may explain the gating mechanism of lysenin at a molecular level.
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The intriguing effect of ethanol and nicotine on acetylcholine-sensitive potassium current IKAch: Insight from a quantitative model.
Šimurda, J, Šimurdová, M, Bébarová, M
PloS one. 2019;(10):e0223448
Abstract
Recent experimental work has revealed unusual features of the effect of certain drugs on cardiac inwardly rectifying potassium currents, including the constitutively active and acetylcholine-induced components of acetylcholine-sensitive current (IKAch). These unusual features have included alternating susceptibility of the current components to activation and inhibition induced by ethanol or nicotine applied at various concentrations, and significant correlation between the drug effect and the current magnitude measured under drug-free conditions. To explain these complex drug effects, we have developed a new type of quantitative model to offer a possible interpretation of the effect of ethanol and nicotine on the IKAch channels. The model is based on a description of IKAch as a sum of particular currents related to the populations of channels formed by identical assemblies of different α-subunits. Assuming two different channel populations in agreement with the two reported functional IKAch-channels (GIRK1/4 and GIRK4), the model was able to simulate all the above-mentioned characteristic features of drug-channel interactions and also the dispersion of the current measured in different cells. The formulation of our model equations allows the model to be incorporated easily into the existing integrative models of electrical activity of cardiac cells involving quantitative description of IKAch. We suppose that the model could also help make sense of certain observations related to the channels that do not show inward rectification. This new ionic channel model, based on a concept we call population type, may allow for the interpretation of complex interactions of drugs with ionic channels of various types, which cannot be done using the ionic channel models available so far.
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Challenges and advances in atomistic simulations of potassium and sodium ion channel gating and permeation.
DeMarco, KR, Bekker, S, Vorobyov, I
The Journal of physiology. 2019;(3):679-698
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Abstract
Ion channels are implicated in many essential physiological events such as electrical signal propagation and cellular communication. The advent of K+ and Na+ ion channel structure determination has facilitated numerous investigations of molecular determinants of their behaviour. At the same time, rapid development of computer hardware and molecular simulation methodologies has made computational studies of large biological molecules in all-atom representation tractable. The concurrent evolution of experimental structural biology with biomolecular computer modelling has yielded mechanistic details of fundamental processes unavailable through experiments alone, such as ion conduction and ion channel gating. This review is a short survey of the atomistic computational investigations of K+ and Na+ ion channels, focusing on KcsA and several voltage-gated channels from the KV and NaV families, which have garnered many successes and engendered several long-standing controversies regarding the nature of their structure-function relationship. We review the latest advancements and challenges facing the field of molecular modelling and simulation regarding the structural and energetic determinants of ion channel function and their agreement with experimental observations.
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10.
Analyses of epithelial Na+ channel variants reveal that an extracellular β-ball domain critically regulates ENaC gating.
Wang, X, Chen, J, Shi, S, Sheng, S, Kleyman, TR
The Journal of biological chemistry. 2019;(45):16765-16775
Abstract
Epithelial Na+ channel (ENaC)-mediated Na+ transport has a key role in the regulation of extracellular fluid volume, blood pressure, and extracellular [K+]. Among the thousands of human ENaC variants, only a few exist whose functional consequences have been experimentally tested. Here, we used the Xenopus oocyte expression system to investigate the functional roles of four nonsynonymous human ENaC variants located within the β7-strand and its adjacent loop of the α-subunit extracellular β-ball domain. αR350Wβγ and αG355Rβγ channels exhibited 2.5- and 1.8-fold greater amiloride-sensitive currents than WT αβγ human ENaCs, respectively, whereas αV351Aβγ channels conducted significantly less current than WT. Currents in αH354Rβγ-expressing oocytes were similar to those expressing WT. Surface expression levels of three mutants (αR350Wβγ, αV351Aβγ, and αG355Rβγ) were similar to that of WT. However, three mutant channels (αR350Wβγ, αH354Rβγ, and αG355Rβγ) exhibited a reduced Na+ self-inhibition response. Open probability of αR350Wβγ was significantly greater than that of WT. Moreover, other Arg-350 variants, including αR350G, αR350L, and αR350Q, also had significantly increased channel activity. A direct comparison of αR350W and two previously reported gain-of-function variants revealed that αR350W increases ENaC activity similarly to αW493R, but to a much greater degree than does αC479R. Our results indicate that αR350W along with αR350G, αR350L, and αR350Q, and αG355R are novel gain-of-function variants that function as gating modifiers. The location of these multiple functional variants suggests that the αENaC β-ball domain portion that interfaces with the palm domain of βENaC critically regulates ENaC gating.