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

2020 ◽  
Author(s):  
Joseph W. Nors ◽  
Shipra Gupta ◽  
Marcel P. Goldschen-Ohm
Keyword(s):  
Psychotropic Drugs ◽  
Channel Gating ◽  
Side Chain ◽  
Inhibitory Neurotransmitter ◽  
Channel Pore ◽  
Critical Residue ◽  
Positive Modulator ◽  
The Central Nervous System ◽  
Pore Opening ◽  
Important Residue

AbstractBenzodiazepines (BZDs) are a class of widely prescribed psychotropic drugs. Their anxiolytic and sedative effects are conferred by modulating the activity of GABAA receptors (GABAARs), which are the primary inhibitory neurotransmitter receptors throughout the central nervous system. However, the physical mechanism by which BZDs exert their effects on the receptor is poorly understood. In particular, BZDs require coapplication with an agonist to effectively open the channel pore, making it difficult to dissect whether the drug has altered either agonist binding or channel gating as these two processes are intimately coupled. To isolate effects on gating we used a spontaneously active gain of function mutant (α1L9’Tβ2γ2L) that is directly gated by BZDs alone in the absence of agonist. In the α1L9’T background we explored effects of alanine substitutions throughout the α1M2-M3 linker on modulation of the channel pore by the BZD positive modulator diazepam (DZ). The M2-M3 linker is known to be an important element for channel activation. Linker mutations generally impaired unliganded pore opening, indicating that side chain interactions are important for channel gating in the absence of bound agonist. All but one mutation had no effect on the transduction of chemical energy from DZ binding to pore gating. Strikingly, α1V279A doubles DZ’s energetic contribution to gating, whereas larger side chains at this site do not. In a wild-type background α1V279A enhances DZ-potentiation of currents evoked by saturating GABA, consistent with a direct effect on the pore closed/open equilibrium. Our observations identify an important residue regulating coupling between the BZD site and the pore gate, thereby shedding new light on the molecular mechanism of a frequently prescribed class of psychotropic drugs.

scholarly journals A critical residue in the α1M2–M3 linker regulating mammalian GABAA receptor pore gating by diazepam

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Joseph W Nors ◽  
Shipra Gupta ◽  
Marcel P Goldschen-Ohm
Keyword(s):  
Ion Channels ◽  
Synaptic Transmission ◽  
Molecular Mechanism ◽  
Psychotropic Drugs ◽  
Wild Type ◽  
Critical Residue ◽  
Positive Modulator ◽  
Pore Opening ◽  
Important Residue

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'Tβ2γ2L) 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.

scholarly journals Gating Competence of Constitutively Open CLC-0 Mutants Revealed by the Interaction with a Small Organic Inhibitor

2003 ◽  
Vol 122 (3) ◽  
pp. 295-306 ◽  
Author(s):  
Sonia Traverso ◽  
Laura Elia ◽  
Michael Pusch
Keyword(s):  
Chloride Channels ◽  
Bound State ◽  
Side Chain ◽  
Pore Channel ◽  
Kinetic Properties ◽  
Wild Type ◽  
High Affinity ◽  
Critical Residue ◽  
Fast Gating

Opening of CLC chloride channels is coupled to the translocation of the permeant anion. From the recent structure determination of bacterial CLC proteins in the closed and open configuration, a glutamate residue was hypothesized to form part of the Cl−-sensitive gate. The negatively charged side-chain of the glutamate was suggested to occlude the permeation pathway in the closed state, while opening of a single protopore of the double-pore channel would reflect mainly a movement of this side-chain toward the extracellular pore vestibule, with little rearrangement of the rest of the channel. Here we show that mutating this critical residue (Glu166) in the prototype Torpedo CLC-0 to alanine, serine, or lysine leads to constitutively open channels, whereas a mutation to aspartate strongly slowed down opening. Furthermore, we investigated the interaction of the small organic channel blocker p-chlorophenoxy-acetic acid (CPA) with the mutants E166A and E166S. Both mutants were strongly inhibited by CPA at negative voltages with a >200-fold larger affinity than for wild-type CLC-0 (apparent KD at −140 mV ∼4 μM). A three-state linear model with an open state, a low-affinity and a high-affinity CPA-bound state can quantitatively describe steady-state and kinetic properties of the CPA block. The parameters of the model and additional mutagenesis suggest that the high-affinity CPA-bound state is similar to the closed configuration of the protopore gate of wild-type CLC-0. In the E166A mutant the glutamate side chain that occludes the permeation pathway is absent. Thus, if gating consists only in movement of this side-chain the mutant E166A should not be able to assume a closed conformation. It may thus be that fast gating in CLC-0 is more complex than anticipated from the bacterial structures.

scholarly journals An Inactivation Stabilizer of the Na+ Channel Acts as an Opportunistic Pore Blocker Modulated by External Na+

2005 ◽  
Vol 125 (5) ◽  
pp. 465-481 ◽  
Author(s):  
Ya-Chin Yang ◽  
Chung-Chin Kuo
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Dissociation Constants ◽  
Channel Gating ◽  
Structural Motif ◽  
Na Channel ◽  
Channel Pore ◽  
Conduction Pathway ◽  
Gating Process ◽  
Pore Blocker

The Na+ channel is the primary target of anticonvulsants carbamazepine, phenytoin, and lamotrigine. These drugs modify Na+ channel gating as they have much higher binding affinity to the inactivated state than to the resting state of the channel. It has been proposed that these drugs bind to the Na+ channel pore with a common diphenyl structural motif. Diclofenac is a widely prescribed anti-inflammatory agent that has a similar diphenyl motif in its structure. In this study, we found that diclofenac modifies Na+ channel gating in a way similar to the foregoing anticonvulsants. The dissociation constants of diclofenac binding to the resting, activated, and inactivated Na+ channels are ∼880 μM, ∼88 μM, and ∼7 μM, respectively. The changing affinity well depicts the gradual shaping of a use-dependent receptor along the gating process. Most interestingly, diclofenac does not show the pore-blocking effect of carbamazepine on the Na+ channel when the external solution contains 150 mM Na+, but is turned into an effective Na+ channel pore blocker if the extracellular solution contains no Na+. In contrast, internal Na+ has only negligible effect on the functional consequences of diclofenac binding. Diclofenac thus acts as an “opportunistic” pore blocker modulated by external but not internal Na+, indicating that the diclofenac binding site is located at the junction of a widened part and an acutely narrowed part of the ion conduction pathway, and faces the extracellular rather than the intracellular solution. The diclofenac binding site thus is most likely located at the external pore mouth, and undergoes delicate conformational changes modulated by external Na+ along the gating process of the Na+ channel.

scholarly journals Neurological Syndromes Associated with Anti-GAD Antibodies

2020 ◽  
Vol 21 (10) ◽  
pp. 3701 ◽  
Author(s):  
Maëlle Dade ◽  
Giulia Berzero ◽  
Cristina Izquierdo ◽  
Marine Giry ◽  
Marion Benazra ◽  
...  
Keyword(s):  
Unmet Need ◽  
Acid Decarboxylase ◽  
Gamma Aminobutyric Acid ◽  
Gad Antibodies ◽  
Intracellular Enzyme ◽  
Inhibitory Neurotransmitter ◽  
Predictors Of Response ◽  
Physiologic Function ◽  
The Central Nervous System ◽  
Relative Rarity

Glutamic acid decarboxylase (GAD) is an intracellular enzyme whose physiologic function is the decarboxylation of glutamate to gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter within the central nervous system. GAD antibodies (Ab) have been associated with multiple neurological syndromes, including stiff-person syndrome, cerebellar ataxia, and limbic encephalitis, which are all considered to result from reduced GABAergic transmission. The pathogenic role of GAD Ab is still debated, and some evidence suggests that GAD autoimmunity might primarily be cell-mediated. Diagnosis relies on the detection of high titers of GAD Ab in serum and/or in the detection of GAD Ab in the cerebrospinal fluid. Due to the relative rarity of these syndromes, treatment schemes and predictors of response are poorly defined, highlighting the unmet need for multicentric prospective trials in this population. Here, we reviewed the main clinical characteristics of neurological syndromes associated with GAD Ab, focusing on pathophysiologic mechanisms.

scholarly journals Mutations Causing Slow-Channel Myasthenia Reveal That a Valine Ring in the Channel Pore of Muscle AChR is Optimized for Stabilizing Channel Gating

2016 ◽  
Vol 37 (10) ◽  
pp. 1051-1059 ◽  
Author(s):  
Xin-Ming Shen ◽  
Tatsuya Okuno ◽  
Margherita Milone ◽  
Kenji Otsuka ◽  
Koji Takahashi ◽  
...  
Keyword(s):  
Channel Gating ◽  
Channel Pore ◽  
Slow Channel

scholarly journals Permeability Properties of Enac Selectivity Filter Mutants

2001 ◽  
Vol 118 (6) ◽  
pp. 679-692 ◽  
Author(s):  
Stephan Kellenberger ◽  
Muriel Auberson ◽  
Ivan Gautschi ◽  
Estelle Schneeberger ◽  
Laurent Schild
Keyword(s):  
Amino Acid ◽  
Transmembrane Domain ◽  
Ion Selectivity ◽  
Critical Role ◽  
Selectivity Filter ◽  
Side Chain ◽  
Na Channel ◽  
Ionic Selectivity ◽  
Channel Pore ◽  
Subunit Interface

The epithelial Na+ channel (ENaC), located in the apical membrane of tight epithelia, allows vectorial Na+ absorption. The amiloride-sensitive ENaC is highly selective for Na+ and Li+ ions. There is growing evidence that the short stretch of amino acid residues (preM2) preceding the putative second transmembrane domain M2 forms the outer channel pore with the amiloride binding site and the narrow ion-selective region of the pore. We have shown previously that mutations of the αS589 residue in the preM2 segment change the ion selectivity, making the channel permeant to K+ ions. To understand the molecular basis of this important change in ionic selectivity, we have substituted αS589 with amino acids of different sizes and physicochemical properties. Here, we show that the molecular cutoff of the channel pore for inorganic and organic cations increases with the size of the amino acid residue at position α589, indicating that αS589 mutations enlarge the pore at the selectivity filter. Mutants with an increased permeability to large cations show a decrease in the ENaC unitary conductance of small cations such as Na+ and Li+. These findings demonstrate the critical role of the pore size at the αS589 residue for the selectivity properties of ENaC. Our data are consistent with the main chain carbonyl oxygens of the αS589 residues lining the channel pore at the selectivity filter with their side chain pointing away from the pore lumen. We propose that the αS589 side chain is oriented toward the subunit–subunit interface and that substitution of αS589 by larger residues increases the pore diameter by adding extra volume at the subunit–subunit interface.

scholarly journals The structure of MgtE in the absence of magnesium provides new insights into channel gating

PLoS Biology ◽  
2021 ◽  
Vol 19 (4) ◽  
pp. e3001231
Author(s):  
Fei Jin ◽  
Minxuan Sun ◽  
Takashi Fujii ◽  
Yurika Yamada ◽  
Jin Wang ◽  
...  
Keyword(s):  
Channel Gating ◽  
Channel Activity ◽  
State Structure ◽  
Closed State ◽  
Channel Inactivation ◽  
Cryo Electron Microscopy ◽  
Pore Opening ◽  
Tm Domain ◽  
Functional Analyses ◽  
Cytoplasmic Side

MgtE is a Mg2+ channel conserved in organisms ranging from prokaryotes to eukaryotes, including humans, and plays an important role in Mg2+ homeostasis. The previously determined MgtE structures in the Mg2+-bound, closed-state, and structure-based functional analyses of MgtE revealed that the binding of Mg2+ ions to the MgtE cytoplasmic domain induces channel inactivation to maintain Mg2+ homeostasis. There are no structures of the transmembrane (TM) domain for MgtE in Mg2+-free conditions, and the pore-opening mechanism has thus remained unclear. Here, we determined the cryo-electron microscopy (cryo-EM) structure of the MgtE-Fab complex in the absence of Mg2+ ions. The Mg2+-free MgtE TM domain structure and its comparison with the Mg2+-bound, closed-state structure, together with functional analyses, showed the Mg2+-dependent pore opening of MgtE on the cytoplasmic side and revealed the kink motions of the TM2 and TM5 helices at the glycine residues, which are important for channel activity. Overall, our work provides structure-based mechanistic insights into the channel gating of MgtE.

Effects of Novel 6-Desfluoroquinolones and Classic Quinolones on Pentylenetetrazole-Induced Seizures in Mice

1999 ◽  
Vol 43 (7) ◽  
pp. 1729-1736 ◽  
Author(s):  
A. De Sarro ◽  
V. Cecchetti ◽  
V. Fravolini ◽  
F. Naccari ◽  
O. Tabarrini ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Chemical Structure ◽  
Aminobutyric Acid ◽  
Side Chain ◽  
Amino Group ◽  
Principal Mechanism ◽  
The Central Nervous System ◽  
Clonic Seizures ◽  
The Relationship ◽  
Quinolone Derivatives

ABSTRACT There have been several reports that convulsions, although rare, occur in patients who receive fluoroquinolones. In this study, the proconvulsant effects exhibited by a novel series of 6-desfluoroquinolones and some classic quinolones on pentylenetetrazole (PTZ)-induced seizures in mice were evaluated and compared. Animals were intraperitoneally injected with vehicle or quinolone derivatives (5 to 100 μg/g of body weight) 30 min before the subcutaneous (s.c.) administration of PTZ (40 μg/g). In each experiment, mice were then observed for 1 h to monitor for the incidence and onset of clonic seizures. The order of proconvulsant activity in our epileptic model was MF5184 > MF5187 > pefloxacin > MF5189 > ofloxacin > ciprofloxacin > MF5140 > MF5181 > MF5137 > rufloxacin > MF5143 > MF5158 > MF5191 > MF5128 > MF5138 > cinoxacin > MF5142 > norfloxacin > nalidixic acid. The relationship between the chemical structure and the proconvulsant activity of 6-desfluoroquinolone derivatives was studied. We observed that, in terms of toxicity to the central nervous system (CNS), besides the heterocyclic side chain (moiety) at the C-7 position, the C-6 substituent also appears to play an important role. In particular, a hydrogen at the C-6 position seemed to be responsible for major neurotoxic activity in comparison to an amino group located in the same position. The relationship between lipophilicity and proconvulsant activity was also investigated. We did not find any clear relationship between a higher level of lipophilicity and major proconvulsant properties. Although the principal mechanism by which quinolones induce potentiation of the proconvulsant effects of PTZ cannot be easily determined, it is possible that the convulsions are caused by drug interactions, because both PTZ and quinolones are believed to increase excitation of the CNS by inhibition of γ-aminobutyric acid binding to receptors.

scholarly journals An Updated Review on Pharmaceutical Properties of Gamma-Aminobutyric Acid

Molecules ◽  
2019 ◽  
Vol 24 (15) ◽  
pp. 2678 ◽  
Author(s):  
Dai-Hung Ngo ◽  
Thanh Sang Vo
Keyword(s):  
Neuronal Development ◽  
Aminobutyric Acid ◽  
Gamma Aminobutyric Acid ◽  
Peripheral Tissues ◽  
Inhibitory Neurotransmitter ◽  
Anti Cancer ◽  
The Central Nervous System ◽  
Potential Alternative ◽  
Pharmaceutical Properties ◽  
Anti Inflammation

Gamma-aminobutyric acid (Gaba) is a non-proteinogenic amino acid that is widely present in microorganisms, plants, and vertebrates. So far, Gaba is well known as a main inhibitory neurotransmitter in the central nervous system. Its physiological roles are related to the modulation of synaptic transmission, the promotion of neuronal development and relaxation, and the prevention of sleeplessness and depression. Besides, various pharmaceutical properties of Gaba on non-neuronal peripheral tissues and organs were also reported due to anti-hypertension, anti-diabetes, anti-cancer, antioxidant, anti-inflammation, anti-microbial, anti-allergy, hepato-protection, reno-protection, and intestinal protection. Therefore, Gaba may be considered as potential alternative therapeutics for prevention and treatment of various diseases. Accordingly, this updated review was mainly focused to describe the pharmaceutical properties of Gaba as well as emphasize its important role regarding human health.

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