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1

Mutation in F64 position of GABAAR alpha subunit affects the receptor gating

100%
Czyzewska M. M., Szczot M., Kisiel M., Mozrzymas J. W.
Acta Neurobiologiae Experimentalis
|
2013
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tom 73
|
nr Suppl.1
GABAA receptor (GABAAR) is a pentamer, formed by 2α, 2β and γ subunit. GABA binding site is localized at the interface between α and β subunits. Our aim was to characterize how mutation localized at the binding pocket (α1F64) influences agonist binding and conformational transitions between bound receptor states (gating). We used patchclamp technique with ultrafast perfusion system and HEK 293 cells expressing native or mutated GABAARs. All mutations (α1F64C/L/A) right-shifted the dose-dependent curve and accelerated current deactivation, indicating impairment of binding. Reduction of fast desensitization, which in the case of α1F64C was complete, indicates changes in gating. Moreover, the mutation decreased the maximum open channel probability, a key feature of receptor gating. Experiments performed with different agonists confirmed mutation-induced changes in the channel’s opening/closing transitions (gating). Quantitative analysis based on model simulations indicated that this mutation mostly affected the channel state which precedes opening and is interpreted as a macromolecule destabilization (“priming” or “flipping”) following agonist binding, whereas desensitization or efficacy are affected to a smaller extent. Our data thus suggest that mutation of α1F64 residue affects the “transition wave” from binding sites to the channel gate.
2

Hydrophobic residue in GABAA receptor ligand binding site strongly influences receptor gating

100%
Szczot M., Kisiel M., Czyzewska M. M., Mozrzymas J. W.
Acta Neurobiologiae Experimentalis
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2012
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tom 72
|
nr 2
Despite broad knowledge on GABAA receptors (GABAARs) structure, the mechanism of ligand induced conformational transitions remains poorly understood. To address this issue we have examined the activity of a recombinant α1/β1/γ2 GABAAR with point mutation introduced at the ligand binding site. Currents were elicited by ultrafast GABA applications and measured using patch-clamp technique. We show that cystein mutation of single hydrophobic residue not only weakened the agonist binding but also abolished fast desensitization and slowed the onset of currents evoked by saturating GABA. Non-stationary variance analysis showed that the mutation does not affect single channel conductance, but reduces maximal open probability, further indicating a change in gating properties. Ratio of current amplitudes elicited by pentobarbital (which activates GABAAR by different pathway than GABA), and by GABA was higher for mutant receptors, supporting interference with receptor gating. Our data show that the considered residue may strongly influence conformational transitions of GABAARs thus indicating this residue as a key element in transduction of free energy supplied by agonist binding to the conformational transitions.
3

Alpha1F64 influences GABAAR gating through flipping mechanism

88%
Kisiel M., Szczot M., Czyzewska M. M., Jatczak M., Mozrzymas J. W.
Acta Neurobiologiae Experimentalis
|
2014
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tom 74
|
nr 3
GABAARs are crucial for neuronal inhibition. Using patch-clamp technique with ultrafast perfusion we found that mutations of hydrophobic residue at GABA-binding site affected not only binding affinity but also kinetics of macroscopic desensitization. Nonstationary variance analysis indicated that α1F64C mutation reduces maximum open probability. To obtain further information about the role of α1F64 we used two different agonists. Experiments with a partial agonist, P4S, suggested an impact of α1F64C mutation on the channel gating efficacy. Application of muscimol (with higher affinity than GABA) entailed a partial rescue of rapid desensitization in α1F64Lβ1γ2 receptors but in cysteine mutants – did not. Model simulations show that observed effects result from changes in flipping mechanism which links binding and gating. We conclude that α1F64 plays a crucial role in signal transduction from binding site to the channel gate. Supported by NCN Grant 350/B/P01/2011/40 to JWM
4

Mutation at GABA binding site (alpha1F64) affects both binding and gating properties of GABAA receptors

88%
Mozrzymas J. W., Kisiel M., Czyzewska M. M., Jatczak M., Szczot M.
Acta Neurobiologiae Experimentalis
|
2014
|
tom 74
|
nr 3
In spite of broad knowledge of GABAAR pharmacokinetics, molecular mechanisms of conformation transitions remain elusive. Intriguingly, GABA binding site is distant from the channel gate (ca. 5 nm). In this study we searched for residues at GABA binding site involved in conveying binding energy to the channel gate of α1β2γ2 GABAARs. Mutation at α1F64 decreased the receptor affinity (shifted dose-response and reduced binding rate in racing protocol) and, in addition, strongly influenced onset, deactivation and desensitization of currents elicited by saturating agonist, indicating gating modification. Non-stationary variance analysis of GABA- or pentobarbital-evoked currents showed that the cysteine mutation strongly decreased the open channel probability confirming its impact on receptor gating. We conclude that α1F64 residue, although located at the binding site, strongly affects gating properties of GABAA receptors. Supported by NCN Grant 350/B/P01/2011/40 to JWM.
5

Absinthe ingredient monoterpenoid alfa-thujone is a modulator of neuronal GABAA receptors

88%
Czyzewska M. M., Szczot M., Pollastro F., Appendino G., Mozrzymas J. W.
Acta Neurobiologiae Experimentalis
|
2011
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tom 71
|
nr S
Monoterpenoid α-thujone is a compound found in absinthe, alcoholic beverage commonly abused (often by famous artists) in late XIX and early XX century. It has been long speculated that α-thujone is responsible for some adverse effects of this liquor including seizures. It has been investigated that the effect of α-thujone is related to its action on GABAA receptors but a precise pharmacological analysis is lacking. In the present work we investigated mechanism of α-thujone action on GABAergic currents (current responses to exogenous GABA and miniature synaptic currents) in cultured hippocampal neurons. We found that high concentrations (100 - 300 μM) of α-thujone have only modest effect on amplitude of responses elicited by low (3 μM) [GABA], but it prolonged the current rise-time by nearly fivefold and significantly decreased the current fading during prolonged GABA application. At saturating [GABA] (10 mM), the amplitude of current response, elicited by rapid agonist applications, was significantly reduced by 300 µM α-thujone and current onset was also slowed down almost three times but this effect was markedly smaller than for currents evoked by low [GABA]. To assess the impact of α-thujone on desensitization, the time course of currents elicited by prolonged applications of saturating [GABA] were analyzed. In the presence of α-thujone the steady-state to peak value was markedly increased indicating a decrease in the extent of macroscopic desensitization. To check for the effect of α-thujone on synaptic transmission, we measured miniature inhibitory currents (mIPSC) and found that at 300 μM of α-thujone, amplitude and frequency of mIPSC were significantly reduced. In conclusion, our results suggest that α-thujone may act as a low potency allosteric modulator of GABAARs. Preliminary kinetic analysis suggests that this compound affects both binding and gating of neuronal GABAARs. This study was funded by the FNP award Mistrz (contract No 7/2008).
6

Different pharmacological profile in alpha1-gamma2 and alpha1-beta2-gamma2 GABAA receptors

76%
Brodzki M., Czyzewska M. M., Rutkowski R., Kisiel M., Jatczak M., Mozrzymas J. W.
Acta Neurobiologiae Experimentalis
|
2015
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tom 75
|
nr Supl.
BACKGROUND AND AIMS: GABAA receptors (GABAAR) mediate the main component of ionotropic inhibitory transmission in adult mammalian brain. These receptors are heteropentamers and are strongly diversified throughout the CNS but the most frequent subunit composition is alpha1-beta2-gamma2. It has been reported that alpha1-gamma2 receptors can be potently expressed in recombinant GABAAR model (Verdoorn et al. 1990). In this study we aimed to characterize the kinetic and pharmacological profile of these receptors in comparison to alpha1-beta2-gamma2 ones. METHODS: We used patch-clamp technique with ultrafast (~10e–4 s) solution exchange based on theta-glass capillaries driven by a piezoelectric translator. We used HEK293 cells which were transiently transfected by GABAAR subunit cDNA using standard calcium phosphate method. Modulators used were zinc ions, low extracellular pH, flurazepam and pentobarbital. RESULTS: Zinc ions inhibited current responses more strongly for alpha1-gamma2 GABAARs, however, zinc effect on desensitization onset was observed only for alpha1-beta2-gamma2 receptors. In the case of responses mediated by alpha1-beta2-gamma2 receptors, lowering extracellular pH enhanced current amplitudes and prolonged deactivation time course of currents elicited by short and saturating GABA pulses to a much larger extent than for alpha1-gamma2 ones. Saturating GABA elicited responses mediated by alpha1-beta2-gamma2 receptors were slightly inhibited by flurazepam, whereas in the case of alpha1-gamma2 receptors, currents were significantly potentiated. Activation by high concentrations of pentobarbital yielded similar rebound current amplitudes in both receptor subtypes. CONCLUSIONS: We found that although alpha1-gamma2 receptors show a similar kinetic profile to alpha1-beta2-gamma2 receptors they are characterized by pharmacological properties that are substantially different. Supported by NCN grant DEC-2013/11/B/NZ3/00983.
7

GABAA receptor binding site residue Beta2 glutamate 155:possible role in channel preactivation

76%
Kisiel M., Jatczak M., Czyzewska M. M., Brodzki M., Cajkowski C., Mozrzymas J. W.
Acta Neurobiologiae Experimentalis
|
2015
|
tom 75
|
nr Supl.
BACKGROUND AND AIMS: The GABAA receptor is the main mediator responsible for inhibitory transmission in the brain. In our previous work (Szczot et al. 2014), we demonstrated for α1β2γ2 receptors that “classical” channel gating (opening/closing and desensitization) is preceded by a preactivation step, which is most likely initiated at the agonist-binding site. Here, we investigated the role of β2E155 residue in channel gating focusing on preactivation. Residue β2E155 is located in the GABA-binding site and may directly interact with agonist. Moreover, agonist induced local motions near this residue suggests it is an initial trigger that couples agonist binding to channel gating. METHODS: In this study, we combined ultrafast solution exchange with patch-clamp electrophysiology to record macroscopic currents mediated by wild-type and mutant (β2E155C) α1β2γ2 and α1β2 receptors. RESULTS: Cysteine substitution of β2E155 caused a large right-shift of the dose–response curves for GABA-elicited currents, which was independent of the presence of γ2 subunit. Furthermore, especially for α1β2γ2 receptors, β2E155C slowed down macroscopic desensitization kinetics. The mutant receptors also exhibited spontaneous channel activity. Taken together, the data suggest this mutation alters not only GABA binding but also GABA-mediated gating transitions. Nonstationary noise analysis of variance showed that for α1β2γ2 receptors, the β2E155C mutation significantly decreased maximal open probability without affecting single channel conductance. CONCLUSIONS: Model kinetic simulations of our data indicate that β2E155 is likely involved in preactivation transitions that precede channel opening supporting its role as an initial trigger for coupling binding to gating. This research has been financially supported by grant National Centre of Science grant: DEC-2013/11/B/NZ3/00983 and by ministry grant Pbmn135.
8

Flurazepam modulates gating of spontaneous and agonist-evoked GABAA receptor activity via distinct mechanisms

64%
Jatczak-Sliwa M., Terejko K., Brodzki M., Michalowski M. A., Czyzewska M. M., Nowicka J. M., Andrzejczak A., Srinivasan R., Mozrzymas J. W.
Acta Neurobiologiae Experimentalis
|
2018
|
tom 78
|
nr Suppl.1
GABAA receptors mediate inhibitory transmission in the adult mammalian brain and are modulated by many clinically used drugs such as benzodiazepines. It has pre‑ viously been demonstrated that benzodiazepines affect binding and gating transitions. However, the mechanism of their modulation is still not fully understood. In our present study we address this problem by examining modulation of spontaneous activity by the benzodiaz‑ epine flurazepam and its cross-talk with ligand-evoked activity of wild-type and mutated (at α1F64 position lo‑ cated in the GABA-binding site, shown to affect preac‑ tivation/flipping transition) α1β2γ2 GABAA receptors. We used patch-clamp technique to measure macroscop‑ ic and single-channel currents mediated by wild-type and mutated (Leu, Ala or Cys substitution at the α1F64 position) GABAA receptors. Spontaneous activity was measured using a BioLogic Perfusion System and picro‑ toxin application. We also performed experiments for saturating GABA and partial agonist applications using an ultrafast perfusion system (theta-glass). We used flu‑ razepam pretreatment and co-application (flurazepam with GABA) protocols, which allowed us to observe the cross-talk between spontaneous and ligand-induced ac‑ tivity. Model simulations were performed in ChaneLab software. α1F64 mutants exhibited larger spontaneous activity compared to wild-type receptors and fluraze‑ pam potentiated this activity to the same extent for all considered receptor types. Our single-channel analysis showed prolonged openings upon flurazepam treat‑ ment. For saturating [GABA] applications in a pretreat‑ ment protocol, we found a significant correlation be‑ tween the increase of the overshoot (amplitude above the baseline after agonist removal) and the amplitude of currents upon flurazepam application. Flurazepam po‑ tentiates the amplitude of currents mediated by mutants after GABA and partial agonist application and affects their kinetics. Our model simulations indicate that flu‑ razepam affects opening/closing transitions of sponta‑ neous activity but affects preactivation and desensitiza‑ tion transitions of ligand-induced activity. Flurazepam’s mechanism of GABAA receptor modulation is different for spontaneous and ligand-induced activity. Moreover, spontaneous openings clearly affect agonist-evoked re‑ sponses. Altogether, flurazepam alters the GABAA re‑ ceptor gating transitions in a manner dependent on the receptor ligation. Supported by NCN grants: 2013/11/B/ NZ3/00983 and 2015/18/A/NZ1/00395.
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