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

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Czyzewska M. M. , Szczot M. , Pollastro F. , Appendino G. , Mozrzymas J. W.

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nr S

EN

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).

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

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Brodzki M. , Czyzewska M. M. , Rutkowski R. , Kisiel M. , Jatczak M. , Mozrzymas J. W.

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nr Supl.

EN

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.

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

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Kisiel M. , Jatczak M. , Czyzewska M. M. , Brodzki M. , Cajkowski C. , Mozrzymas J. W.

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nr Supl.

EN

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.

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

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Jatczak-Sliwa M. , Terejko K. , Brodzki M. , Michalowski M. A. , Czyzewska M. M. , Nowicka J. M. , Andrzejczak A. , Srinivasan R. , Mozrzymas J. W.

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nr Suppl.1

EN

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.