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Persistance of some polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans of pentachlorophenol in human adipose tissue

100%
Gorski T., Konopka L., Brodzki M.
Roczniki Państwowego Zakładu Higieny
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1984
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tom 35
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nr 4
2

Loop G of the GABAA receptor orthosteric binding site is involved in the final stages of channel gating

100%
Brodzki M., Michalowski M. A., Gos M., Mozrzymas J. W.
Acta Neurobiologiae Experimentalis
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2019
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tom 79
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nr Suppl.1
INTRODUCTION: Inhibition of neuronal activity is shaped primarily by GABAA receptors. Agonist binding site (BS) at the β+/α‑ intersubunit interface is composed of 7 loops (A‑C from β and D‑G from α subunit), and the Loop G has been reported to play a major role in receptor activation, however the exact mechanism is not clear. α1F45 residue at Loop G has been shown to be engaged in receptor activation despite not directly contacting the agonist, and is well positioned for interactions with other crucial BS residues. Since this loop spans from the BS to the extracellular-transmembrane domain interface, it might have an important role in transferring energy of BS conformational transitions to the pore region. AIM(S): This study aims to reveal the role of loop G in distinct steps of receptor activation. METHOD(S): We used rapid agonist application to elicit macroscopic responses and single-channel recordings of GABA-evoked currents for wild-type (WT) and mutated (α1F45C/L/K/G) receptors. Model simulations of macroscopic and single-channel activity and in silico structural analysis have been performed. RESULTS: Mutated receptors showed a different kinetic profile of macroscopic currents (except α1F45L) with faster deactivation (α1F45C/K/G) and impaired desensitization (α1F45C/G). Single‑channel currents showed profound differences in all mutants; that is, closures were prolonged, openings were shortened, and Popen within bursts was reduced. Model simulations revealed changes primarily in opening/closing transitions. The homology model of WT showed loop G energy minimum at the α1F45 position, underlining its role in loop stability. In α1F45G/K mutants, this minimum declined. In α1F45G mutant, it can be attributed to the BS aromatic box disruption and α1F45K substitution could impair the GABA – α1R66 interaction. CONCLUSIONS: Mutations of the α1F45 residue in loop G of the BS affects final gating stages. This indicates the role of loop G in linking binding and gating processes. FINANCIAL SUPPORT: Supported by NCN grant UMO‑2015/18/A/NZ1/00395.
3

Linking agonist binding to GABAA receptor opening transition

88%
Morrzymas J. W., Kisiel M., Jatczak M., Czyzewska M., Brodzki M.
Acta Neurobiologiae Experimentalis
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2015
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tom 75
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nr Supl.
GABAA receptors are responsible for mediating inhibition in the adult mammalian CNS. These receptors are greatly diversified but the most common type is alpha1beta2gamma2. Intriguingly, GABA binding sites on GABAA receptor are remarkably distant (ca. 5 nm) from the channel gate. This structural feature raises the question about molecular mechanisms underlying the energy transfer from binding process to conformational transitions. Recently, we found that mutation of binding site residue alpha1F64 affects not only binding but also conformational transitions. Extensive experimental data and model simulations indicated that the major mechanism underlying alpha1F64 mutation is to affect so called preopening (channel remains closed but increases its propensity to open) and desensitization (Szczot et al. 2014). Singlechannel revealed that, additionally, this mutation shortens the channel opening time, indicating increase in the closing rate. Interestingly, alpha1F64 mutation was found to affect GABAAR proton sensitivity (Huang et al. 2004). We thus checked the impact of pH changes on WT and mutated alpha1beta2gamma2 receptors and found that protons modulate gating by altering mainly preactivation and desensitization. Kinetic analysis of alpha1beta2gamma2 receptors with mutation at a different location within agonist binding site (beta2E155) suggested again involvement of this residue in preactivation transition. Taking altogether, preactivation transition emerges as a key conformation transition which affects both kinetics and pharmacological sensitivity of currents mediated by alpha1beta2gamma2 GABAA receptors. Supported by NCN grant DEC-2013/11/B/NZ3/00983.
4

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
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2015
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tom 75
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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.
5

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
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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.
6

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
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2018
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tom 78
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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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