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1

GABAA Beta2 subunit loop C shapes binding and gating properties of receptor activation

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Terejko K., Kaczor P. T., Michalowski M. A., Dabrowska A., Mozrzymas J. W.
Acta Neurobiologiae Experimentalis
|
2019
|
tom 79
|
nr Suppl.1
INTRODUCTION: GABAA receptors (GABAARs) are pentameric ligand-gated ion channels that are crucial in fast inhibitory transmission in adult CNS. The activation process of GABAAR couples agonist binding to the binding site in the extracellular domain, with an opening of the channel gate in the far-distant transmembrane domain. GABAAR gating is a complex process that includes preactivation (flipping), which is a step of bound receptor that remains closed and precedes opening. Our recent study shows that flipping is modulated by benzodiazepines. A key element in GABAARs activation is loop C capping, an inward movement of the β2 subunit loop C upon ligand binding; however, the exact role of loop C in relation to GABAAR binding and gating remains elusive. AIM(S): This study aims to explain how a mutation of the β2F200 residue in loop C affects functioning of the GABAAR in reference to receptor binding and gating, including modulation of preactivation by flurazepam (benzodiazepine). METHOD(S): β2F200 mutated receptors (Tyr, Ile, Cys) were expressed in HEK293 cells. Patch clamp was used to record macroscopic currents elicited by saturating [GABA] (combined with ultrafast perfusion system) and single channel currents. Kinetic analysis was preformed and followed by kinetic modeling. RESULTS: β2F200 mutants exhibited a shift in dose‑response relationship. The mutation significantly slowed down current onset and desensitization, but deactivation was accelerated. Flurazepam potentiated currents evoked by saturating [GABA] in contrast to WT receptors. Single‑channel analysis showed a significant change in all shut time distributions components and shortening of open time distributions. CONCLUSIONS: Kinetic modeling of macroscopic and single channel currents confirmed alteration in all considered gating properties. In silico ligand docking indicated a drop in the binding affinity for each mutant. GABAAR loop C plays a critical role in receptor binding and gating. FINANCIAL SUPPORT: NCN grant 2015/18/A/NZ1/ 00395.
2

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.
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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