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1

Molecular mechanisms of proton modulation in the GABAA receptor as compared to low pH activation scheme in the GLIC ion

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Michalowski M. A., Mozrzymas J. W.
Acta Neurobiologiae Experimentalis
|
2019
|
tom 79
|
nr Suppl.1
INTRODUCTION: The GABAA receptor (GABAAR) belongs to a family of pentameric ligand gated ion channels (pLGICs). Another member of this family, bacterial GLIC, is directly activated by protons. Recent studies showed that protonation of GLIC’s E35 residue starts a cascade of interactions that end at the channel pore and lead to its opening. GABAAR preserves sensitivity to protons, and ligand elicited currents can be modulated by them. The exact molecular mechanism of this action is not known. AIM(S): This study aims to elucidate the molecular action and sensitivity of GABAAR to protons, and to answer the question of whether those mechanism are similar to proton activation in GLIC. METHOD(S): Sequences of pLGICs were obtained from the UniProt database and aligned using T‑Coffe. Additional manual refinements and alignment analysis was done in JalView. Structures of GLIC and GABAAR were obtained from RCSB PDB. Homology modeling was done using Modeller. pKa values of GABAAR residues were assessed with PropKa 3. Structure analysis and visualization was done in VMD. RESULTS: Using in silico methods, pKa values of GABAAR residues were assessed, and we determined the positions that are homologous to proton sensitive residues of GLIC. In GABAAR, no proton sensitive residues at positions homologous to GLIC E35 were detected. Instead, residues with pKa values in proximity of physiological values was found at the cys‑loop (e.g. E138 and H142 at α subunit and E150 and H156 at γ subunit), GABA binding site (βE155), upper part of ion pore (βH267 and βE270), and in its bottom part (intracellular part of the receptor, αE303 and βE313). CONCLUSIONS: The molecular scheme of low pH activation of GLIC is not preserved in GABAAR. Lack of proton sensitive residue at positions homologous to E35, and the presence of multiple possibly proton susceptible residues spread within receptor structure indicate a complex and scattered mechanism of modulation. FINANCIAL SUPPORT: Supported by National Science Center grant 2018/29/N/NZ1/02834.
2

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

81%
Brodzki M., Michalowski M. A., Gos M., Mozrzymas J. W.
Acta Neurobiologiae Experimentalis
|
2019
|
tom 79
|
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

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

71%
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.
4

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

51%
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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