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1

Protons affect GABAA receptor gating by altering both preactivation and desensitization transitions

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Kisiel M., Jatczak M., Mozrzymas J. W.
Acta Neurobiologiae Experimentalis
|
2015
|
tom 75
|
nr Supl.
BACKGROUND AND AIMS: GABAA receptors are essential for inhibitory transmission in the adult central nervous system. It has been demonstrated that protons are potent modulators of GABAARs. It is known that α1F64 residue – which plays a role in the receptor preactivation (Szczot et al. 2014) – is also involved in pH sensitivity (Huang et al. 2004). For this reason, we decided to examine whether preactivation transitions are affected by protons. METHODS: To this end we used patch-clamp technique with rapid exchange system, and tested the impact of pH changes on macroscopic and single-channel currents evoked by saturating concentration of full (GABA) or partial (P4S) agonist and mediated by wild type (α1β2γ2) receptors or by α1F64 leucine and cysteine mutants. RESULTS: Acidification (from pH 8.0 to 6.0) caused a significant increase in current amplitude for all used combinations of receptors and agonists. This effect was accompanied by slowing down of desensitization kinetics (especially for currents elicited by GABA in non-mutated). Surprisingly, protons differently influenced deactivation kinetics in WT and mutated receptors. Kinetic simulations suggest that the mechanism of GABAARs modulation by pH changes includes both modifications in preactivation and in one of the classical gating components (opening or desensitization). Single-channel recordings for non-mutated receptors and for cysteine mutants indicated no effect of pH changes on closing/opening transitions suggesting thus the lack of protons impact on channel efficacy. Moreover, we observed that acidification caused prolongation of bursts in WT receptors and the longest component of closure dwell times in cysteine mutants. CONCLUSION: We conclude that protons modulate GABAARs by the impact on gating transitions involving both preactivation and microscopic desensitization. This work has been supported by grant DEC-2013/11/B/NZ3/00983 of National Centre of Science and ministry grant Pbmn135.
2

Linking agonist binding to GABAA receptor opening transition

88%
Morrzymas J. W., Kisiel M., Jatczak M., Czyzewska M., Brodzki M.
Acta Neurobiologiae Experimentalis
|
2015
|
tom 75
|
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.
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
|
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

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
|
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.
6

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.
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