GABAA Receptor ß2E155 Residue Located at the Agonist-Binding Site Is Involved in the Receptor Gating.

GABAA receptors (GABAARs) play a crucial role in mediating inhibition in the adult brain. In spite of progress in describing (mainly) the static structures of this receptor, the molecular mechanisms underlying its activation remain unclear. It is known that in the α1ß2γ2L receptors, the mutation of the ß2E155 residue, at the orthosteric binding site, strongly impairs the receptor activation, but the molecular and kinetic mechanisms of this effect remain elusive. Herein, we investigated the impact of the ß2E155C mutation on binding and gating of the α1ß2γ2L receptor. To this end, we combined the macroscopic and single-channel analysis, the use of different agonists [GABA and muscimol (MSC)] and flurazepam (FLU) as a modulator. As expected, the ß2E155C mutation caused a vast right shift of the dose-response (for GABA and MSC) and, additionally, dramatic changes in the time course of current responses, indicative of alterations in gating. Mutated receptors showed reduced maximum open probability and enhanced receptor spontaneous activity. Model simulations for macroscopic currents revealed that the primary effect of the mutation was the downregulation of the preactivation (flipping) rate. Experiments with MSC and FLU further confirmed a reduction in the preactivation rate. Our single-channel analysis revealed the mutation impact mainly on the second component in the shut times distributions. Based on model simulations, this finding further confirms that this mutation affects mostly the preactivation transition, supporting thus the macroscopic data. Altogether, we provide new evidence that the ß2E155 residue is involved in both binding and gating (primarily preactivation).

Protons modulate gating of recombinant α1ß2γ2 GABAA receptor by affecting desensitization and opening transitions.

Protons are potent modulators of GABAA receptors (GABAARs) and α1Phe64 residue was implicated in their pH sensitivity. Recently, we have demonstrated that this residue is involved in flipping transitions which precede channel opening. We thus re-addressed the mechanism of GABAAR modulation by protons by considering the gating scheme extended by flipping. The impact of pH changes was examined on currents mediated by wild-type α1ß2γ2 receptors or by their α1Phe64Leu or α1Phe64Cys mutants and elicited by saturating concentrations of full (GABA) or partial (piperidine-4-sulfonic acid) agonists. To describe the impact of extracellular pH on receptor gating, we combined macroscopic analysis of currents elicited by rapid agonist applications with single-channel studies. Acidification (pH 6.0) increased current amplitudes (in the case of leucine mutants effect was stronger when P4S was used) and decreased the rate and the extent of desensitization whereas alkalization (pH 8.0) had the opposite but weaker effect. Deactivation kinetics for wild-type receptors was slowed down by acidification while in the case of mutants this effect was observed upon alkalization. Moreover, α1Phe64 mutations enhanced GABAAR sensitivity to alkaline pH. Single-channel analysis revealed that acidification prolonged burst durations and affected shut but not open time distributions. Model simulations for macroscopic and single-channel activity indicated a novel mechanism in which protons primarily affected opening and desensitization rates but not flipping/unflipping. This evidence for the impact of protons on the receptor gating together with previously demonstrated effect on the agonist binding, point to a complex effect of extracellular pH on GABAAR macromolecule.

Distinct Modulation of Spontaneous and GABA-Evoked Gating by Flurazepam Shapes Cross-Talk Between Agonist-Free and Liganded GABAA Receptor Activity.

GABAA receptors (GABAARs) play a crucial inhibitory role in the CNS. Benzodiazepines (BDZs) are positive modulators of specific subtypes of GABAARs, but the underlying mechanism remains obscure. Early studies demonstrated the major impact of BDZs on binding and more recent investigations indicated gating, but it is unclear which transitions are affected. Moreover, the upregulation of GABAAR spontaneous activity by BDZs indicates their impact on receptor gating but the underlying mechanisms remain unknown. Herein, we investigated the effect of a BDZ (flurazepam) on the spontaneous and GABA-induced activity for wild-type (WT, α1ß2γ2) and mutated (at the orthosteric binding site α1F64) GABAARs. Surprisingly, in spite of the localization at the binding site, these mutations increased the spontaneous activity. Flurazepam (FLU) upregulated this activity for mutants and WT receptors to a similar extent by affecting opening/closing transitions. Spontaneous activity affected GABA-evoked currents and is manifested as an overshoot after agonist removal that depended on the modulation by BDZs. We explain the mechanism of this phenomenon as a cross-desensitization of ligand-activated and spontaneously active receptors. Moreover, due to spontaneous activity, FLU-pretreatment and co-application (agonist + FLU) protocols yielded distinct results. We provide also the first evidence that GABAAR may enter the desensitized state in the absence of GABA in a FLU-dependent manner. Based on our data and model simulations, we propose that FLU affects agonist-induced gating by modifying primarily preactivation and desensitization. We conclude that the mechanisms of modulation of spontaneous and ligand-activated GABAAR activity concerns gating but distinct transitions are affected in spontaneous and agonist-evoked activity.

Fractured Superior Mesenteric Artery Stent With Stent Displacement Leading to Recurrent Symptoms of Superior Mesenteric Ischemia.

We report a case of a patient with recurrent symptoms of superior mesenteric ischemia 7 months after successful percutaneous angioplasty and implantation of a stent in the superior mesenteric artery (SMA). Stent fracture and stent displacement were observed. To the best of our knowledge, this is the first report of SMA stent fracture with stent displacement.