α1F64 Residue at GABA(A) receptor binding site is involved in gating by influencing the receptor flipping transitions.

GABA receptors (GABAARs) mediate inhibition in the adult brain. These channels are heteropentamers and their ligand binding sites are localized at the ß+ / α- interfaces. As expected, mutations of binding-site residues affect binding kinetics but accumulating evidence indicates that gating is also altered, although the underlying mechanisms are unclear. We investigated the impact of the hydrophobic box residue localized at α1(-), F64 (α1F64), on the binding and gating of rat recombinant α1ß1γ2 receptors. The analysis of current responses to rapid agonist applications confirmed a marked effect of α1F64 mutations on agonist binding and revealed surprisingly strong effects on gating, including the disappearance of rapid desensitization, the slowing of current onset, and accelerated deactivation. Moreover, nonstationary variance analysis revealed that the α1F64C mutation dramatically reduced the maximum open probability without altering channel conductance. Interestingly, for wild-type receptors, responses to saturating concentration of a partial agonist, P4S, showed no rapid desensitization, similar to GABA-evoked responses mediated by α1F64C mutants. For the α1F64L mutation, the application of the high-affinity agonist muscimol partially rescued rapid desensitization compared with responses evoked by GABA. These findings suggest that α1F64 mutations do not disrupt desensitization mechanisms but rather affect other gating features that obscure it. Model simulations indicated that all of our observations related to α1F64 mutations could be properly reproduced by altering the flipped state transitions that occurred after agonist binding but preceded opening. In conclusion, we propose that the α1F64 residue may participate in linking binding and gating by influencing flipping kinetics.

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.

Spontaneous activity, singly bound states and the impact of alpha1Phe64 mutation on GABAAR gating in the novel kinetic model based on the single-channel recordings.

GABAA receptor is the primary mediator of inhibition in the adult mammalian brain. Our recent studies revealed that a classic gating scheme for GABAAR needed to be updated with an intermediate step (flipping) and that the α1Phe64 mutation at the GABA binding site affects this transition. However, description of flipping at the single-channel level remains incomplete. In particular, its role in singly-bound and spontaneous activity remains unknown. We have performed thus single-channel recordings over wide range of agonist concentration for wild-type α1ß2γ2L receptors and α1Phe64 mutants. For WT receptors we observed relatively frequent brief spontaneous openings which were also present at low [GABA]. However, closed times distributions for spontaneous activity and at low [GABA] were clearly different indicating that a proportion of short-lived openings were due to liganded, most likely singly bound receptors. Increasing [GABA] resulted in prolongation of bursts and increased occurrence of bursts with long openings and short closures. Mutations of α1Phe64 residue dramatically affected the open and closed time distributions at high and saturating [GABA], especially in the case of cysteine mutants. However, this mutation weakly affected spontaneous or singly bound activity. Model fitting of our single-channel data led us to propose a novel and, to our knowledge, most complete GABAAR kinetic model in which flipping occurs in singly and doubly bound states. However, spontaneous activity did not reveal involvement of flipping. Moreover, we report that α1Phe64 mutation affects not only the flipping but also the opening/closing transitions indicating its generalized impact on the receptor gating.

Comparison of kinetic and pharmacological profiles of recombinant α1γ2L and α1ß2γ2L GABAA receptors - A clue to the role of intersubunit interactions.

The fastest inhibitory mechanism in the CNS is mediated by ionotropic GABAA receptors and it is known that subunit composition critically determines their properties. While a typical GABAA receptor consists of two α, two ß and one γ/δ subunit, there are some exceptions, e.g. αß receptors. Functional α1γ2 GABAA receptors can be expressed in recombinant model (Verdoorn et al., 1990) and although their role remains unknown, it seems appealing to extend their characterization to further explore the structure-function relationship of GABAA receptors. Intriguingly, this receptor is lacking canonical GABA binding sites but it can be activated by GABA and dose-response relationships for α1ß2γ2L and α1γ2L receptors overlap. Deactivation kinetics was similar for both receptors but the percentage of the fast component was smaller in the case of α1γ2L receptors and, consequently, the mean deactivation time constant was slower. The rate and extent of macroscopic desensitization were smaller in the case of α1γ2L receptors but they showed slower recovery. Both receptor types had a similar proton sensitivity showing only subtle but significant differences in pH effects on deactivation. Flurazepam exerted a similar effect on both receptors but the rapid deactivation components were differently affected and an opposite effect was observed on desensitization extent. Rebound currents evoked by pentobarbital were undistinguishable for both receptor types. Taking altogether, although some significant differences were found, α1ß2γ2L and α1γ2L receptors showed unforeseen similarity. We propose that functioning of GABAA receptors might rely on subunit-subunit cooperative interactions to a larger extent than believed so far.

CD44: a novel synaptic cell adhesion molecule regulating structural and functional plasticity of dendritic spines.

Synaptic cell adhesion molecules regulate signal transduction, synaptic function, and plasticity. However, their role in neuronal interactions with the extracellular matrix (ECM) is not well understood. Here we report that the CD44, a transmembrane receptor for hyaluronan, modulates synaptic plasticity. High-resolution ultrastructural analysis showed that CD44 was localized at mature synapses in the adult brain. The reduced expression of CD44 affected the synaptic excitatory transmission of primary hippocampal neurons, simultaneously modifying dendritic spine shape. The frequency of miniature excitatory postsynaptic currents decreased, accompanied by dendritic spine elongation and thinning. These structural and functional alterations went along with a decrease in the number of presynaptic Bassoon puncta, together with a reduction of PSD-95 levels at dendritic spines, suggesting a reduced number of functional synapses. Lack of CD44 also abrogated spine head enlargement upon neuronal stimulation. Moreover, our results indicate that CD44 contributes to proper dendritic spine shape and function by modulating the activity of actin cytoskeleton regulators, that is, Rho GTPases (RhoA, Rac1, and Cdc42). Thus CD44 appears to be a novel molecular player regulating functional and structural plasticity of dendritic spines.