GABA receptors (GABA A Rs) mediate inhibition in the adult brain. These channels are heteropentamers and their ligand binding sites are localized at the ␤(ϩ)/␣(Ϫ) 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 ␣ 1 (Ϫ), F64 (␣ 1 F64), on the binding and gating of rat recombinant ␣ 1 ␤ 1 ␥ 2 receptors. The analysis of current responses to rapid agonist applications confirmed a marked effect of ␣ 1 F64 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 ␣ 1 F64C 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 ␣ 1 F64C mutants. For the ␣ 1 F64L mutation, the application of the high-affinity agonist muscimol partially rescued rapid desensitization compared with responses evoked by GABA. These findings suggest that ␣ 1 F64 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 ␣ 1 F64 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 ␣ 1 F64 residue may participate in linking binding and gating by influencing flipping kinetics.

CD44 is a novel molecular player that regulates structure and function of the synapse. It affects excitatory synaptic transmission, dendritic spine shape, number of functional synapses, and activity-dependent neuronal plasticity. These functions are exerted via the regulation of small Rho GTPases.

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GABA A receptors (GABA A Rs) 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 α 1 β 2 γ 2L receptors, the mutation of the β 2 E155 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 β 2 E155C mutation on binding and gating of the α 1 β 2 γ 2L 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 β 2 E155C 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 β 2 E155 residue is involved in both binding and gating (primarily preactivation).