α₁F64 Residue at GABA_AReceptor Binding Site Is Involved in Gating by Influencing the Receptor Flipping Transitions

Abstract

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.

Contrasting actions of a convulsant barbiturate and its anticonvulsant enantiomer on the α1β3γ2L GABAA receptor account for their in vivo effects

“…; Szczot et al . ). In GABA A Rs, GABA activates three open states, but their interconnectivity is not well established.…”

GABAA Receptor β2E155 Residue Located at the Agonist-Binding Site Is Involved in the Receptor Gating

“…50 Å) indicating that the mechanism of activation must be very complex, and it may comprise vast parts of the receptor macromolecule (Chakrapani et al, 2004;Miller and Aricescu, 2014). It has been already shown for GABA A Rs (Gielen et al, 2012;Szczot et al, 2014;Dixon et al, 2015;Kisiel et al, 2018) and previously also for other receptors of the Cys-loop family (Burzomato et al, 2004;Lape et al, 2008;Mukhtasimova et al, 2009;Jadey and Auerbach, 2012;Corradi and Bouzat, 2014) that, after agonist binding, a key transition prior to channel opening is the preactivation/flipped transition. The preactivated (flipped) state may be thought of as a still closed conformation at which the receptor is poised to undergo an opening transition.…”

“…For the expression of recombinant GABA A Rs, human embryonic kidney cells were used (HEK293 cell line, Sigma-Aldrich), cultured as previously described (Szczot et al, 2014). To transiently transfect the cells, the calcium phosphate precipitation method (Chen and Okayama, 1987) was used.…”

“…The preactivated (flipped) state may be thought of as a still closed conformation at which the receptor is poised to undergo an opening transition. Interestingly, at least some key structural determinants (e.g., α 1 F64) of these gating transitions are located very closely to the agonist-binding site (Szczot et al, 2014). It seems thus interesting to pursue the issue of how ''strategically'' located residues close to the orthosteric binding sites impact the channel gating.…”

“…Multiple amino acid residues, located on loops A, B, and C of the β subunit and loops D, E, and F of the α subunit, have been identified as important determinants for the GABA-binding site (Wagner et al, 2004;Miller and Smart, 2010;Sander et al, 2011;Miller and Aricescu, 2014). Besides critical involvement of α 1 F64 from loop D in receptor gating (Szczot et al, 2014;Kisiel et al, 2018), there are also some hints (Newell et al, 2004;Mortensen et al, 2014) suggesting that β 2 E155 residue in loop B, may also be involved in channel gating properties, which is clearly manifested by enhancement of the spontaneous activity when mutating it. This residue has been previously shown by the homology modeling of GABA A R and ligand docking (Mortensen et al, 2014) to interact with the positively charged moiety of GABA, most probably by anchoring the GABA amino nitrogen end .…”