Mutation of valine 53 at the interface between extracellular and transmembrane domains of the ß2 principal subunit affects the GABAA receptor gating.

GABAA receptors (gamma-aminobutyric acid type A receptors) are pentameric ligand-gated ion channels mediating inhibition in adult mammalian brains. Their static structure has been intensely studied in the past years but the underlying molecular activatory mechanisms remain obscure. The interface between extracellular and transmembrane domains has been recognized as a key player in the receptor gating. However, the role of the valine 53 in the ß1-ß2 loop of the principal subunit (ß2) remains controversial showing differences compared to homologous residues in some cys-loop counterparts such as nAChR. To address the role of the ß2V53 residue in the α1ß2γ2L receptor gating, we performed high resolution macroscopic and single-channel recordings. To explore underlying molecular mechanisms a variety of substituting amino acids were investigated: Glutamate and Lysine (different electric charge), Alanine (aliphatic, larger than Valine) and Histidine (same residue as in homologous α1H55). We report that mutation of the ß2V53 residue results in alterations of nearly all gating transitions including opening/closing, preactivation and desensitization. A dramatic gating impairment was observed for glutamate substitution (ß2V53E) but ß2V53K mutation had a weak effect. The impact of histidine substitution was also small while ß2V53A markedly affected the receptor but to a smaller extent than ß2V53E. Considering available structures in desensitized and bicuculline blocked shut states we propose that strongly detrimental effect of ß2V53E mutation on receptor activation results from electrostatic interaction between the glutamate and ß2K274 on the loop M2-M3 which stabilizes the receptor in the shut state. We conclude that ß2V53 is strongly involved in mechanisms underlying the receptor gating.

α1 Proline 277 Residues Regulate GABAAR Gating through M2-M3 Loop Interaction in the Interface Region.

Cys-loop receptors are a superfamily of transmembrane, pentameric receptors that play a crucial role in mammalian CNS signaling. Physiological activation of these receptors is typically initiated by neurotransmitter binding to the orthosteric binding site, located at the extracellular domain (ECD), which leads to the opening of the channel pore (gate) at the transmembrane domain (TMD). Whereas considerable knowledge on molecular mechanisms of Cys-loop receptor activation was gathered for the acetylcholine receptor, little is known with this respect about the GABAA receptor (GABAAR), which mediates cellular inhibition. Importantly, several static structures of GABAAR were recently described, paving the way to more in-depth molecular functional studies. Moreover, it has been pointed out that the TMD-ECD interface region plays a crucial role in transduction of conformational changes from the ligand binding site to the channel gate. One of the interface structures implicated in this transduction process is the M2-M3 loop with a highly conserved proline (P277) residue. To address this issue specifically for α1ß2γ2L GABAAR, we choose to substitute proline α1P277 with amino acids with different physicochemical features such as electrostatic charge or their ability to change the loop flexibility. To address the functional impact of these mutations, we performed macroscopic and single-channel patch-clamp analyses together with modeling. Our findings revealed that mutation of α1P277 weakly affected agonist binding but was critical for all transitions of GABAAR gating: opening/closing, preactivation, and desensitization. In conclusion, we provide evidence that conservative α1P277 at the interface is strongly involved in regulating the receptor gating.

The ß2 subunit E155 residue as a proton sensor at the binding site on GABA type A receptors.

GABA type A receptor plays a key role in inhibitory signaling in the adult central nervous system. This receptor can be modulated by protons but the underlying molecular mechanisms have not been fully explored. To find possible pH-sensor residues, a comparative study for proton-activated GLIC channel and α1ß2γ2 GABA receptor was performed and pK 's of respective residues were estimated by numerical algorithms which consider local interactions. ß E155, located at the GABA binding site, showed pKa values close to physiological values and dependence on the receptor state and ligation, suggesting a role in modulation by pH. To validate this prediction, pH sensitivity of current responses to GABA was investigated using patch-clamp technique for WT and mutated (ß2E155[C, S, Q, L]) GABA receptors. Cysteine mutation preserved pH sensitivity. However, for remaining mutants, the sensitivity to acidification (pH = 6.0) was reduced becoming not statistically significant. The effect of alkaline pH (8.0) was maintained for all mutants with exception for ß2E155L for which it was nearly abolished. To further explore the impact of considered mutations, molecular docking was performed which indicated that pH modulation is probably affected by interplay between binding site residues, zwitterion GABA and protons. These data, altogether, indicate that mutation of ß2E155 to hydrophobic residue (L) maximally impaired pH modulation while for polar substitutions the effect was smaller. In conclusion, our data provide evidence that a key binding site residue ß2E155 plays an important role in proton sensitivity of GABA receptor.

Mutations at the M2 and M3 Transmembrane Helices of the GABAARs α1 and ß2 Subunits Affect Primarily Late Gating Transitions Including Opening/Closing and Desensitization.

GABA type A receptors (GABAARs) belong to the pentameric ligand-gated ion channel (pLGIC) family and play a crucial role in mediating inhibition in the adult mammalian brain. Recently, a major progress in determining the static structure of GABAARs was achieved, although precise molecular scenarios underlying conformational transitions remain unclear. The ligand binding sites (LBSs) are located at the extracellular domain (ECD), very distant from the receptor gate at the channel pore. GABAAR gating is complex, comprising three major categories of transitions: openings/closings, preactivation, and desensitization. Interestingly, mutations at, e.g., the ligand binding site affect not only binding but often also more than one gating category, suggesting that structural determinants for distinct conformational transitions are shared. Gielen and co-workers (2015) proposed that the GABAAR desensitization gate is located at the second and third transmembrane segment. However, studies of our and others' groups indicated that other parts of the GABAAR macromolecule might be involved in this process. In the present study, we asked how selected point mutations (ß2G254V, α1G258V, α1L300V, and ß2L296V) at the M2 and M3 transmembrane segments affect gating transitions of the α1ß2γ2 GABAAR. Using high resolution macroscopic and single-channel recordings and analysis, we report that these substitutions, besides affecting desensitization, also profoundly altered openings/closings, having some minor effect on preactivation and agonist binding. Thus, the M2 and M3 segments primarily control late gating transitions of the receptor (desensitization, opening/closing), providing a further support for the concept of diffuse gating mechanisms for conformational transitions of GABAAR.

Interaction between GABAA receptor α1 and ß2 subunits at the N-terminal peripheral regions is crucial for receptor binding and gating.

Pentameric ligand gated ion channels (pLGICs) are crucial in electrochemical signaling but exact molecular mechanisms of their activation remain elusive. So far, major effort focused on the top-down molecular pathway between the ligand binding site and the channel gate. However, recent studies revealed that pLGIC activation is associated with coordinated subunit twisting in the membrane plane. This suggests a key role of intersubunit interactions but the underlying mechanisms remain largely unknown. Herein, we investigated a "peripheral" subunit interface region of GABAA receptor where structural modeling indicated interaction between N-terminal α1F14 and ß2F31 residues. Our experiments underscored a crucial role of this interaction in ligand binding and gating, especially preactivation and opening, showing that the intersubunit cross-talk taking place outside (above) the top-down pathway can be strongly involved in receptor activation. Thus, described here intersubunit interaction appears to operate across a particularly long distance, affecting vast portions of the macromolecule.

Mutations of α1F45 residue of GABAA receptor loop G reveal its involvement in agonist binding and channel opening/closing transitions.

GABAA receptors (GABAARs) mediate inhibitory neurotransmission in the mammalian brain. Recently, numerous GABAAR static structures have been published, but the molecular mechanisms of receptor activation remain elusive. Loop G is a rigid ß-strand belonging to an extensive ß-sheet that spans the regions involved in GABA binding and the interdomain interface which is important in receptor gating. It has been reported that loop G participates in ligand binding and gating of GABAARs, however, it remains unclear which specific gating transitions are controlled by this loop. Analysis of macroscopic responses revealed that mutation at the α1F45 residue (loop G midpoint) resulted in slower macroscopic desensitization and accelerated deactivation. Single-channel analysis revealed that these mutations also affected open and closed times distributions and reduced open probability. Kinetic modeling demonstrated that mutations affected primarily channel opening/closing and ligand binding with a minor effect on preactivation. Thus, α1F45 residue, in spite of its localization close to binding site, affects late gating transitions. In silico structural analysis suggested an important role of α1F45 residue in loop G stability and rigidity as well as in general structure of the binding site. We propose that the rigid ß-sheet comprising loop G is well suited for long range communication within GABAAR but this mechanism becomes impaired when α1F45 is mutated. In conclusion, we demonstrate that loop G is crucial in controlling both binding and gating of GABAARs. These data shed new light on GABAAR activation mechanism and may also be helpful in designing clinically relevant modulators.

The C loop at the orthosteric binding site is critically involved in GABAA receptor gating.

GABAA receptors (GABAARs) play a crucial role in mammalian adult brain inhibition. The dysfunction of GABAergic drive is related to such disorders as epilepsy, schizophrenia, and depression. Substantial progress has recently been made in describing the static structure of GABAARs, but the molecular mechanisms that underlie the activation process remain elusive. The C loop of the GABAAR structure shows the largest movement upon ligand binding to the orthosteric binding site, a phenomenon that is referred to as "capping." The C loop is known to be involved in agonist binding, but its role in the gating of Cys-loop receptors is still debated. Herein, we investigated this issue by analyzing the impact of a ß2F200 residue mutation of the C loop on gating properties of α1ß2γ2 GABAARs. Extensive analyses and the modeling of current responses to saturating agonist application demonstrated that this mutation strongly affected preactivation, opening, closing and desensitization, i.e. all considered gating steps. Single-channel analysis revealed that the ß2F200 mutation slowed all shut time components, and open times were shortened. Model fitting of these single-channel data further confirmed that the ß2F200 mutation strongly affected all of the gating characteristics. We also found that this mutation altered receptor sensitivity to the benzodiazepine flurazepam, which was attributable to a change in preactivation kinetics. In silico analysis indicated that the ß2F200 mutation resulted in distortion of the C loop structure, causing the movement of its tip from the binding site. Altogether, we provide the first evidence that C loop critically controls GABAAR gating.

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.