Influences on blockade by <i>t</i>‐butylbicyclo‐phosphoro‐thionate of GABA<sub>A</sub> receptor spontaneous gating, agonist activation and desensitization

106

122

Pentameric ligand-gated ion channels (pLGICs) mediate fast neurotransmission in the nervous system. Their dysfunction is associated with psychiatric, neurological and neurodegenerative disorders such as schizophrenia, epilepsy and Alzheimer's disease. Understanding their biophysical and pharmacological properties, at both the functional and the structural level, thus holds many therapeutic promises. In addition to their agonist-elicited activation, most pLGICs display another key allosteric property, namely desensitization, in which they enter a shut state refractory to activation upon sustained agonist binding. While the activation mechanisms of several pLGICs have been revealed at near-atomic resolution, the structural foundation of desensitization has long remained elusive. Recent structural and functional data now suggest that the activation and desensitization gates are distinct, and are located at both sides of the ion channel. Such a 'dual gate mechanism' accounts for the marked allosteric effects of channel blockers, a feature illustrated herein by theoretical kinetics simulations. Comparison with other classes of ligand- and voltage-gated ion channels shows that this dual gate mechanism emerges as a common theme for the desensitization and inactivation properties of structurally unrelated ion channels.

Section: Box 2 Pore-blockers As Allosteric Modulators Differentiallymentioning

confidence: 84%

The dual‐gate model for pentameric ligand‐gated ion channels activation and desensitization

Gielen

Corringer

2018

106

122

“…The α1(K278M) substitution, which inhibits GABA and propofol efficacy, also reduces spontaneous gating (Othman et al . ).…”

Section: Discussionmentioning

confidence: 97%

“…For outside‐out patch recordings, maximally efficacious and saturating concentrations of agonists were applied also using the Perfusion Fast Step system, with heat pulled and bevelled (Narashige, London, UK) theta and three‐barrelled pipes (Othman et al . ). This allows rapid solution exchange consistently of < 500 μs around an open pipette tip (Hinkle & Macdonald, ).…”

Section: Methodsmentioning

confidence: 97%

Loop G in the GABA_A receptor α1 subunit influences gating efficacy

Baptista‐Hon

Gulbinaite

Hales

2017

Self Cite

Key points summary The functional importance of residues in loop G of the GABA A receptor has not been investigated. D43 and T47 in the α1 subunit are of particular significance as their structural modification inhibits activation by GABA. While the T47C substitution had no significant effect, non-conservative substitution of either residue (D43C and T47R) reduced the apparent potency of GABA. Propofol potentiated maximal GABA-evoked currents mediated by α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors. Non-stationary variance analysis revealed a reduction in maximal GABA-evoked P open , suggesting impaired agonist efficacy. Further analysis of α1(T47R)β2γ2 receptors revealed that the efficacy of the partial agonist THIP relative to GABA was impaired. GABA-, THIP-and propofol-evoked currents mediated by α1(T47R)β2γ2 receptors deactivated faster than those mediated by α1β2γ2 receptors indicating that the mutation impairs agonist-evoked gating.This article is protected by copyright. All rights reserved.2  Spontaneous gating caused by the β2(L285R) mutation was also reduced in α1(T47R)β2(L285R)γ2 compared to α1β2(L285R)γ2 receptors confirming that α1(T47R) impairs gating independently of agonist activation.This article is protected by copyright. All rights reserved. 3 AbstractThe modification of Cys residues (substituted for D43 and T47) by 2-aminoethyl methanethiosulfonate in the GABA A α1 subunit loop G impairs activation of α1β2γ2 receptors by GABA and propofol (Baptista-Hon et al., 2016). While the T47C substitution had no significant effect, non-conservative substitution of either residue (D43C and T47R) reduced the apparent potency of GABA. Propofol (1 µM), which potentiates sub-maximal, but not maximal GABA-evoked currents mediated by α1β2γ2 receptors, also potentiated maximal currents mediated by α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors. Furthermore, the peak open probabilities of α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors were reduced. The kinetics of macroscopic currents mediated by α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors were characterised by slower desensitisation and faster deactivation. Similar changes in macroscopic current kinetics, together with a slower activation rate,were observed with the loop D α1(F64C) substitution, known to impair both efficacy and agonist binding, and when the partial agonist THIP was used to activate WT or α1(T47R)β2γ2 receptors.Propofol-evoked currents mediated by α1(T47R)β2γ2 and α1(F64C)β2γ2 receptors also exhibited faster deactivation than their WT counterparts revealing that these substitutions impair gating through a mechanism independent of orthosteric binding. Spontaneous gating caused by the introduction of the β2(L285R) mutation was also reduced in α1(T47R)β2(L285R)γ2 compared to

“…The same mutation also reduced the level of spontaneous gating of α1(K278M)β2γ2 GABA A receptors consistent with a global reduction in gating (Othman et al . ). Comparison of the open (ivermectin and glutamate bound) and closed ( apo ) structures of GluCl also reveals movement in this region associated with channel opening (Althoff et al .…”

Section: Discussionmentioning

confidence: 97%

A role for loop G in the β1 strand in GABA_A receptor activation

Baptista‐Hon

Krah

Zachariae

et al. 2016

Self Cite

Key points summary The role of the 1 strand in GABA A receptor function is unclear. It lies anti-parallel to the 2 strand, which is known to participate in receptor activation. Molecular dynamics simulation revealed solvent accessible residues within the β1 strand of the GABA A β3 homopentamer that might be amenable to analysis using the substituted Cys accessibility method. Cys substitutions from Asp43 to Thr47, in the GABA A α1 subunit showed that D43C and T47C reduced apparent potency of GABA. F45C caused a bi-phasic GABA concentration-response relationship and increased spontaneous gating. Cys43 and Cys47 were accessible to MTSEA modification, while Cys45 was not. Both GABA and the allosteric agonist propofol reduced MTSEA modification of Cys43 and Cys47. By contrast, modification of Cys64 in the 2 strand loop D was impeded by GABA but unaffected by propofol. These data reveal movement of 1 strand loop G residues during agonist activation of the GABA A receptor. 3 AbstractThe GABA A receptor α subunit β1 strand runs anti-parallel to the 2 strand, which contains loop D, known to participate in receptor activation and agonist binding. However, a role for the β1 strand has yet to be established. We used molecular dynamics simulation to quantify the solvent accessible surface area (SASA) of β1 strand residues in the GABA A β3 homopentamer structure. Residues in the complementary interface equivalent to those between Asp43 and Thr47 in the 1 subunit have an alternating pattern of high and low SASA consistent with a β strand structure. We investigated the functional role of these β1 strand residues in the α1 subunit by individually replacing them with Cys residues. D43C and T47C substitutions reduced the apparent potency of GABA at α1β2γ2 receptors by around 50-fold and 8-fold, respectively, whereas the F45C substitution caused a biphasic GABA concentration-response relationship and increased spontaneous gating. Receptors with D43C or T47C substitutions were sensitive to MTSEA modification. However, GABA-evoked currents mediated by α1(F45C)β2γ2 receptors were unaffected by MTSEA, suggesting that this residue is inaccessible. Both GABA and the allosteric agonist propofol reduced MTSEA modification of α1(D43C)β2γ2 and α1(T47C)β2γ2 receptors indicating movement of the β1 strand even during allosteric activation. This is in contrast to α1(F64C)β2γ2 receptors where only GABA, but not propofol reduced MTSEA modification. These findings provide the first functional evidence for movement of the β1 strand during gating of the receptor and identify residues that are critical for maintaining GABA A receptor function.