Details

Autor(en) / Beteiligte

• Baptista-Hon, Daniel T
• Krah, Alexander
• Zachariae, Ulrich
• Hales, Tim G

Titel

A role for loop G in the [beta]1 strand in GABAA receptor activation

Ist Teil von

• The Journal of physiology, 2016-10, Vol.594 (19), p.5555

Ort / Verlag

London: Wiley Subscription Services, Inc

Erscheinungsjahr

2016

Quelle

Wiley Online Library

Beschreibungen/Notizen

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