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 GABA sub(A) receptor activation

Ist Teil von

• The Journal of physiology, 2016-10, Vol.594 (19), p.5555-5571

Erscheinungsjahr

2016

Quelle

Electronic Journals Library

Beschreibungen/Notizen

• Key points * The role of the beta 1 strand in GABA sub(A) 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 GABA sub(A) beta 3 homopentamer that might be amenable to analysis using the substituted Cys accessibility method. * Cys substitutions from Asp43 to Thr47 in the GABA sub(A) 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 GABA sub(A) receptor. The GABA sub(A) 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 GABA sub(A) 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 GABA sub(A) receptor function. Key points * The role of the beta 1 strand in GABA sub(A) 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 GABA sub(A) beta 3 homopentamer that might be amenable to analysis using the substituted Cys accessibility method. * Cys substitutions from Asp43 to Thr47 in the GABA sub(A) 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 GABA sub(A) receptor.