A role for loop G in the β1 strand in GABAA receptor activation. (27th June 2016)

• Record Type: Journal Article
• Authors: Baptista‐Hon, Daniel T.; Krah, Alexander; Zachariae, Ulrich; Hales, Tim G.
• Is Part Of: Journal of physiology. Volume 594:Number 19(2016:Oct.)
• Page Start: 5555
• Page End: 5571
• Publication Date: 2016-06-27
• Subjects: Physiology -- Periodicals; 612.005
• DOI: 10.1113/JP272463
• Languages: English
• ISSNs: 0022-3751

Abstract

Abstract : Key points: The role of the β1 strand in GABAA receptor function is unclear. It lies anti‐parallel to the β2 strand, which is known to participate in receptor activation. Molecular dynamics simulation revealed solvent accessible residues within the β1 strand of the GABAA β3 homopentamer that might be amenable to analysis using the substituted Cys accessibility method. Cys substitutions from Asp43 to Thr47 in the GABAA α1 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 β2 strand loop D was impeded by GABA but unaffected by propofol. These data reveal movement of β1 strand loop G residues during agonist activation of the GABAA receptor. Abstract: The GABAA receptor α subunit β1 strand runs anti‐parallel to the β2 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 GABAA β3 homopentamer structure. Residues in the complementary interface equivalent to those between Asp43 and Thr47 in the α1 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 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 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 GABAA receptor function. Key points: The role of the β1 strand in GABAA receptor function is unclear. It lies anti‐parallel to the β2 strand, which is known to participate in receptor activation. Molecular dynamics simulation revealed solvent accessible residues within the β1 strand of the GABAA β3 homopentamer that might be amenable to analysis using the substituted Cys accessibility method. Cys substitutions from Asp43 to Thr47 in the GABAA α1 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 β2 strand loop D was impeded by GABA but unaffected by propofol. These data reveal movement of β1 strand loop G residues during agonist activation of the GABAA receptor.