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A role for loop G in the β1 strand in GABA A receptor activation
Author(s) -
BaptistaHon Daniel T.,
Krah Alexander,
Zachariae Ulrich,
Hales Tim G.
Publication year - 2016
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jp272463
Subject(s) - agonist , allosteric regulation , gabaa receptor , protein subunit , gating , biophysics , loop (graph theory) , receptor , chemistry , stereochemistry , biochemistry , biology , mathematics , combinatorics , gene
Key points The role of the β1 strand in GABA A 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 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 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 GABA A receptor.Abstract The GABA A 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 GABA A 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 GABA A receptor function.