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

Abstract

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.

Insights into the ion-coupling mechanism in the MATE transporter NorM-VC

“…We therefore conclude that protonation events can occur directly between water and either residue. We ascribe the high hydration level observed in all simulations to the presence of at least one charged carboxylate group, which has previously been reported to increase the local hydration level in different proteins [65,66]. In addition, continuous solvation allows the ion to enter the binding site from the bulk solution in all possible protonation states of E255 and D371 (see Figure 5, below).…”

Loop G in the GABAA receptor α1 subunit influences gating efficacy

“…Transfections were performed by calcium phosphate precipitation, using 1 μg total cDNA per dish, as described previously (Baptista‐Hon et al . ). cDNAs encoding wild type (WT) and mutant mouse GABA A subunits were in the pRK5 mammalian expression vector.…”

“…We recently demonstrated the involvement of specific GABA A receptor α1 subunit loop G residues in function using cysteine‐scanning mutagenesis (Baptista‐Hon et al . ). D43C was the only one of five loop G substituents to reduce the apparent potency of GABA.…”

“…Cysteines substituted at three other positions in loop G of the α1 subunit (residues 44, 45 and 46) either were inaccessible to MTSEA or their modification was without functional consequence (Baptista‐Hon et al . ).…”

“…To replicate MTSEA‐modified α1(T47C), which impairs function (Baptista‐Hon et al . ), we replaced Thr47 by Arg. We compared the efficacy of GABA as an activator of α1(D43C)β2γ2, α1(T47R)β2γ2 and α1β2γ2 receptors using analysis of channel open probability ( P open ) and potentiation by the positive allosteric modulator propofol.…”