A role for loop G in the ß1 strand in GABAA receptor activation.

KEY POINTS: The role of the ß1 strand in GABAA receptor function is unclear. It lies anti-parallel to the ß2 strand, which is known to participate in receptor activation. Molecular dynamics simulation revealed solvent accessible residues within the ß1 strand of the GABAA ß3 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 ß2 strand loop D was impeded by GABA but unaffected by propofol. These data reveal movement of ß1 strand loop G residues during agonist activation of the GABAA receptor. ABSTRACT: The GABAA receptor α subunit ß1 strand runs anti-parallel to the ß2 strand, which contains loop D, known to participate in receptor activation and agonist binding. However, a role for the ß1 strand has yet to be established. We used molecular dynamics simulation to quantify the solvent accessible surface area (SASA) of ß1 strand residues in the GABAA ß3 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 ß strand structure. We investigated the functional role of these ß1 strand residues in the α1 subunit by individually replacing them with Cys residues. D43C and T47C substitutions reduced the apparent potency of GABA at α1ß2γ2 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)ß2γ2 receptors were unaffected by MTSEA, suggesting that this residue is inaccessible. Both GABA and the allosteric agonist propofol reduced MTSEA modification of α1(D43C)ß2γ2 and α1(T47C)ß2γ2 receptors, indicating movement of the ß1 strand even during allosteric activation. This is in contrast to α1(F64C)ß2γ2 receptors, where only GABA, but not propofol, reduced MTSEA modification. These findings provide the first functional evidence for movement of the ß1 strand during gating of the receptor and identify residues that are critical for maintaining GABAA receptor function.

The molecular mechanism of toxin-induced conformational changes in a potassium channel: relation to C-type inactivation.

Recently, a solid-state NMR study revealed that scorpion toxin binding leads to conformational changes in the selectivity filter of potassium channels. The exact nature of the conformational changes, however, remained elusive. We carried out all-atom molecular dynamics simulations that enabled us to cover the complete pathway of toxin approach and binding, and we validated our simulation results by using solid-state NMR data and electrophysiological measurements. Our structural model revealed a mechanism of cooperative toxin-induced conformational changes that accounts both for the signal changes observed in solid-state NMR and for the tight interaction between KcsA-Kv1.3 and Kaliotoxin. We show that this mechanism is structurally and functionally closely related to recovery from C-type inactivation. Furthermore, our simulations indicate heterogeneity in the binding modes of Kaliotoxin, which might serve to enhance its affinity for KcsA-Kv1.3 further by entropic stabilization.