Activation of the human β3 GABAA receptor by combinations of receptor protonation with chemical agonists and gain-of-function mutations

Native GABA A receptors are continuously exposed to numerous active compounds, including protons, whose net activating and modulatory effects determine receptor function and underlie the inhibitory tone in the brain. Here, we have investigated the interactions and energetic additivity between activation of the homomeric β3 GABA A receptor due to protonation and its activation by standard chemical agonists or gain-of-function mutations. Energetic additivity results from combined actions of agonists or mutations with independent sites and mechanisms, and manifests as apparent synergistic potentiation. It is therefore a clinically important mode of manipulation of receptor function. Our findings indicate a lack of strict energetic additivity in nearly each test, although the underlying mechanisms were different. Activation of the wild-type and tested mutant β3 receptors by the orthosteric agonist histamine was associated with reduced energetic contribution at higher [H + ]. Mutational studies revealed that protonation of the β3(H107) residue, located within ∼ 8 Å of its binding site, impairs activation by histamine. Mutation of this residue preventing its protonation revealed energetic additivity between activation by histamine and protonation at β3(H267). Activation by the allosteric agonist propofol exhibited reduced energetic impact at an increased level of background activity regardless of extracellular pH. Finally, the gating efficacy of H + was reduced in two of the tested gain-of-function mutants. Overall, the results indicate a complex interplay between protonation and activation of the β3 receptor by other agents or mutations. It can reflect apparent direct interactions between protonation- and activation-sites, interactions mediated by changes in baseline activity or energetic activity.