Abstract

The GABA_A receptor is a multisubunit protein that transduces the binding of a neurotransmitter at an intersubunit interface into the opening of a central ion channel. The structural components that mediate the steps involved in this action are poorly defined. A large amount of work has focused on clarifying the specific functions and interactions of residues believed to surround the GABA binding pocket. Here, we explored two charged residues (β₂Asp163 and α₁Arg120), which have been suggested by homology models to participate in a salt-bridge interaction. When mutated to alanine, both single mutants, as well as the double mutant, increase EC_50-GABA, decrease the GABA binding rate, and accelerate deactivation and GABA unbinding rates. Double-mutant cycle analysis demonstrates that the effects of each alanine mutation on the GABA binding rate were additive and independent. In contrast, a significant coupling energy was found during an analysis of deactivation time constants. Using kinetic modeling, we further demonstrated that the GABA unbinding rates, in particular, are strongly coupled. These data suggest that β₂Asp163 and α₁Arg120 form a state-dependent salt bridge, interacting when GABA is bound to the receptor but not when the receptor is in the unbound state.