Locating the carboxylate group of GABA in the homomeric rho GABA A receptor ligand-binding pocket

Abstract

γ-Aminobutyric acid, typeA(GABAA) receptors, of which the GABA C receptor family is a subgroup, are members of the Cys loop family of neurotransmitter receptors. Homology modeling of the extracellular domain of these proteins has revealed many molecular details, but it is not yet clear how GABA is orientated in the binding pocket. Here we have examined the role of arginine residues that the homology model locates in or close to the binding site of the GABAC receptor (Arg-104, Arg-170, Arg-158, and Arg-249) using mutagenesis and functional studies. The data suggest that Arg-158 is critical for GABA binding and/or function; substitution with Lys, Ala, or Glu resulted in nonfunctional receptors, and modeling placed the carboxylate ofGABAwithin 3Å of this residue. Substitution of Arg-104 with Ala or Glu resulted in >10,000-fold increases in EC50 values compared with wild type receptors, and modeling indicated a role of this residue both in binding GABA and in the structure of the binding pocket. Substitution of Arg-170 with Asp or Ala yielded nonfunctional receptors, whereas Lys caused an ∼10-fold increase in EC50. Arg-249 was substituted with Ala, Glu, or Asp with relatively small (∼4-30-fold) changes in EC50. These and data from other residues that the model suggested could interact with GABA (His-105, Ser-168, and Ser-243) support a location for GABA in the binding site with its carboxylate pincered between Arg-158 and Arg-104, with Arg-104, Arg-170, and Arg-249 contributing to the structure of the binding pocket through salt bridges and/or hydrogen bonds. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.