Abstract
gamma-Aminobutyric acid, type A (GABA(A)) 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 GABA(C) 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 of GABA within 3A of this residue. Substitution of Arg-104 with Ala or Glu resulted in >10,000-fold increases in EC(50) 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 approximately 10-fold increase in EC(50). Arg-249 was substituted with Ala, Glu, or Asp with relatively small ( approximately 4-30-fold) changes in EC(50). 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.
Highlights
GABAC receptor-mediated responses are not inhibited by bicuculline nor induced by baclofen
There is clearly some variation in the structures of the different GABA-binding sites contained in these receptors, the sequence homology between GABAA and GABAC receptor subunit sequences (Ͼ25%) indicates that their basic structures will be similar and will be similar to other members of the Cys loop family [4, 5]
This approach has been made possible by the availability of the high resolution structure of the acetylcholine binding protein (AChBP), which is homologous to the extracellular domain of the nicotinic ACh receptor [6]
Summary
GABAC receptor-mediated responses are not inhibited by bicuculline (the classic competitive GABAA receptor antagonist) nor induced by baclofen (the classic GABAB receptor agonist). This approach has been made possible by the availability of the high resolution structure of the acetylcholine binding protein (AChBP), which is homologous to the extracellular domain of the nicotinic ACh receptor [6] Using this as a template, computer-generated models of ligandbinding pockets of Cys loop receptors, combined with previous data from structure-activity studies, have identified important features of these pockets and on the orientation of agonists and antagonists when located in their binding sites [7,8,9,10,11,12,13]. The data have allowed us to define the probable orientation of GABA
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