Locating the Carboxylate Group of GABA in the Homomeric rho GABAA Receptor Ligand-binding Pocket

Abstract

GABA3 is the major inhibitory neurotransmitter in the mammalian central nervous system (1). It mediates its effects via both ionotropic (GABA A ) and metabotropic (GABA B ) receptors. GABA C receptors are a subfamily of GABA A receptors and are sometimes referred to as GABA receptors, because the first subunit identified from this class of receptor was called 1 (2). These receptors have been separated from "classic" GABA A receptors because of their distinct pharmacological properties;GABA C receptor-mediated responses are not inhibited by bicuculline (the classic competitive GABA A receptor antagonist) nor induced by baclofen (the classic GABA B receptor agonist). In addition, 3-aminopropylphosphinic acid, a potent competitive GABA C receptor antagonist, acts as an agonist at the GABA B receptor and is inactive at the GABA A receptor, and GABA and trans-4-aminocrotonic acid are more potent agonists at GABA C receptors than GABA A or GABA B receptors (3). Thus there is clearly some variation in the structures of the different GABA-binding sites contained in these receptors, although the sequence homology between GABA A and GABA C receptor subunit sequences (Ͼ25%) indicates that their basic structures will be similar (structural homology is estimated at close to 80%) and will be similar to other members of the Cys loop family (4, 5).The lack of detailed structural information available for Cys loop receptors means that homology modeling is currently one of the best approaches to investigate possible molecular interactions between binding site residues and ligands. 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). Generating an accurate representation of the orientation of GABA in the GABA C receptor-binding site could identify the processes involved in ligand recognition at this receptor and would also assist in drug design; current data indicate that drugs acting on GABA C receptors could possibly be used to treat visual, sleep, and cognitive disorders (4,14).It has recently been shown that Tyr-198 forms a cationinteraction with the positive amine of GABA (15), and thus we can confidently locate this part of the GABA molecule close to this residue. The aim of this study was to locate the other end of GABA, the carboxylate group, in the binding site. This negatively charged end of GABA has been shown to interact with one or more positively charged residues in the GABA A receptor-binding pocket (16), and here we examine the role of positively charged and polar residues that are in or close to GABA in the GABA C receptor-binding...

Differentiating Enantioselective Actions of GABOB: A Possible Role for Threonine 244 in the Binding Site of GABAC ρ1 Receptors

“…32 Mutation of serine 243 to alanine (S243A) afforded mutant receptors that were functional, with GABA potency reduced only by approximately 2-fold compared to ρ 1 wild-type. 32 The lack of a hydroxyl group in the residue at 243 did not strongly change the potency of GABA.…”

“…32 Mutation of serine 243 to alanine (S243A) afforded mutant receptors that were functional, with GABA potency reduced only by approximately 2-fold compared to ρ 1 wild-type. 32 The lack of a hydroxyl group in the residue at 243 did not strongly change the potency of GABA. However, our results confirm the fact that removal of the hydroxyl group at T244 was not tolerated, 27 indicating that the T244 has a more important role in ρ 1 receptor GABA mediated channel gating than S243.…”

Structural model of ρ1 GABAC receptor based on evolutionary analysis: Testing of predicted protein–protein interactions involved in receptor assembly and function

“…This absence of responsiveness is perhaps not surprising, as both GABA and muscimol are zwitterionic at neutral pH, and the charged residue R158 presumably plays an important role in agonist binding and positioning. 39 The findings that removing arginine in the R158A mutant yields a 200-fold increase in EC 50 and that the double mutant R158D-D204R is inactive together suggest that the absence of a positively charged residue specifically at Position 158 strongly diminishes the binding affinity of the GABA and muscimol.…”

“…Remarkably, experimental data obtained from the literature demonstrated that mutations at positions with the lowest x-values usually had significant effects on the receptor function or expression. [15][16][17]39,40,42 Additionally, we have assessed the advantage of using evolutionary information in improving the quality of the homology model of GABA C receptor. We have compared our model with the model obtained using standard programs with default settings and default search results (as described in ''Methods'').…”

“…15,17,18 Figure 3 illustrates details of one of these intersubunit pockets, and standard state in tripeptide Gly-X-Gly. 41 A literature search revealed that mutation data on 30 out of 45 pocket residues are available, 15,17,[37][38][39][40] as listed in Table I.…”