Mapping the ρ1 GABAC Receptor Agonist Binding Pocket

Abstract

␥-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian brain. The GABA receptor type C (GABA C ) is a ligand-gated ion channel with pharmacological properties distinct from the GABA A receptor. To date, only three binding domains in the recombinant 1 GABA C receptor have been recognized among six potential regions. In this report, using the substituted cysteine accessibility method, we scanned three potential regions previously unexplored in the 1 GABA C receptor, corresponding to the binding loops A, E, and F in the structural model for ligand-gated ion channels. The cysteine accessibility scanning and agonist/antagonist protection tests have resulted in the identification of residues in loops A and E, but not F, involved in forming the GABA C receptor agonist binding pocket. Three of these newly identified residues are in a novel region corresponding to the extended stretch of loop E. In addition, the cysteine accessibility pattern suggests that part of loop A and part of loop E have a ␤-strand structure, whereas loop F is a random coil. Finally, when all of the identified ligand binding residues are mapped onto a three-dimensional homology model of the amino-terminal domain of the 1 GABA C receptor, they are facing toward the putative binding pocket. Combined with previous findings, a complete model of the GABA C receptor binding pocket was proposed and discussed in comparison with the GABA A receptor binding pocket. GABA A1 and GABA C receptors are both GABA-gated chloride channels but with very different pharmacological properties. In fact, that is the basis for their classification. Although both types of receptors can be activated by GABA, the GABA A receptor can be specifically antagonized by bicuculline. In contrast, the GABA C receptor is insensitive to bicuculline but can be selectively antagonized by 1,2,5,6-tetrahydropyridine-4-yl)-methylphosphinic acid (1). The functional properties of these two types of GABA receptors are also different. For example, the GABA C receptor has higher GABA sensitivity but slower activation and deactivation kinetics than the GABA A receptor (2). The GABA C receptor also shows almost no desensitization (2), whereas the GABA A receptor exhibits strong desensitization (3). Although the functional differences could arise from differences in the structural architecture of the GABA binding pockets, distinct antagonist profiles of GABA A and GABA C receptors indicate that their agonist/antagonist binding pockets are not the same. Studies over the last 15 years have shaped a relatively complete model for the GABA A receptor (4 -9). In contrast, structural information on the GABA C receptor agonist binding pocket is far from complete. Some candidate binding residues in several potential "binding loops" are still undefined.GABA-gated ion channels belong to the ligand-gated ion channel family, which also includes nicotinic acetylcholine receptors, serotonin-3 receptors, and glycine receptors. The study of nicotinic receptors in the past two decades h...

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

“…This hypothesis is not inconsistent with the data, because we observed functional receptors with Lys, which could compensate for both of these bonding requirements, but not Ala or Asp. These data might also suggest that a positive charge is necessary for receptor function at position 170, but such a hypothesis is not supported by the results of Sedelnikova et al (5), in which a cysteine substitution yielded a functional receptor. The EC 50 for the R170C mutant was increased ϳ10-fold compared with wild type, a shift similar to that we observed for the R170K receptor.…”

“…Because Cys can form hydrogen bonds, it could compensate to some extent for the loss of Arg. It is not possible to tell from our data whether binding affinity or functional efficacy is being affected, but the study of Sedelnikova et al (5) indicated that this is a ligandbinding residue, because the Cys mutant at this position was protected from modification by both GABA and a competitive GABA C receptor antagonist. Further evidence comes from GABA A receptor work, where the equivalent residue (Arg-131) has been proposed to form part of a crown of arginines (in addition to Arg-207 and Arg-66) that stabilize the GABA carboxylate (16).…”

“…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 structural basis of function in Cys-loop receptors

“…For example, molecular dynamic studies examining GABA binding to the GABA C R show that GABA appears to ' flip ' from one orientation to another during the simulation, although there is currently only experimental data to support one of the orientations (Melis et al 2008), and in silico predictions in the 5-HT 3 R have concluded that there are two possible orientations for both mCPBG and granisetron Schulte et al 2006). Comprehensive reports can be found elsewhere on the binding sites of 5-HT 3 , nACh (Romanelli et al 2007), Gly (Lynch, 2004) and GABA receptors (Abdel-Halim et al 2008 ;Huang et al 2006 ;Korpi et al 2002 ;Sedelnikova et al 2005).…”

Allosteric activation mechanism of the cys-loop receptors

“…In the case of the ρ1 GABA C receptor, a mutation of the homologous residue (Y102S) in loop D created spontaneously opening channels [59] , further suggesting the importance of this aro- matic residue in loop D in initial conformational changes induced by an agonist. Similarly, in the ρ1 GABA C receptor, a mutation (F146C) in loop A and a mutation in loop E (Q160C) also create spontaneously opening channels [60] . Unlike AChBP, the amino-terminal domain of a cys-loop receptor is coupled to transmembrane domain.…”