Comparative Models of GABA_A Receptor Extracellular and Transmembrane Domains: Important Insights in Pharmacology and Function

Abstract

Comparative models of the extracellular and transmembrane domains of GABA A receptors in the agonist-free state were generated based on the recently published structures of the nicotinic acetylcholine receptor. The models were validated by computational methods, and their reliability was estimated by analyzing conserved and variable elements of the cys-loop receptor topology. In addition, the methodological limits in the interpretation of such anion channel receptor models are discussed. Alignment ambiguities in the helical domain were resolved for helix 3 by placing two gaps into the linker connecting helices 2 and 3. The resulting models were shown to be consistent with a wide range of pharmacological and mutagenesis data from GABA A and glycine receptors. The loose packing of the models results in a large amount of solvent-accessible space and offers a natural explanation for the rich pharmacology and the great flexibility of these receptors that are known to exist in numerous drug-induced conformational states. Putative drug binding pockets found within and between subunits are described, and amino acid residues important for the action and subtype selectivity of volatile and intravenous anesthetics, barbiturates, and furosemide are shown to be part of these pockets. The entire helical domain, however, seems to be crucial not only for binding of drugs but also for transduction of binding to gating or of allosteric modulation. These models can now be used to design new experiments for clarification of pharmacological and structural questions as well as for investigating and visualizing drug induced conformational changes.GABA A receptors mediate a large part of the fast inhibitory transmission in the central nervous system and are the targets for many clinically important drugs, such as sedatives, hypnotics, anxiolytics, anticonvulsives, muscle relaxants, and anesthetics (Sieghart, 1995). They are composed of five subunits that can belong to different homologous subunit classes and form a chloride channel that can be opened by GABA. Individual neurons can express many different subunits, resulting in the formation of a large variety of functionally different receptor subtypes (Sieghart and Sperk, 2002). Depending on the subunit composition, these receptors exhibit a distinct pharmacology (Sieghart, 1995).The subunit organization with the extracellular ligand binding domain containing the "signature" disulfide, four transmembrane segments, and a large variable cytoplasmic domain (termed the "cytoplasmic loop") of unknown structure, as well as the receptor organization as a pentamer, are hallmarks of the superfamily of cys-loop receptors (pentameric ligand-gated ion channels) comprising the cation-conducting nicotinic acetylcholine (nACh) and serotonin type 3 (5HT 3 -) receptors and the anion-conducting GABA A and glycine receptors.In 2001, the X-ray crystallographic structure of acetylcholine binding protein (AChBP) has revealed the fold in which the ␤-strand rich "extracellular domain" of the superfam...

State-Dependent Cross-Linking of the M2 and M3 Segments: Functional Basis for the Alignment of GABAAand Acetylcholine Receptor M3 Segments

“…1, 6). Thus, despite the cogent rationale offered recently for a two residue gap in the M2-M3 loop (Ernst et al, 2005), it is not supported by our current experiments. Furthermore, our results imply that the relative depths of the M2 and M3 segments in the 2BG9 AChR structure are correct (Unwin, 2005).…”

“…Recently, GABA A receptor homology models (Cromer et al, 2002;Trudell and Bertaccini, 2004;Ernst et al, 2005) were constructed based on the acetylcholine binding protein and Torpedo ACh receptor structures (Brejc et al, 2001;Miyazawa et al, 2003;Celie et al, 2004;Unwin, 2005). Homology models depend on the correct alignment of subunit sequences from different superfamily members.…”

“…Alignment of ACh and GABA A M1 and M2 transmembrane segments is facilitated by highly conserved residues corresponding to GABA A ␣1R221, ␣1P233, ␣1F245, ␣1R255, ␣1L264, and ␣1P278 ( Fig. 1) (Le Novere and Changeux, 1999;Ernst et al, 2005). M3 segment alignment is more complicated, because they are mostly hydrophobic with very limited sequence identity (Fig.…”

The major central endocannabinoid directly acts at GABA A receptors

“…Although allosteric effects by the mutations cannot be fully excluded, we consider this possibility unlikely. Both AARs are predicted to be located in cytoplasmic leaflet of the M4 α-helix, in an angle of approximately 60°and a distance in the direction of the α-helix of 4.5 Å. Intriguingly, in a homology model described by Ernst et al (28), all four mentioned residues face the same cavity in the receptor.…”

Identification of novel positive allosteric modulators and null modulators at the GABAA receptor α+β− interface

“…However, a modelling study (Ernst et al, 2005) indicated the presence of multiple solvent accessible pockets within the GABAA receptor that could function as possible drug binding sites. Simultaneous drug interaction with several of these binding sites can explain the extremely complex pharmacology of these receptors.…”