Chloride Channels of Glycine and GABA Receptors with Blockers: Monte Carlo Minimization and Structure-Activity Relationships

AbstractGABA and glycine receptors (GlyRs) are pentameric ligand-gated ion channels that respond to the inhibitory neurotransmitters by opening a chloride-selective central pore lined with five M2 segments homologous to those of α1 GlyR/ ARVG2′LGIT6′TVLTMTTQSSGSR. The activity of cyanotriphenylborate (CTB) and picrotoxinin (PTX), the best-studied blockers of the Cl− pores, depends essentially on the subunit composition of the receptors, in particular, on residues in positions 2′ and 6′ that form the pore-facing rings R2′ and R6′. Thus, CTB blocks α1 and α1/β, but not α2 GlyRs (Rundström, N., V. Schmieden, H. Betz, J. Bormann, and D. Langosch. 1994. Proc. Natl. Acad. Sci. U.S.A. 91:8950–8954). PTX blocks homomeric receptors (α1 GlyR and rat ρ1 GABAR), but weakly antagonizes heteromeric receptors (α1/β GlyR and ρ1/ρ2 GABAR) (Pribilla, I., T. Takagi, D. Langosch, J. Bormann, and H. Betz. 1992. EMBO J. 11:4305–4311; Zhang D., Z. H. Pan, X. Zhang, A. D. Brideau, and S. A. Lipton. 1995. Proc. Natl. Acad. Sci. U.S.A. 92:11756–11760). Using as a template the kinked-helices model of the nicotinic acetylcholine receptor in the open state (Tikhonov, D. B., and B. S. Zhorov. 1998. Biophys. J. 74:242–255), we have built homology models of GlyRs and GABARs and calculated Monte Carlo-minimized energy profiles for the blockers pulled through the pore. The profiles have shallow minima at the wide extracellular half of the pore, a barrier at ring R6′, and a deep minimum between rings R6′ and R2′ where the blockers interact with five M2s simultaneously. The star-like CTB swings necessarily on its way through ring R6′ and its activity inversely correlates with the barrier at R6′: Thr6′s and Ala2′s in α2 GlyR confine the swinging by increasing the barrier, while Gly2′s in α1 GlyR and Phe6′s in β GlyR shrink the barrier. PTX has an egg-like shape with an isopropenyl group at the elongated end and the rounded end trimmed by ether and carbonyl oxygens. In the optimal binding mode to α1 GlyR and ρ1 GABAR, the rounded end of PTX accepts several H-bonds from Thr6′s, while the elongated end enters ring R2′. The lack of H-bond donors on the side chains of Phe6′s (β GlyR) and Met6′s (ρ2 GABAR) deteriorates the binding. The hydrophilic elongated end of picrotin does not fit the hydrophobic ring of Pro2′s/Ala2′s in GABARs, but fit a more hydrophilic ring with Gly2′s in GlyRs. This analysis provides explanations for structure-activity relationships of noncompetitive agonists and predicts a narrow pore of LGICs in agreement with experimental data on the permeation of organic cations