beta-Carbolines are potent modulators of GABA type A receptors and they have recently been shown to inhibit glycine receptors in a subunit-specific manner. The present study screened four structurally similar beta-carbolines, 1,2,3,4-tetrahydronorharmane, norharmane, harmane and 6-methoxyharmalan, at recombinantly expressed alpha1, alpha1beta, alpha2 and alpha3 glycine receptors with the aims of identifying structural elements of both the receptor and the compounds that are important for binding and subunit specificity. The four compounds exhibited only weak subunit specificity, rendering them unsuitable as pharmacological probes. Because they displayed competitive antagonist activity, we investigated the roles of known glycine binding residues in coordinating the four compounds. The structural similarity of the compounds, coupled with the differential effects of C-loop mutations (T204A, F207Y) on compound potency, implied direct interactions between variable beta-carboline groups and mutated residues. Mutant cycle analysis employing harmane and norharmane revealed a strong pairwise interaction between the harmane methyl group and the C-loop in the region T204 and F207. These results which define the orientation of the bound beta-carbolines were supported by molecular docking simulations. The information may also be relevant to understanding the mechanism beta-carboline of binding to GABA type A receptors where they are potent pharmacological probes.
Posts tegged as 'Cys-loop Receptor'
Ivermectin is an anthelminthic drug that works by activating glutamate-gated chloride channel receptors (GluClRs) in nematode parasites. GluClRs belong to the Cys-loop receptor family that also includes glycine receptor (GlyR) chloride channels. GluClRs and A288G mutant GlyRs are both activated by low nanomolar ivermectin concentrations. The crystal structure of the C. elegans α GluClR complexed with ivermectin has recently been published. Here we probed ivermectin sensitivity determinants on the α1 GlyR using site-directed mutagenesis and
electrophysiology. Based on a mutagenesis screen of transmembrane residues, we identified A288 and P230 as crucial sensitivity determinants. A comparison of the actions of selamectin and ivermectin suggested the benzofuran C05-OH was required for high efficacy. When taken together with docking simulations, these results supported a GlyR ivermectin binding orientation similar to that seen in the GluClR crystal structure. However, whereas the crystal structure shows that ivermectin interacts with the α GluClR via H-bonds with L218, S260 and T285 (α GluClR numbering), our data indicate that Hbonds with residues homologous to S260 and T285 are not important for high ivermectin sensitivity or direct agonist efficacy in A288G α1 GlyRs or three other GluClRs. Our data also suggest that van der Waals interactions between the ivermectin disaccharide and GlyR M2-M3 loop residues are unimportant for high ivermectin sensitivity. Thus, although our results corroborate the ivermectin binding orientation as revealed by the crystal structure, they demonstrate that some of the binding interactions revealed by this structure do not pertain to other highly ivermectin-sensitive Cys-loop receptors.