A Unified Model of the GABA(A) Receptor Comprising Agonist and Benzodiazepine Binding Sites

We present a full-length α(1)β(2)γ(2) GABA receptor model optimized for agonists and benzodiazepine (BZD) allosteric modulators. We propose binding hypotheses for the agonists GABA, muscimol and THIP and for the allosteric modulator diazepam (DZP). The receptor model is primarily based on the glutamate-gated chloride channel (GluCl) from C. elegans and includes additional structural information from the prokaryotic ligand-gated ion channel ELIC in a few regions. Available mutational data of the binding sites are well explained by the model and the proposed ligand binding poses. We suggest a GABA binding mode similar to the binding mode of glutamate in the GluCl X-ray structure. Key interactions are predicted with residues α(1)R66, β(2)T202, α(1)T129, β(2)E155, β(2)Y205 and the backbone of β(2)S156. Muscimol is predicted to bind similarly, however, with minor differences rationalized with quantum mechanical energy calculations. Muscimol key interactions are predicted to be α(1)R66, β(2)T202, α(1)T129, β(2)E155, β(2)Y205 and β(2)F200. Furthermore, we argue that a water molecule could mediate further interactions between muscimol and the backbone of β(2)S156 and β(2)Y157. DZP is predicted to bind with interactions comparable to those of the agonists in the orthosteric site. The carbonyl group of DZP is predicted to interact with two threonines α(1)T206 and γ(2)T142, similar to the acidic moiety of GABA. The chlorine atom of DZP is placed near the important α(1)H101 and the N-methyl group near α(1)Y159, α(1)T206, and α(1)Y209. We present a binding mode of DZP in which the pending phenyl moiety of DZP is buried in the binding pocket and thus shielded from solvent exposure. Our full length GABA(A) receptor is made available as Model S1

Concentration dependent effect of GsMTx4 on mechanosensitive channels of small conductance in E. coli spheroplasts

The spider peptide GsMTx4, at saturating concentration of 5 muM, is an effective and specific inhibitor for stretch-activated mechanosensitive (MS) channels found in a variety of eukaryotic cells. Although the structure of the peptide has been solved, the mode of action remains to be determined. Because of its amphipathic structure, the peptide is proposed to interact with lipids at the boundaries of the MS channel proteins. In addition, GsMTx4 has antimicrobial effects, inhibiting growth of several species of bacteria in the range of 5-64 microM. Previous studies on prokaryotic MS channels, which serve as model systems to explore the principle of MS channel gating, have shown that various amphipathic compounds acting at the protein-lipid interface affect MS channel gating. We have therefore analyzed the effect of different concentrations of extracellular GsMTx4 on MS channels of small conductance, MscS and MscK, in the cytoplasmic membrane of wild-type E. coli spheroplasts using the patch-clamp technique. Our study shows that the peptide GsMTx4 exhibits a biphasic response in which peptide concentration determines inhibition or potentiation of activity in prokaryotic MS channels. At low peptide concentrations of 2 and 4 microM the gating of the prokaryotic MS channels was hampered, manifested by a decrease in pressure sensitivity. In contrast, application of peptide at concentrations of 12 and 20 microM facilitated prokaryotic MS channel opening by increasing the pressure sensitivity

Data from: Hybridisation and genetic diversity in introduced Mimulus (Phrymaceae)

Hybridisation among taxa with different ploidy levels is often associated with hybrid sterility. Clonal reproduction can stabilise these hybrids, but pervasive clonality may have a profound impact on the distribution of genetic diversity in natural populations. Here we investigate a widespread triploid taxon resulting from hybridisation between diploid Mimulus guttatus and tetraploid Mimulus luteus, two species that were introduced into the United Kingdom (UK) in the nineteenth century. This hybrid, Mimulus x robertsii, is largely sterile but capable of prolific vegetative propagation and has been recorded in the wild since 1872. We surveyed 40 Mimulus populations from localities across the UK to examine the current incidence of hybrids, and selected seventeen populations for genetic analysis using codominant markers. Cluster analyses revealed two main groups of genetically distinct individuals, corresponding to either diploid (M. guttatus) or polyploid (M. luteus and M. x robertsii) samples. Triploid hybrids were found in around 50% of sampled sites, sometimes coexisting with one of the parental species (M. guttatus). The other parent, M. luteus, was restricted to a single locality. Individual populations of M. x robertsii were genetically variable, containing multiple, highly heterozygous clones, with the majority of genetic variation distributed among- rather than within populations. Our findings demonstrate that this largely sterile, clonal taxon can preserve non-negligible amounts of genetic variation. The presence of genetically variable hybrid populations may provide the material for the continued success of asexual taxa in diverse environments