Characterizing Ligand-Gated Ion Channel Receptors with Genetically Encoded Ca++ Sensors

We present a cell based system and experimental approach to characterize agonist and antagonist selectivity for ligand-gated ion channels (LGIC) by developing sensor cells stably expressing a Ca²⁺ permeable LGIC and a genetically encoded Förster (or fluorescence) resonance energy transfer (FRET)-based calcium sensor. In particular, we describe separate lines with human α7 and human α4β2 nicotinic acetylcholine receptors, mouse 5-HT_3A serotonin receptors and a chimera of human α7/mouse 5-HT_3A receptors. Complete concentration-response curves for agonists and Schild plots of antagonists were generated from these sensors and the results validate known pharmacology of the receptors tested. Concentration-response relations can be generated from either the initial rate or maximal amplitudes of FRET-signal. Although assaying at a medium throughput level, this pharmacological fluorescence detection technique employs a clonal line for stability and has versatility for screening laboratory generated congeners as agonists or antagonists on multiple subtypes of ligand-gated ion channels. The clonal sensor lines are also compatible with in vivo usage to measure indirectly receptor activation by endogenous neurotransmitters.     

TAK1 promotes BMP4/Smad1 signaling via inhibition of erk MAPK: a new link in the FGF/BMP regulatory network

FGFs and BMPs act in concert to regulate a wide range of processes in vertebrate development. In most cases, FGFs and BMPs have opposing effects, and specific developmental outcomes arise out of a balance between the two growth factors. We and others have previously demonstrated that signaling pathways activated by FGFs and BMPs interact via inhibitory crosstalk. Here we demonstrate a role for the BMP effector TGF-β Activated Kinase 1 (TAK1) in the maintenance of Smad1 activity in Xenopus embryos, via the inhibition of erk MAPK. Up- or downregulation of TAK1 levels produces an inverse alteration in the amount of activated erk MAPK. The inhibition of erk MAPK by TAK1 is mediated by p38 and a corresponding decrease in phosphorylation of MEK. TAK1 morphant embryos show a decrease in the nuclear accumulation of Smad1. Conversely, reduction of erk MAPK activity via overexpression of MAP Kinase Phosphatase1 (MKP1) leads to an increase in nuclear Smad1. Both TAK1 morphant ectoderm and ectoderm treated with FGF show a decrease in the expression of several Smad1-inducible genes. Neural-specific gene expression is inhibited in isolated ectoderm coexpressing noggin and TAK1, suggesting that TAK1 is sufficient to inhibit neural specification. Introduction of TAK1 morpholino oligonucleotide expands the expression of organizer genes, disrupts formation of the boundary between organizer and non-organizer mesoderm, and increases the spatial range of MAPK activation in response to localized FGF. Our results indicate that inhibitory interactions between FGF and BMP4 effector pathways increase the robustness of BMP signaling via a feed-forward mechanism.     

Succession of Phenotypic, Genotypic, and Metabolic Community Characteristics during In Vitro Bioslurry Treatment of Polycyclic Aromatic Hydrocarbon-Contaminated Sediments

Dredged harbor sediment contaminated with polycyclic aromatic hydrocarbons (PAHs) was removed from the Milwaukee Confined Disposal Facility and examined for in situ biodegradative capacity. Molecular techniques were used to determine the successional characteristics of the indigenous microbiota during a 4-month bioslurry evaluation. Ester-linked phospholipid fatty acids (PLFA), multiplex PCR of targeted genes, and radiorespirometry techniques were used to define in situ microbial phenotypic, genotypic, and metabolic responses, respectively. Soxhlet extractions revealed a loss in total PAH concentrations of 52%. Individual PAHs showed reductions as great as 75% (i.e., acenapthene and fluorene). Rates of ¹⁴C-PAH mineralization (percent/day) were greatest for phenanthrene, followed by pyrene and then chrysene. There was no mineralization capacity for benzo[a]pyrene. Ester-linked phospholipid fatty acid analysis revealed a threefold increase in total microbial biomass and a dynamic microbial community composition that showed a strong correlation with observed changes in the PAH chemistry (canonical r² of 0.999). Nucleic acid analyses showed copies of genes encoding PAH-degrading enzymes (extradiol dioxygenases, hydroxylases, and meta-cleavage enzymes) to increase by as much as 4 orders of magnitude. Shifts in gene copy numbers showed strong correlations with shifts in specific subsets of the extant microbial community. Specifically, declines in the concentrations of three-ring PAH moieties (i.e., phenanthrene) correlated with PLFA indicative of certain gram-negative bacteria (i.e., Rhodococcus spp. and/or actinomycetes) and genes encoding for naphthalene-, biphenyl-, and catechol-2,3-dioxygenase degradative enzymes. The results of this study suggest that the intrinsic biodegradative potential of an environmental site can be derived from the polyphasic characterization of the in situ microbial community.     

Contemporary paradigms for cholinergic ligand design guided by biological structure

The identification of the various nicotinic receptor subtypes, when coupled with the recent development of three-dimensional structures of surrogate extracellular receptor domains, offers new opportunities to design nicotinic ligands. Conformation and fluctuations in receptor structure are critical to ligand selectivity, and we present here how a flexible receptor template can be used in the development of selective ligands affecting cholinergic neurotransmission.     

Female preference for male saliva: implications for sexual isolation of Mus musculus subspecies

We studied the effects of a single genetic change on a complex mammalian behavior using animals congenic for two variants of Abpa, the gene for the alpha subunit of mouse salivary androgen-binding protein (ABP), in two-way preference tests. Females exhibited a preference for investigating salivas of males of their own genetic type of ABP but not for urines of either type of male. This preference behavior is consistent for samples of mice from geographically diverse populations of Mus musculus domesticus and M. m. musculus. These findings provide an explanation for the observation that this gene is evolving under strong selection.     

Tryptophan fluorescence reveals conformational changes in the acetylcholine binding protein

The recent characterization of an acetylcholine binding protein (AChBP) from the fresh water snail, Lymnaea stagnalis, shows it to be a structural homolog of the extracellular domain of the nicotinic acetylcholine receptor (nAChR). To ascertain whether the AChBP exhibits the recognition properties and functional states of the nAChR, we have expressed the protein in milligram quantities from a synthetic cDNA transfected into human embryonic kidney (HEK) cells. The protein secreted into the medium shows a pentameric rosette structure with ligand stoichiometry approximating five sites per pentamer. Surprisingly, binding of acetylcholine, selective agonists, and antagonists ranging from small alkaloids to larger peptides results in substantial quenching of the intrinsic tryptophan fluorescence. Using stopped-flow techniques, we demonstrate rapid rates of association and dissociation of agonists and slow rates for the alpha-neurotoxins. Since agonist binding occurs in millisecond time frames, and the alpha-neurotoxins may induce a distinct conformational state for the AChBP-toxin complex, the snail protein shows many of the properties expected for receptor recognition of interacting ligands. Thus, the marked tryptophan quenching not only documents the importance of aromatic residues in ligand recognition, but establishes that the AChBP will be a useful functional as well as structural surrogate of the nicotinic receptor.     

Modulation of TASK-1 (Kcnk3) and TASK-3 (Kcnk9) potassium channels: volatile anesthetics and neurotransmitters share a molecular site of action

TASK-1 and TASK-3, members of the two-pore-domain channel family, are widely expressed leak potassium channels responsible for maintenance of cell membrane potential and input resistance. They are sites of action for a variety of modulatory agents, including volatile anesthetics and neurotransmitters/hormones, the latter acting via mechanisms that have remained elusive. To clarify these mechanisms, we generated mutant channels and found that alterations disrupting anesthetic (halothane) activation of these channels also disrupted transmitter (thyrotropin-releasing hormone, TRH) inhibition and did so to a similar degree. For both TASK-1 and TASK-3, mutations (substitutions with corresponding residues from TREK-1) in a six-residue sequence at the beginning of the cytoplasmic C terminus virtually abolished both anesthetic activation and transmitter inhibition. The only sequence motif identified with a classical signaling mechanism in this region is a potential phosphorylation site; however, mutation of this site failed to disrupt modulation. TASK-1 and TASK-3 differed insofar as a large portion of the C terminus was necessary for the full effects of halothane and TRH on TASK-3 but not on TASK-1. Finally, tandem-linked TASK-1/TASK-3 heterodimeric channels were fully modulated by anesthetic and transmitter, and introduction of the identified mutations either into the TASK-1 or the TASK-3 portion of the channel was sufficient to disrupt both effects. Thus, both anesthetic activation and transmitter inhibition of these channels require a region at the interface between the final transmembrane domain and the cytoplasmic C terminus that has not been associated previously with receptor signal transduction. Our results also indicate a close molecular relationship between these two forms of modulation, one endogenous and the other clinically applied.