Susan M.J. Dunn

Bioactive Contaminants Leach from Disposable Laboratory Plasticware

Disposable plasticware such as test tubes, pipette tips, and multiwell assay or culture plates are used routinely in most biological research laboratories. Manufacturing of plastics requires the inclusion of numerous chemicals to enhance stability, durability, and performance. Some lubricating (slip) agents, exemplified by oleamide, also occur endogenously in humans and are biologically active, and cationic biocides are included to prevent bacterial colonization of the plastic surface. We demonstrate that these manufacturing agents leach from laboratory plasticware into a standard aqueous buffer, dimethyl sulfoxide, and methanol and can have profound effects on proteins and thus on results from bioassays of protein function. These findings have far-reaching implications for the use of disposable plasticware in biological research.

Molecular Neurobiology of the GabaA Receptor

Publisher Summary This chapter discusses the molecular neurobiology of the GABAA receptor. The GABAA receptor is responsible for the majority of neuronal inhibition in the vertebrate CNS. The ubiquitous distribution of the GABAA receptor in the mammalian CNS is revealed by the use of [3H]GABA radioligand binding techniques, whereas autoradiographic studies have demonstrated their distinct topographical localization. There is evidence that the receptor is not only expressed in regions of the cell that receive GABAergic input, but also, for example, on Golgi cell somata where no synaptic contacts are found. With the two monoclonal antibodies used in this study, no immunoreactivity is detected on axons, nerve terminals, or glial cells. The distribution of the GABAA receptor at both synaptic and nonsynaptic sites is quite distinct from that of the closely associated glycine receptor, which appears to be mainly associated with synapses. Initial electrophysiological studies of the GABAA receptor demonstrated that its activation by GABA resulted in an increased chloride conductance of the supporting neuronal membrane.

Endogenous cannabinoid anandamide directly inhibits voltage-dependent Ca2+ fluxes in rabbit t-tubule membranes

The effect of the endogenous cannabinoid, anandamide on Ca(2+) flux responses mediated by voltage-dependent Ca(2+) channels was studied in transverse tubule membrane vesicles from rabbit skeletal muscle. Vesicles were loaded with 45Ca(2+) and membrane potentials were generated by establishing K(+) gradients across the vesicle using the ionophore, valinomycin. Anandamide, in the range of 1-100 microM, inhibited depolarization-induced efflux responses. Anandamide also functionally modulated the effects of nifedipine (1-10 microM) and Bay K 8644 (1 microM) on Ca(2+) flux responses. Pretreatment with the specific cannabinoid receptor antagonist, SR141716A (1 microM), pertussis toxin (5 microg/ml), the amidohydrolase inhibitor, phenylmethylsulfonyl fluoride (0.2 mM) or the cyclooxygenase inhibitor, indomethacin (5 microM) did not alter the inhibition of efflux responses by anandamide. Arachidonic acid (10-100 microM) also effectively inhibited 45Ca(2+) efflux from membrane vesicles. In radioligand binding studies, it was found that both anandamide and arachidonic acid inhibited the specific binding of [3H]PN 200-110 to transverse tubule membranes with IC(50) values of 4.4+/-0. 7 and 13.4+/-3.5 microM, respectively. These results indicate that anandamide, independent of cannabinoid receptor activation, directly inhibits the function of voltage-dependent calcium channels and modulates the specific binding of calcium channel ligands of the dihydropyridine class.

Structural Requirements for Ligand Interactions at the Benzodiazepine Recognition Site of the GABAA Receptor

Abstract: His101 of the GABAA receptor α1 subunit is an important determinant of benzodiazepine recognition and a major site of photolabeling by [3H]flunitrazepam. To investigate further the chemical specificity of the residue in this position, we substituted it with phenylalanine, tyrosine, lysine, glutamate, glutamine, or cysteine. The mutant α subunits were coexpressed with the rat β2 and γ2 subunits in TSA201 cells, and the effects of the substitutions on the binding of benzodiazepine site ligands were examined. [3H]Ro 15‐4513 bound to all mutant receptors with equal or greater affinity than to the wild‐type receptor. However, flunitrazepam and ZK93423 recognition was adversely affected by substitutions of the amino acid in this position. The binding of the antagonists, Ro 15‐1788 and ZK93426, was also sensitive to the mutations, with the largest decreases in affinity occurring with the tyrosine, lysine, and glutamate substitutions. In all mutants that recognized flunitrazepam, GABA potentiated the binding of this ligand to a similar extent, suggesting that it is a full agonist at these receptors. The effects of GABA on the binding of Ro 15‐1788 and Ro 15‐4513 suggest that their efficacies may have been changed by some of the substitutions. This study further emphasizes the importance of the residue at position 101 in both ligand recognition and pharmacological effect.

Identification of a residue in the γ-aminobutyric acid type A receptor α subunit that differentially affects diazepam-sensitive and -insensitive benzodiazepine site binding

GABAA receptors that contain either the α4‐ or α6‐subunit isoform do not recognize classical 1,4‐benzodiazepines (BZDs). However, other classes of BZD site ligands, including β‐carbolines, bind to these diazepam‐insensitive receptor subtypes. Some β‐carbolines [e.g. ethyl β‐carboline‐3‐carboxylate (β‐CCE) and methyl 6,7‐dimethoxy‐4‐ethyl‐β‐carboline‐3‐carboxylate (DMCM)] display a higher affinity for α4‐ compared to α6‐containing receptors. In order to identify the structural determinants that underlie these affinity differences, we constructed chimeric α6/α4 subunits and co‐expressed these with wild‐type rat β2 and γ2L subunits in tsA201 cells for radioligand binding analysis. After identification of candidate regions, site‐directed mutagenesis was used to narrow the ligand selectivity to a single amino acid residue (α6N204/α4I203). Substitutions at α6N204 did not alter the affinity of the imidazobenzodiazepine Ro15‐4513. A homologous mutation in the diazepam‐sensitive α1 subunit (S205N) resulted in a 7–8‐fold reduction in affinity for the β‐carbolines examined. Although the binding of the classical agonist flunitrazepam was relatively unaffected by this mutation in the α1 subunit, the affinity for Ro15‐1788 and Ro15‐4513 was decreased by ∼19‐fold and ∼38‐fold respectively. The importance of this residue, located in the Loop C region of the extracellular N‐terminus of the subunit protein, emphasizes the differential interaction of ligands with the α subunit in diazepam‐sensitive and ‐insensitive receptors.

Identification of a domain in the δ subunit (S238-V264) of the α4β3δ GABAA receptor that confers high agonist sensitivity

We have expressed the α4β3δ and α4β3γ2L subtypes of the rat GABAA receptor in Xenopus oocytes and have investigated their agonist activation properties. GABA was a more potent agonist of the α4β3δ receptor (EC50u2003≈u20031.4u2003μmol/L) than of the α4β3γ2L subtype (EC50u2003≈u200327.6u2003μmol/L). Other GABAA receptor agonists (muscimol, 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridin‐3‐ol, imidazole‐4‐amino acid) displayed similar subtype selectivity. The structural determinants underlying these differences have been investigated by co‐expressing chimeric δ/γ2L subunits with α4 and β3 subunits. A stretch of amino acids in the δ subunit, S238‐V264, is shown to play an important role in determining both agonist potency and the efficacies of full or partial agonists. This segment includes transmembrane domain 1 and the short intracellular loop that leads to the second transmembrane domain. The effects of the competitive antagonists, bicuculline and SR95531, and the channel blocker, picrotoxin, were not significantly affected by the incorporation of chimeric subunits. As the δ and γ2L subunits have not been previously implicated directly in agonist binding, we suggest that the effects are likely to arise from changes in the transduction mechanisms that link agonist binding to channel activation.

Isomerization of the proline in the M2-M3 linker is not required for activation of the human 5-HT3A receptor.

Each subunit of the cation‐selective members of the Cys‐loop family of ligand‐gated ion channels contains a conserved proline residue in the extracellular loop between the second and third transmembrane domains. In the mouse homomeric 5‐hydroxytryptamine type 3A (5‐HT3A) receptor, the effects of substitution of this proline by unnatural amino acids led to the suggestion that trans‐cis isomerization of the protein backbone at this position is integral to agonist‐induced channel opening [Nature (2005) vol. 438, pp. 248–252]. We explored the generality of this conclusion using natural amino acid mutagenesis of the homologous human 5‐HT3A receptor. The conserved proline (P303) was substituted by either a histidine or tryprophan and the mutant receptors were expressed in Xenopus oocytes. These mutations did not significantly affect the magnitude of agonist‐mediated currents, compromise channel gating by 5‐HT or inhibition of 5‐HT‐induced currents by either picrotoxin or d‐tubocurarine. The mutations did, however, result in altered dependence on extracellular Ca2+ concentration and a 10‐fold increase in the rate of receptor desensitization. These results demonstrate an important role for P303 in 5‐HT3A receptor function but indicate that trans‐cis isomerization at this proline is unlikely to be a general mechanism underlying the gating process.

Mapping of GABAA receptor sites that are photoaffinity-labelled by [3H]flunitrazepam and [3H]Ro 15-4513

The GABAA receptor in brain membranes prepared from bovine cerebral cortex and cerebellum has been photoaffinity-labelled by the classical benzodiazepine agonist, [3H]flunitrazepam, or by the partial inverse agonist [3H]Ro 15-4513. Following solubilization and precipitation with trichloroacetic acid, the photoaffinity-labelled receptor preparations were subjected to specific chemical cleavage using hydroxylamine, a reagent which cleaves specifically at a relatively rare Asn-Gly bond. The resulting peptides were resolved by denaturing polyacrylamide gel electrophoresis and mapping of these peptides to the known amino acid sequences of the GABAA receptor subunits has localized the photoaffinity-labelling sites for these two ligands to distinct portions of the alpha subunits. It is shown that the site for [3H]flunitrazepam photoaffinity-labelling in the receptor populations of both the cerebral cortex and cerebellum occurs within residues 1-103 of the bovine alpha 1 subunit sequence (or within analogous segments of homologous alpha subunits). In contrast, the site of photoaffinity-labelling by [3H]Ro 15-4513 in the cerebral cortex and in the diazepam-sensitive GABAA receptor population of the cerebellum lies between residues 104 and the carboxy-terminus of the bovine alpha 1 or homologous alpha subunits. However, the [3H]Ro 15-4513 photoaffinity-labelling site for the diazepam-insensitive receptors of the cerebellum is shown to occur within residues 1-101 (alpha 6 subunit numbering). These results demonstrate that the photoaffinity-labelling sites for [3H]flunitrazepam and [3H]Ro 15-4513 on the GABAA receptor are localized to distinct domains of the alpha 1 subunit and that [3H]Ro 15-4513 photoaffinity labels a site on the alpha 6 subunit that is unique from its site of labelling on the alpha 1 subunit.

Insights into the structure and pharmacology of GABA(A) receptors.

GABA is the major inhibitory neurotransmitter in the adult mammalian CNS. The ionotropic GABA type A receptors (GABA(A)Rs) belong to the Cys-loop family of receptors. Each member of the family is a large pentameric protein in which each subunit traverses the cell membrane four times. Within this family, the GABA type A receptors are particularly important for their rich pharmacology as they are targets for a range of therapeutically important drugs, including the benzodiazepines, barbiturates, neuroactive steroids and anesthetics. This review discusses new insights into receptor properties that allow us to begin to relate the structure of an individual receptor to its functional and pharmacological properties.

Identification of Domains in Human Recombinant GABAA Receptors that are Photoaffinity Labelled by [3H]Flunitrazepam and [3H]Ro15-4513

We have used [3H]flunitrazepam and [3H]Ro15-4513 as photoaffinity labelling agents in combination with a chemical cleavage technique to localize the benzodiazepine recognition sites of specific human recombinant alpha 1 beta 1 gamma 2, alpha 1 beta 3 gamma 2 and alpha 6 beta 3 gamma 2 GABAA receptor subtypes. The chemical agent utilized was hydroxylamine, whose substrate is a relatively rare asparagine-glycine amide bond that occurs only in the alpha subunits of the receptors examined in this study. Cleavage products were resolved using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The results of these experiments show that, in the alpha 1 subunit-containing receptors, incorporation of [3H]flunitrazepam occurs within residues 1-103 of the alpha 1 subunit, while incorporation of [3H]Ro15-4513 occurs within the region of the alpha 1 subunit that lies between residue 104 and the C-terminus. Photolabelling of membranes prepared from the alpha 6 beta 3 gamma 2-expressing cell line with [3H]Ro15-4513 resulted in the incorporation of radiolabel into two major protein species of M(r) 56,000 and M(r) 48,000, indicating incorporation into the alpha 6 subunit and possibly also the gamma 2 subunit. Hydroxylamine cleavage of alpha 6-containing receptors labelled with [3H]Ro15-4513 produced a gel profile consistent with the incorporation of the label occurring between residue 125 and the C-terminal. Thus, we have shown that the recognition sites for the agonist [3H]flunitrazepam and the inverse agonist [3H]Ro15-4513 occur within distinct domains of the human GABAA receptor.