Triton X-100 inhibits L-type voltage-operated calcium channels.

Triton X-100 (TX-100) is a nonionic detergent frequently used at millimolar concentrations to disrupt cell membranes and solubilize proteins. At low micromolar concentrations, TX-100 has been reported to inhibit the function of potassium channels. Here, we have used electrophysiological and functional techniques to examine the effects of TX-100 on another class of ion channels, L-type voltage-operated calcium channels (VOCCs). TX-100 (30 nmol·L(-1) to 3 µmol·L(-1)) caused reversible concentration-dependent inhibition of recombinant L-type VOCC (CaV 1.2) currents and of native L-type VOCC currents recorded from rat vascular smooth muscle cells and cardiac myocytes, and murine and human pancreatic ß-cells. In functional studies, TX-100 (165 nmol·L(-1) to 3.4 µmol·L(-1)) caused concentration-dependent relaxation of rat isolated mesenteric resistance arteries prestimulated with phenylephrine or KCl. This effect was independent of the endothelium. TX-100 (1.6 µmol·L(-1)) inhibited depolarization-induced exocytosis in both murine and human isolated pancreatic ß-cells. These data indicate that at concentrations within the nanomolar to low micromolar range, TX-100 significantly inhibits L-type VOCC activity in a number of cell types, an effect paralleled by inhibition of cell functions dependent upon activation of these channels. This inhibition occurs at concentrations below those used to solubilize proteins and may compromise the use of solutions containing TX-100 in bioassays.

The Cys-loop pentameric ligand-gated ion channel receptors: 50 years on.

This year, 2011, the Department of Pharmacology at the University of Alberta celebrated its 50th anniversary. This timeframe covers nearly the entire history of Cys-loop pentameric ligand-gated ion channel (pLGIC) research. In this review we consider how major technological advancements affected our current understanding of pLGICs, and highlight the contributions made by members of our department. The individual at the center of our story is Susan Dunn; her passing earlier this year has robbed the Department of Pharmacology and the research community of a most insightful colleague. Her dissection of ligand interactions with the nAChR, together with their interpretation, was the hallmark of her extensive collaborations with Michael Raftery. Here, we highlight some electrophysiological studies from her laboratory over the last few years, using the technique that she introduced to the department in Edmonton, the 2-electrode voltage-clamp of Xenopus oocytes. Finally, we discuss some single-channel studies of the anionic GlyR and GABA(A)R that prefaced the introduction of this technique to her laboratory.

On the disruption of biochemical and biological assays by chemicals leaching from disposable laboratory plasticware.

Plastic consumables, used universally in bioscience laboratories, are presumed inert with respect to bioassay outcomes. However, it is clear that many pipette tips, microfuge tubes, and other plastic disposables leach bioactive compounds into assay solutions, profoundly affecting data and experimental interpretation. In this paper we discuss the nature and sources of leachates and review several examples of compromised bioassay data that speak to the probable widespread nature of this largely unrecognised source of error. Strategies for minimizing leachate interferences are discussed.

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.

Benzodiazepine modulation of the rat GABAA receptor α4ß3γ2L subtype expressed in Xenopus oocytes.

The effects of benzodiazepines on GABA(A) receptors are dependent largely on the particular α subunit isoform that is present in the receptor pentamer. The inclusion of either the α4 or α6 subunit is generally thought to render the receptor insensitive to classical benzodiazepines. We expressed the rat α4ß3γ2L subtype in Xenopus oocytes and observed that both diazepam and flunitrazepam significantly potentiated GABA-gated currents. This potentiation occurred at nanomolar concentrations similar to those seen at the most abundant "diazepam-sensitive" receptor i.e., the α1ß2γ2 subtype. In the α4ß3γ2L receptor, the effects of diazepam and flunitrazepam were inhibited by nanomolar concentrations of the benzodiazepine site antagonists, Ro15-1788 and ZK93426. The presence of the ß3 subunit appears to be important for this modulation since diazepam did not affect GABA responses mediated by recombinant α4ß1γ2L or α4ß2γ2L receptors. Interestingly, when the α4ß3γ2L receptor was expressed in HEK293 cells, diazepam and flunitrazepam displaced the relatively non-selective benzodiazepine site ligand, [(3)H]Ro15-4513, only at high concentrations (>10 µM) demonstrating a lack of high affinity binding for these classical benzodiazepines. Functional studies of the cell-expressed receptors using whole cell recording techniques showed that neither diazepam nor flunitrazepam potentiated GABA-evoked currents although currents were enhanced by nanomolar concentrations of Ro15-4513. These results suggest that the observed benzodiazepine modulation of the α4ß3γ2L subtype depends on the expression system used and may be specific for expression in Xenopus oocytes.