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.
Background and PurposeIt has been proposed that arginine residues lining the intracellular portals of the homomeric 5-HT3A receptor cause electrostatic repulsion of cation flow, accounting for a single-channel conductance substantially lower than that of the 5-HT3AB heteromer. However, comparison of receptor homology models for wild-type pentamers suggests that salt bridges in the intracellular domain of the homomer may impart structural rigidity, and we hypothesized that this rigidity could account for the low conductance.Experimental ApproachMutations were introduced into the portal region of the human 5-HT3A homopentamer, such that putative salt bridges were broken by neutralizing anionic partners. Single-channel and whole cell currents were measured in transfected tsA201 cells and in Xenopus oocytes respectively. Computational simulations of protein flexibility facilitated comparison of wild-type and mutant receptors.Key ResultsSingle-channel conductance was increased substantially, often to wild-type heteromeric receptor values, in most 5-HT3A mutants. Conversely, introduction of arginine residues to the portal region of the heteromer, conjecturally creating salt bridges, decreased conductance. Gating kinetics varied significantly between different mutant receptors. EC50 values for whole-cell responses to 5-HT remained largely unchanged, but Hill coefficients for responses to 5-HT were usually significantly smaller in mutants. Computational simulations suggested increased flexibility throughout the protein structure as a consequence of mutations in the intracellular domain.Conclusions and ImplicationsThese data support a role for intracellular salt bridges in maintaining the quaternary structure of the 5-HT3 receptor and suggest a role for the intracellular domain in allosteric modulation of cooperativity and agonist efficacy.Linked ArticleThis article is commented on by Vardy and Kenakin, pp. 1614–1616 of volume 171 issue 7. To view this commentary visit http://dx.doi.org/10.1111/bph.12550.
The 5-hydroxytryptamine type 3 (5-HT 3 ) receptor belongs to the Cys-loop family of ligand-gated ion channels (LGICs) that includes the nicotinic acetylcholine, GABA A and glycine receptors (Reeves and Lummis 2002;Lester et al. 2004). Each member of the family is a pentamer of homologous subunits that assemble to form a central ion pore. All subunits share a common transmembrane topology i.e., a large extracellular N-terminal domain that contributes to the formation of binding sites for agonists and competitive antagonists followed by four transmembrane (M) domains, the second of which (M2) lines the ion channel. Studies of most members of the LGIC family are confounded by their extensive subunit heterogeneity and the complexities that arise from the presence of dissimilar receptor subunits within the pentamer. The 5-HT 3 receptors appear to be less heterogeneous and although five gene products (5-HT3A to 5-HT3E) have been identified (Neisler et al. 2007), the presence of the 5-HT3A subunit appears to be obligatory for the formation of a functional receptor. Thus far, only two receptor subtypes have been studied in significant detail i.e., the homomeric 5-HT 3 A receptor and the heteromeric 5-HT 3 AB receptor (Maricq et al. 1991;Davies et al. 1999). The 5-HT 3 A receptor, like its homomeric nicotinic a7 and glycine a1 or a2 counterparts, provides a relatively simple system to explore detailed mechanisms of receptor activation.Although no crystal structure is available for any member of the LGIC family, considerable structural information has come recently from high resolution electron microscopy images of the prototypical Torpedo nicotinic receptor AbstractEach 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-HT 3 A) 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-HT 3 A 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 Ca 2+ concentration and a 10-fold increase in the rate of receptor desensitization. These results demonstrate an important role for P303 in 5-HT 3 A receptor function but indicate that trans-cis isomerization at this proline is unlikely to be a general mechanism underlying the gating process.
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