ABSTRACTlonotropic receptors for y-aminobutyric acid (GABA) are important to inhibitory neurotransmission in the mammalian retina, mediating GABAA and GABAc responses. In many species, these responses are blocked by the convulsant picrotoxinin (PTX), although the mechanism of block is not fully understood. In contrast, GABAC responses in the rat retina are extremely resistant to PTX. We hypothesized that this difference could be explained by molecular characterization of the receptors underlying the GABAC response. Here we report the cloning of two rat GABA receptor subunits, designated rpl and rp2 after their previously identifled human homologues. When coexpressed in Xenopus oocytes, rpl/rp2 heteromeric receptors mimicked PTX-resistant GABAC responses of the rat retina. PTX resistance is apparently conferred in native heteromeric receptors by rp2 subunits since homomeric rpl receptors were sensitive to PTX; rp2 subunits alone were unable to form functional homomeric receptors. Site-directed mutagenesis confirmed that a single amino acid residue in the second membrane-spanning region (a methionine in rp2 in place of a threonine in rpl) is the predominant determinant of PTX resistance in the rat receptor. This study reveals not only the molecular mechanism underlying PTX blockade of GABA receptors but also the heteromeric nature of native receptors in the rat retina that underlie the PTX-resistant GABAC response.

Ligand-gated ion channels display a fundamental property-channels remain virtually closed at rest and open upon agonist binding. Here we show that substituting alanines for either of two amino acid residues (T314 or L317) in the M2 region of the ␥-aminobutyric acid (GABA) 1 subunit results in spontaneous channel opening in the absence of ligand. Surprisingly, for two single point mutants (T314A or L317A), application of very low concentrations of agonist partially suppressed this spontaneous current, while higher concentrations re-activated the receptors. When both of these sites were mutated (T314A͞L317A), GABA nearly completely suppressed the constitutive current and did not re-activate the current even at very high concentrations. This study provides important new insights into the structure-function relationship of ligand-gated ion channels, where modification of the structure of the channel pore region not only alters the allosteric transition of the receptor protein but also reverses the polarity of agonist regulation of channel gating. Our results suggest that the sites where these two residues are located are structurally critical for channel gating.Members of the ligand-gated ion channel superfamily share certain structural and functional similarities (1-5) and include the following receptors: nicotinic acetylcholine (nACh), glycine, 5-hydroxytryptamine type 3, and ␥-aminobutyric acid (GABA, A and C subtypes). In this family, the receptor is apparently composed of five subunits, and the second membrane-spanning segment (M2) forms the pore of the channel (6-8). Typically, the channel is closed at rest and opens upon agonist binding to its receptor site, located at some distance from the pore region. Channel activity is thought to be controlled by a gate inside the pore (7-9). The structure of the nACh receptor (nAChR) channel in the closed and open states has been characterized at 9-Å resolution in electron crystallographic studies (9, 10). Receptor protein function has been interpreted in terms of an allosteric transition model (11,12). However, the nature of the channel gate as well as the exact mechanism of ligand regulation of gating are still not clear.The GABA subunits are known to comprise, at least in part, the recently described GABA C receptor (5, 13-17). A threonine residue lies at position 314 in the M2 domain of the 1 subunit and is conserved in most known GABA and glycine receptor subunits (Fig. 1). Previously, we reported that a naturally occurring mutation (a methionine instead of a threonine) at the corresponding site of the 2 subunit is responsible for the resistance to picrotoxinin (PTX) blockade of native GABA C receptors in the rat retina (16). To explore further the role of this and surrounding sites on receptor-channel function of GABA C receptors, we made a series of additional substitutions by site-directed mutagenesis, taking advantage of the observation that recombinant 1 subunits are in general capable of forming functional homomeric receptors (5, 16). Here we report t...

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Background/Aims: Rheumatoid arthritis (RA) is a systemic chronic inflammatory disease characterised by prominent synoviocyte hyperplasia and a potential imbalance between the growth and death of fibroblast-like synoviocytes (FLS). Mitomycin C (MMC) has previously been demonstrated to inhibit fibroblast proliferation and to induce fibroblast apoptosis. However, the effects of MMC on the proliferation and apoptosis of human RA FLS and the potential mechanisms underlying its effects remain unknown. Methods: Cell viability was determined using the Cell Counting Kit-8 assay. Apoptotic cell death was analysed via Annexin V-FITC/PI double staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling. The production of intracellular reactive oxygen species (ROS) was assessed via flow cytometry, and the changes in mitochondrial membrane potential (ΔΨm) were visualized based on JC-1 staining via fluorescence microscopy. The expression of apoptosis-related proteins was determined via Western blot. Results: Treatment with MMC significantly reduced cell viability and induced apoptosis in RA FLS. Furthermore, MMC exposure was found to stimulate the production of ROS and to disrupt the ΔΨm compared to the control treatment. Moreover, MMC increased the release of mitochondrial cytochrome c, the ratio of Bax/Bcl-2, the activation of caspase-9 and caspase-3, and the subsequent cleavage of poly(ADP-ribose) polymerase. Conclusion: Our findings suggest that MMC inhibits cell proliferation and induces apoptosis in RA FLS, and the mechanism underlying this MMC-induced apoptosis may involve a mitochondrial signalling pathway.