The effects of the antihelmintic, ivermectin, were investigated in recombinantly expressed human ␣ 1 homomeric and ␣ 1 ␤ heteromeric glycine receptors (GlyRs). At low (0.03 M) concentrations ivermectin potentiated the response to sub-saturating glycine concentrations, and at higher (>0.03 M) concentrations it irreversibly activated both ␣ 1 homomeric and ␣ 1 ␤ heteromeric GlyRs. Relative to glycine-gated currents, ivermectin-gated currents exhibited a dramatically reduced sensitivity to inhibition by strychnine, picrotoxin, and zinc. The insensitivity to strychnine could not be explained by ivermectin preventing the access of strychnine to its binding site. Furthermore, the elimination of a known glycine-and strychnine-binding site by site-directed mutagenesis had little effect on ivermectin sensitivity, demonstrating that the ivermectin-and glycine-binding sites were not identical. Ivermectin strongly and irreversibly activated a fast-desensitizing mutant GlyR after it had been completely desensitized by a saturating concentration of glycine. Finally, a mutation known to impair dramatically the glycine signal transduction mechanism had little effect on the apparent affinity or efficacy of ivermectin. Together, these findings indicate that ivermectin activates the GlyR by a novel mechanism.

This study investigated the residues responsible for the reduced picrotoxin sensitivity of the ab heteromeric glycine receptor relative to the a homomeric receptor. By analogy with structurally related receptors, the b subunit M2 domain residues P278 and F282 were considered the most likely candidates for mediating this effect. These residues align with G254 and T258 of the a subunit. The T258A, T258C and T258F mutations dramatically reduced the picrotoxin sensitivity of the a homomeric receptor. Furthermore, the converse F282T mutation in the b subunit increased the picrotoxin sensitivity of the ab heteromeric receptor. The P278G mutation in the b subunit did not affect the picrotoxin sensitivity of the ab heteromer. Thus, a ring of ®ve threonines at the M2 domain depth corresponding to a subunit T258 is speci®cally required for picrotoxin sensitivity. Mutations to a subunit T258 also profoundly in¯uenced the apparent glycine af®nity. A substituted cysteine accessibility analysis revealed that the T258C sidechain increases its pore exposure in the channel open state. This provides further evidence for an allosteric mechanism of picrotoxin inhibition, but renders it unlikely that picrotoxin (as an allosterically acting`competitive' antagonist) binds to this residue.

A cortical-basal ganglia network involving, particularly, the posterior region of dorsomedial striatum (DMS) has been implicated in the acquisition of goal-directed actions; however, no direct evidence of learning-related plasticity in this striatal region has been reported, nor is it known whether, or which, specific cell types are involved in this learning process. The striatum is primarily composed of two classes of spiny projection neurons (SPNs): the striatonigral and striatopallidal SPNs, which express dopamine D1 and D2 receptors, respectively. Here we establish that, in mice, the acquisition of goal-directed actions induced plasticity in both D1-and D2-SPNs specifically in the DMS and, importantly, that these changes were in opposing directions; after learning, AMPA/NMDA ratios were increased in D1-SPNs and reduced in the D2-SPNs in the DMS. Such opposing plasticity could provide the basis for rapidly rebiasing the control of task-specific actions, and its dysregulation could underlie disorders associated with striatal function.

Lycopene plays an important role as an antioxidative and anticancer agent, and is an increasingly valuable commodity in the global market. Rhodobacter sphaeroides, a carotenogenic and phototrophic bacterium, is an efficient and practical host for carotenoid production. Herein, we explored the potential of metabolically engineered Rb. sphaeroides as a novel platform to produce lycopene. The basal lycopene-producing strain was generated by introducing an exogenous crtI from Rhodospirillum rubrum to replace the native crtI and deleting crtC in Rb. sphaeroides. Furthermore, knocking out zwf blocked the competitive pentose phosphate pathway and improved the lycopene content by 88%. Finally, the methylerythritol phosphate pathway was reinforced by integration of dxs combined with zwf deletion, which further increased the lycopene content. The final engineered strain produced lycopene to 10.32 mg/g dry cell weight. This study describes a new lycopene producer and provides insight into a photosynthetic bacterium as a host for lycopene biosynthesis.

Inflammatory pain sensitization is initiated by prostaglandin-induced phosphorylation of α3 glycine receptors (GlyRs) that are specifically located in inhibitory synapses on spinal pain sensory neurons. Phosphorylation reduces the magnitude of glycinergic synaptic currents, thereby disinhibiting nociceptive neurons. Although α1 and α3 subunits are both expressed on spinal nociceptive neurons, α3 is a more promising therapeutic target as its sparse expression elsewhere implies a reduced risk of side-effects. Here we compared glycine-mediated conformational changes in α1 and α3 GlyRs to identify structural differences that might be exploited in designing α3-specific analgesics. Using voltage-clamp fluorometry, we show that glycine-mediated conformational changes in the extracellular M2-M3 domain were significantly different between the two GlyR isoforms. Using a chimeric approach, we found that structural variations in the intracellular M3-M4 domain were responsible for this difference. This prompted us to test the hypothesis that phosphorylation of S346 in α3 GlyR might also induce extracellular conformation changes. We show using both voltage-clamp fluorometry and pharmacology that Ser346 phosphorylation elicits structural changes in the α3 glycine-binding site. These results provide the first direct evidence for phosphorylation-mediated extracellular conformational changes in pentameric ligand-gated ion channels, and thus suggest new loci for investigating how phosphorylation modulates structure and function in this receptor family. More importantly, by demonstrating that phosphorylation alters α3 GlyR glycinebinding site structure, they raise the possibility of developing analgesics that selectively target inflammation-modulated GlyRs. KEYWORDS: pLGIC, Cys-loop receptor, inflammatory pain, glycinergic synapse, electrophysiology, protein conformation M embers of the pentameric ligand-gated ion channel (pLGIC) receptor family mediate fast synaptic transmission in the nervous system. The cation-permeable nicotinic acetylcholine receptor (nAChR) is the most intensively studied member of this family, with other members including the anion-permeable glycine and GABA type-A receptors (GlyRs and GABA A Rs) and the cation-permeable serotonin type-3 receptor (5-HT 3 R).1 Functional pLGICs comprise an assembly of five homologous membrane-spanning subunits arranged symmetrically around a central pore. All subunits incorporate large N-terminal ligand-binding domains that form neurotransmitter-binding sites at the interface of adjacent domains. The eponymous extracellular Cys-loop is conserved among eukaryotic members of this family. In addition, GlyRs incorporate a second Cys-loop that forms the C loop ligandbinding domain that is crucial for glycine binding.2 The ligandbinding domain is followed by four transmembrane α-helices, termed M1−M4, that each span the entire thickness of the cell membrane. Each subunit contributes an M2 domain to the lining of the axial water-filled pore. To facilitate comparison of por...