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...

Although molecular mechanisms for hippocampus-dependent memory have been extensively studied, much less is known about signaling events important for remote memory. Here we report that mice lacking type 1 adenylyl cyclase (AC1) are able to establish and retrieve remote contextual memory but unable to sustain it as long as wild-type mice. Interestingly, mice overexpressing AC1 show superior remote contextual memory even though they exhibit normal hippocampus-dependent contextual memory. These data illustrate that calcium coupling to cAMP contributes to the stability of remote memory and identifies AC1 as a potential drug target site to improve long-term remote memory.

In instrumental conditioning, newly acquired actions are generally goal‐directed and are mediated by the relationship between the action and its consequences or outcome. With continued training, however, the performance of such actions can become automatic, reflexive or habitual and under the control of antecedent stimuli rather than their consequences. Recent evidence suggests that habit learning is mediated by plasticity in the dorsolateral striatum (DLS). To date, however, no direct evidence of learning‐related plasticity associated with overtraining has been reported in this region, 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, the striatonigral and striatopallidal spiny projection neurons, which express dopamine D1 and D2 receptors, and control direct and indirect pathways, respectively. Here we found evidence of a post‐synaptic depression in DLS striatopallidal projecting neurons in the indirect pathway during habit learning in mice. Moreover, this training‐induced depression occluded post‐synaptic depression induced by co‐activation of D2 receptors and transient receptor potential vanilloid 1 (TRPV1) channels, implying that this pathway is involved in habit learning. This hypothesis was further tested by disrupting this signal pathway by knocking out TRPV1 channels, resulting in compromised habit learning. Our findings suggest that post‐synaptic plasticity at D2 neurons in the DLS mediates habit learning and, by implicating an interaction between the D2 receptor and TRPV1 channel activity, provide a potential drug target for influencing habitual action control.

The substituted cysteine accessibility method was used to probe the surface exposure of a pore-lining threonine residue (T6) common to both the glycine receptor (GlyR) and ␥-aminobutyric acid, type A receptor (GABA A R) chloride channels. This residue lies close to the channel activation gate, the ionic selectivity filter, and the main pore blocker binding site. Despite their high amino acid sequence homologies and common role in conducting chloride ions, recent studies have suggested that the GlyRs and GABA A Rs have divergent open state pore structures at the 6 position. When both the human ␣1 T6C homomeric GlyR and the rat ␣1 T6C ␤1 T6C heteromeric GABA A R were expressed in human embryonic kidney 293 cells, their 6 residue surface accessibilities differed significantly in the closed state. However, when a soluble cysteine-modifying compound was applied in the presence of saturating agonist concentrations, both receptors were locked into the open state. This action was not induced by oxidizing agents in either receptor. These results provide evidence for a conserved pore opening mechanism in anion-selective members of the ligand-gated ion channel family. The results also indicate that the GABA A R pore structure at the 6 level may vary between different expression systems. The ligand-gated ion channel (LGIC)1 superfamily includes the nicotinic acetylcholine receptor (nAChR), serotonin type 3 receptor (5HT 3 R), GABA A receptor (GABA A R), and glycine receptor (GlyR), as well as invertebrate glutamate and histidine receptors (1). Functional receptors of this family comprise five homologous subunits arranged in a ring to form a central ion-conducting pore. Each subunit is composed of a large extracellular ligand-binding N-terminal domain, four membranespanning segments (M1-M4), and a large intracellular domain between M3 and M4.The pore-lining, second transmembrane (M2) domain has an ␣-helical secondary structure that undergoes a conformational change as the channel is opened (2). To investigate this process in detail, state-dependent differences in the surface exposure of M2 domain residues can be assayed using the substituted cysteine accessibility method (3). In this technique, residues are mutated individually to cysteines, and changes in their reactivity rates with soluble cysteine-reactive reagents can identify structural changes between different functional states. As expected for receptors belonging to the same family, this technique has generally yielded a good correlation between the open state M2 domain secondary structures of the nAChR (4 -7), GABA A R (8), and 5HT 3 R (9, 10).The M2 domain 6Ј residue, which is a threonine in the GlyR ␣1 subunit and the GABA A R ␣1 and ␤1 subunits (see Fig. 1A), lines a critical part of the pore. It is close to the activation gate (6,11,12) and the ionic selectivity filter (13-15) and forms the main pore blocker binding site (reviewed in Ref. 16). Therefore, structural differences at this level may be expected to have significant functional consequences. In the homo...