Loop 2 Structure in Glycine and GABAA Receptors Plays a Key Role in Determining Ethanol Sensitivity

The present study tests the hypothesis that the structure of extracellular domain Loop 2 can markedly affect ethanol sensitivity in glycine receptors (GlyRs) and γ-aminobutyric acid type A receptors (GABA_ARs). To test this, we mutated Loop 2 in the α1 subunit of GlyRs and in the γ subunit of α1β2γ2GABA_ARs and measured the sensitivity of wild type and mutant receptors expressed in Xenopus oocytes to agonist, ethanol, and other agents using two-electrode voltage clamp. Replacing Loop 2 of α1GlyR subunits with Loop 2 from the δGABA_AR (δL2), but not the γGABA_AR subunit, reduced ethanol threshold and increased the degree of ethanol potentiation without altering general receptor function. Similarly, replacing Loop 2 of the γ subunit of GABA_ARs with δL2 shifted the ethanol threshold from 50 mm in WT to 1 mm in the GABA_A γ-δL2 mutant. These findings indicate that the structure of Loop 2 can profoundly affect ethanol sensitivity in GlyRs and GABA_ARs. The δL2 mutations did not affect GlyR or GABA_AR sensitivity, respectively, to Zn²⁺ or diazepam, which suggests that these δL2-induced changes in ethanol sensitivity do not extend to all allosteric modulators and may be specific for ethanol or ethanol-like agents. To explore molecular mechanisms underlying these results, we threaded the WT and δL2 GlyR sequences onto the x-ray structure of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel homologue (GLIC). In addition to being the first GlyR model threaded on GLIC, the juxtaposition of the two structures led to a possible mechanistic explanation for the effects of ethanol on GlyR-based on changes in Loop 2 structure.Alcohol abuse and dependence are significant problems in our society, with ∼14 million people in the United States being affected (1, 2). Alcohol causes over 100,000 deaths in the United States, and alcohol-related issues are estimated to cost nearly 200 billion dollars annually (2). To address this, considerable attention has focused on the development of medications to prevent and treat alcohol-related problems (3–5). The development of such medications would be aided by a clear understanding of the molecular structures on which ethanol acts and how these structures influence receptor sensitivity to ethanol.Ligand-gated ion channels (LGICs)² have received substantial attention as putative sites of ethanol action that cause its behavioral effects (6–12). Research in this area has focused on investigating the effects of ethanol on two large superfamilies of LGICs: 1) the Cys-loop superfamily of LGICs (13, 14), whose members include nicotinic acetylcholine, 5-hydroxytryptamine₃, γ-aminobutyric acid type A (GABA_A), γ-aminobutyric acid type C, and glycine receptors (GlyRs) (10, 11, 15–20) and 2) the glutamate superfamily, including N-methyl d-aspartate, α-amino-3-hydroxyisoxazolepropionic acid, and kainate receptors (21, 22). Recent studies have also begun investigating ethanol action in the ATP-gated P2X superfamily of LGICs (23–25).A series of studies that employed chimeric and mutagenic strategies combined with sulfhydryl-specific labeling identified key regions within Cys-loop receptors that appear to be initial targets for ethanol action that also can determine the sensitivity of the receptors to ethanol (7–12, 18, 19, 26–30). This work provides several lines of evidence that position 267 and possibly other sites in the transmembrane (TM) domain of GlyRs and homologous sites in GABA_ARs are targets for ethanol action and that mutations at these sites can influence ethanol sensitivity (8, 9, 26, 31).Growing evidence from GlyRs indicates that ethanol also acts on the extracellular domain. The initial findings came from studies demonstrating that α1GlyRs are more sensitive to ethanol than are α2GlyRs despite the high (∼78%) sequence homology between α1GlyRs and α2GlyRs (32). Further work found that an alanine to serine exchange at position 52 (A52S) in Loop 2 can eliminate the difference in ethanol sensitivity between α1GlyRs and α2GlyRs (18, 20, 33). These studies also demonstrated that mutations at position 52 in α1GlyRS and the homologous position 59 in α2GlyRs controlled the sensitivity of these receptors to a novel mechanistic ethanol antagonist (20). Collectively, these studies suggest that there are multiple sites of ethanol action in α1GlyRs, with one site located in the TM domain (e.g. position 267) and another in the extracellular domain (e.g. position 52).Subsequent studies revealed that the polarity of the residue at position 52 plays a key role in determining the sensitivity of GlyRs to ethanol (20). The findings with polarity in the extracellular domain contrast with the findings at position 267 in the TM domain, where molecular volume, but not polarity, significantly affected ethanol sensitivity (9). Taken together, these findings indicate that the physical-chemical parameters of residues at positions in the extracellular and TM domains that modulate ethanol effects and/or initiate ethanol action in GlyRs are not uniform. Thus, knowledge regarding the physical-chemical properties that control agonist and ethanol sensitivity is key for understanding the relationship between the structure and the actions of ethanol in LGICs (19, 31, 34–40).GlyRs and GABA_ARs, which differ significantly in their sensitivities to ethanol, offer a potential method for identifying the structures that control ethanol sensitivity. For example, α1GlyRs do not reliably respond to ethanol concentrations less than 10 mm (32, 33, 41). Similarly, γ subunit-containing GABA_ARs (e.g. α1β2γ2), the most predominantly expressed GABA_ARs in the central nervous system, are insensitive to ethanol concentrations less than 50 mm (42, 43). In contrast, δ subunit-containing GABA_ARs (e.g. α4β3δ) have been shown to be sensitive to ethanol concentrations as low as 1–3 mm (44–51). Sequence alignment of α1GlyR, γGABA_AR, and δGABA_AR revealed differences between the Loop 2 regions of these receptor subunits. Since prior studies found that mutations of Loop 2 residues can affect ethanol sensitivity (19, 20, 39), the non-conserved residues in Loop 2 of GlyR and GABA_AR subunits could provide the physical-chemical and structural bases underlying the differences in ethanol sensitivity between these receptors.The present study tested the hypothesis that the structure of Loop 2 can markedly affect the ethanol sensitivity of GlyRs and GABA_ARs. To accomplish this, we performed multiple mutations that replaced the Loop 2 region of the α1 subunit in α1GlyRs and the Loop 2 region of the γ subunit of α1β2γ2 GABA_ARs with corresponding non-conserved residues from the δ subunit of GABA_AR and tested the sensitivity of these receptors to ethanol. As predicted, replacing Loop 2 of WT α1GlyRs with the homologous residues from the δGABA_AR subunit (δL2), but not the γGABA_AR subunit (γL2), markedly increased the sensitivity of the receptor to ethanol. Similarly, replacing the non-conserved residues of the γ subunit of α1β2γ2 GABA_ARs with δL2 also markedly increased ethanol sensitivity of GABA_ARs. These findings support the hypothesis and suggest that Loop 2 may play a role in controlling ethanol sensitivity across the Cys-loop superfamily of receptors. The findings also provide the basis for suggesting structure-function relationships in a new molecular model of the GlyR based on the bacterial Gloeobacter violaceus pentameric LGIC homologue (GLIC).     

Neurosteroids Allosterically Modulate Binding of the Anesthetic Etomidate to ��-Aminobutyric Acid Type A Receptors

Photoaffinity labeling of γ-aminobutyric acid type A (GABA_A)-receptors (GABA_AR) with an etomidate analog and mutational analyses of direct activation of GABA_AR by neurosteroids have each led to the proposal that these structurally distinct general anesthetics bind to sites in GABA_ARs in the transmembrane domain at the interface between the β and α subunits. We tested whether the two ligand binding sites might overlap by examining whether neuroactive steroids inhibited etomidate analog photolabeling. We previously identified (Li, G. D., Chiara, D. C., Sawyer, G. W., Husain, S. S., Olsen, R. W., and Cohen, J. B. (2006) J. Neurosci. 26, 11599–11605) azietomidate photolabeling of GABA_AR α1Met-236 and βMet-286 (in αM1 and βM3). Positioning these two photolabeled amino acids in a single type of binding site at the interface of β and α subunits (two copies per pentamer) is consistent with a GABA_AR homology model based upon the structure of the nicotinic acetylcholine receptor and with recent αM1 to βM3 cross-linking data. Biologically active neurosteroids enhance rather than inhibit azietomidate photolabeling, as assayed at the level of GABA_AR subunits on analytical SDS-PAGE, and protein microsequencing establishes that the GABA_AR-modulating neurosteroids do not inhibit photolabeling of GABA_AR α1Met-236 or βMet-286 but enhance labeling of α1Met-236. Thus modulatory steroids do not bind at the same site as etomidate, and neither of the amino acids identified as neurosteroid activation determinants (Hosie, A. M., Wilkins, M. E., da Silva, H. M., and Smart, T. G. (2006) Nature 444, 486–489) are located at the subunit interface defined by our etomidate site model.GABA_A³ receptors (GABA_AR) are major mediators of brain inhibitory neurotransmission and participate in most circuits and behavioral pathways relevant to normal and pathological function (1). GABA_AR are subject to modulation by endogenous neurosteroids, as well as myriad clinically important central nervous system drugs including general anesthetics, benzodiazepines, and possibly ethanol (1, 2). The mechanism of GABA_AR modulation by these different classes of drugs is of major interest, including identification of the receptor amino acid residues involved in binding and action of the drugs.In the absence of high resolution crystal structures of drug-receptor complexes, the locations of anesthetic binding sites in GABA_ARs have been predicted based upon analyses of functional properties of point mutant receptors, which identified residues in the α and β subunit M1–M4 transmembrane helices important for modulation by volatile anesthetics (primarily α subunit) and by intravenous agents, including etomidate and propofol (β subunit) (3–5). Position βM2–15, numbered relative to the N terminus of the helix, functions as a major determinant of etomidate and propofol potency as GABA modulators in vitro and in vivo (6–8). By contrast, this residue is not implicated for modulation by the neurosteroids, potent endogenous modulators of GABA_AR (9).Photoaffinity labeling, which allows the identification of residues in proximity to drug binding sites (10, 11), has been used to identify two GABA_AR amino acids covalently modified by the etomidate analog [³H]azietomidate (12): α1Met-236 within αM1 and βMet-286 within βM3. Photolabeling of these residues was inhibited equally by nonradioactive etomidate and enhanced proportionately by GABA present in the assay, consistent with the presence of these two residues in a common drug binding pocket that would be located at the interface between the β and α subunits in the transmembrane domain (12). Mutational analyses identify these positions as etomidate and propofol sensitivity determinants (13–15).A recent mutagenesis study (16) identified two other residues in GABA_AR αM1 and βM3 as critical for direct activation by neurosteroids, αThr-236 (rat numbering, corresponding to α1Thr-237, bovine numbering used here and by Li et al. (12))⁴ and βTyr-284. These residues were also proposed to contribute to a neurosteroid binding pocket in the transmembrane domain at the interface between β and α subunits, based upon their location in an alternative GABA_AR structural model that positioned those amino acids, and not α1Met-236 or βMet-286, at the subunit interface. For GABA_ARs and other members of the Cys-loop superfamily of neurotransmitter-gated ion channels, the transmembrane domain of each subunit is made up of a loose bundle of four α helices (M1–M4), with M2 from each subunit contributing to the lumen of the ion channel and M4 positioned peripherally in greatest contact with lipid, as seen in the structures of the Torpedo nicotinic acetylcholine receptor (nAChR) (17) and in distantly related prokaryote homologs (18). However, uncertainties in the alignment of GABA_AR subunit sequences relative to those of the nAChR result in alternative GABA_AR homology models (12, 19, 20) that differ in the location of amino acids in the M3 and M4 membrane-spanning helices and in the M1 helix in some models (16, 21).If etomidate and neurosteroids both bind at the same β/α interface in the GABA_AR transmembrane domain, the limited space available for ligand binding suggests that their binding pockets might overlap and that ligand binding would be mutually exclusive. To address this question, we photolabeled purified bovine brain GABA_AR with [³H]azietomidate in the presence of different neuroactive steroids and determined by protein microsequencing whether active neurosteroids inhibited labeling of α1Met-236 and βMet-286, as expected for mutually exclusive binding, or resulted in [³H]azietomidate photolabeling of other amino acids, a possible consequence of allosteric interactions. Active steroids failed to inhibit labeling and enhanced labeling of α1Met-236, clearly indicating that the neurosteroid and the etomidate sites are distinct. Our GABA_AR homology model that positions α1Met-236 and βMet-286 at the β/α interface, but not that of Hosie et al. (16), is also consistent with cysteine substitution cross-linking studies (20, 22), which define the proximity relations between amino acids in the αM1, αM2, αM3, and βM3 helices, and these results support the interpretation that the two residues photolabeled by [³H]azietomidate are part of a single subunit interface binding pocket, whereas the steroid sensitivity determinants identified by mutagenesis neither are at the β/α subunit interface nor are contributors to a common binding pocket.     

Protein Kinase C�� Mediates Regulation of Proliferation by the Serotonin 5-Hydroxytryptamine Receptor 2B

Activation of the 5-hydroxytryptamine receptor 2B (5-HT_2B), a G_q/11 protein-coupled receptor, results in proliferation of various cell types. The 5-HT_2B receptor is also expressed on the pacemaker cells of the gastrointestinal tract, the interstitial cells of Cajal (ICC), where activation triggers ICC proliferation. The goal of this study was to characterize the mitogenic signal transduction cascade activated by the 5-HT_2B receptor. All of the experiments were performed on mouse small intestine primary cell cultures. Activation of the 5-HT_2B receptor by its agonist BW723C86 induced proliferation of ICC. Inhibition of phosphatidylinositol 3-kinase by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 decreased base-line proliferation but had no effect on 5-HT_2B receptor-mediated proliferation. Proliferation of ICC through the 5-HT_2B receptor was inhibited by the phospholipase C inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 and by the inositol 1,4,5-trisphosphate receptor inhibitor Xestospongin C. Calphostin C, the α, β, γ, and μ protein kinase C (PKC) inhibitor Gö6976, and the α, β, γ, δ, and ζ PKC inhibitor Gö6983 inhibited 5-HT_2B receptor-mediated proliferation, indicating the involvement of PKC α, β, or γ. Of all the PKC isoforms blocked by Gö6976, PKCγ and μ mRNAs were found by single-cell PCR to be expressed in ICC. 5-HT_2B receptor activation in primary cell cultures obtained from PKCγ^−/− mice did not result in a proliferative response, further indicating the requirement for PKCγ in the proliferative response to 5-HT_2B receptor activation. The data demonstrate that the 5-HT_2B receptor-induced proliferative response of ICC is through phospholipase C, [Ca²⁺]_i, and PKCγ, implicating this PKC isoform in the regulation of cellular proliferation.Tight control of cell proliferation is essential to maintain organ size and function. Proliferation needs to be tightly regulated to maintain a critical mass of a particular cell type while preventing dysplasia or malignancy. Cell proliferation is regulated by a complex interaction between extrinsic and intrinsic factors. Extrinsic factors usually signal through cell surface receptors such as various growth factor receptors. 5-Hydroxytryptamine (5-HT,² serotonin) is well established as a neurotransmitter and a paracrine factor with over 90% of 5-HT produced by the gastrointestinal tract (1, 2). There is now substantial evidence that, together with these established functions, 5-HT is involved in the control of cell proliferation through various 5-HT receptors, in particular the 5-hydroxytryptamine receptor 2B (5-HT_2B (3–9)). The 5-HT_2B receptor is G_q/11 protein-coupled. Activation of the 5-HT_2B receptor regulates cardiac function, smooth muscle contractility, vascular physiology, and mood control. Recently it was demonstrated that activation of the 5-HT_2B receptor also induces proliferation of neurons, retinal cells (3, 4), hepatocytes (5), osteoblasts (8), and interstitial cells of Cajal (ICC) (9). ICC express the 5-HT_2B receptor, and activation by 5-HT induces proliferation of ICC (9). ICC are specialized, mesoderm-derived mesenchymal cells in the gastrointestinal tract. Their best known function is the generation of slow waves (10), but they also conduct and amplify neuronal signals (11, 12), release carbon monoxide to set the intestinal smooth muscle membrane potential gradient (13), and act as mechanosensors (14, 15). Loss of ICC has been associated with pathological conditions such as gastroparesis (16–18), infantile pyloric stenosis (19, 20), pseudo-obstruction (21, 22), and slow transit constipation (23), whereas increased proliferation of ICC or their precursors is associated with gastrointestinal stromal tumors (24).The mechanisms by which activation of the 5-HT_2B receptor results in increased proliferation are not well understood. In cultured cardiomyocytes, stimulation of the 5-HT_2B receptor activated both phosphatidylinositol 3-kinase (PI3′-K)/Akt and ERK1/2/mitogen-activated protein kinase (MAPK) signaling pathways to protect cardiomyocytes from apoptosis (25). On the other hand, the 5-HT2 subfamily of receptors are also known to couple to phospholipase C (PLC) (26–28).The objective of this study was to utilize the known expression of the 5-HT_2B receptor on ICC to determine whether proliferation through the 5-HT_2B receptor required PI3′-K or PLC. This study demonstrates that proliferation mediated by the 5-HT_2B receptor requires PLC, intracellular calcium release, and the ERK/MAPK signaling pathway and identifies the PKC isoform activated by the 5-HT_2B receptor in ICC as PKCγ.     

Dendritic Assembly of Heteromeric ��-Aminobutyric Acid Type B Receptor Subunits in Hippocampal Neurons

Understanding the mechanisms that control synaptic efficacy through the availability of neurotransmitter receptors depends on uncovering their specific intracellular trafficking routes. γ-Aminobutyric acid type B (GABA_B) receptors (GABA_BRs) are obligatory heteromers present at dendritic excitatory and inhibitory postsynaptic sites. It is unknown whether synthesis and assembly of GABA_BRs occur in the somatic endoplasmic reticulum (ER) followed by vesicular transport to dendrites or whether somatic synthesis is followed by independent transport of the subunits for assembly and ER export throughout the somatodendritic compartment. To discriminate between these possibilities we studied the association of GABA_BR subunits in dendrites of hippocampal neurons combining live fluorescence microscopy, biochemistry, quantitative colocalization, and bimolecular fluorescent complementation. We demonstrate that GABA_BR subunits are segregated and differentially mobile in dendritic intracellular compartments and that a high proportion of non-associated intracellular subunits exist in the brain. Assembled heteromers are preferentially located at the plasma membrane, but blockade of ER exit results in their intracellular accumulation in the cell body and dendrites. We propose that GABA_BR subunits assemble in the ER and are exported from the ER throughout the neuron prior to insertion at the plasma membrane. Our results are consistent with a bulk flow of segregated subunits through the ER and rule out a post-Golgi vesicular transport of preassembled GABA_BRs.The efficacy of synaptic transmission depends on the intracellular trafficking of neurotransmitter receptors (1, 2). The trafficking of glutamatergic and GABA_A⁶ receptors has been extensively studied, and their implications for synaptic plasticity have been well documented (3, 4). For example, differential trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors modifies synaptic strength and influences experience-dependent plasticity in vivo (5). The molecular mechanisms that govern the trafficking of metabotropic GABA_BRs and their consequences for synaptic inhibition remain less clear. In particular, limited information is available regarding the relationship between the trafficking of GABA_BRs and the topological complexity of the secretory pathway in neurons.GABA_BRs mediate the slow component of synaptic inhibition by acting on pre- and postsynaptic targets (6–8). They are implicated in epilepsy, anxiety, stress, sleep disorders, nociception, depression, and cognition (9). They also represent attractive targets for the treatment of withdrawal symptoms from drugs of addiction such as cocaine (10). They are obligatory heteromers composed of GABA_BR1 and GABA_BR2 subunits. GABA_BR1 contains an RXR-type sequence in the intracellular C-terminal domain that functions as an ER retention motif (11, 12). The ER retention sequence is masked upon assembly with GABA_BR2 resulting in the appearance of functional receptors at the plasma membrane. Only GABA_BR1 binds GABA with high affinity, whereas G protein signaling is exclusively mediated by the second and third intracellular loops of GABA_BR2 (13–15). GABA_BRs are located in dendrites and axons, but their distribution does not coincide with the active zone or the postsynaptic density. Rather, they are adjacent to both compartments constituting perisynaptic receptors (16, 17).If GABA_BR subunits are synthesized in the soma, at least two possibilities exist for their anterograde transport, assembly, and insertion in dendrites. First, the subunits may be synthesized in the cell body, assembled in the somatic ER, and targeted preassembled in post-Golgi vesicles to their site of insertion in dendrites. Alternatively, they may be synthesized in the soma and transported through the ER membrane as non-heteromeric subunits. In the latter scenario, newly assembled receptors may exit the ER throughout the somatodendritic compartment prior to insertion at the plasma membrane and diffuse laterally for retention at functional sites. No evidence exists to discriminate between these possibilities. We reasoned that a prevalence of associated subunits in post-Golgi vesicles in dendrites would favor the first alternative, whereas the existence of non-associated subunits in intracellular compartments would support a somatodendritic assembly mechanism. Here we explore the presence of associated GABA_BR subunits using fluorescence recovery after photobleaching (FRAP), biochemistry, and quantitative colocalization. In addition, we report for the first time the use of BiFC (18) to study GABA_BR assembly in neurons. Our results demonstrate that GABA_BR subunits are differentially mobile in dendrites and that a high proportion of non-associated subunits prevail in an intracellular fraction of the adult brain. They also show that GABA_BR subunits are heteromeric at the plasma membrane but segregated in intracellular compartments of dendrites of hippocampal neurons. Importantly, treatment with brefeldin A (BFA) or interference of the coatomer protein complex II impair ER export and result in the accumulation of assembled subunits in intracellular compartments throughout the somatodendritic arbor. We conclude that GABA_BR subunits are synthesized in the soma and remain segregated in intracellular compartments prior to somatodendritic assembly. Our observations rule out a post-Golgi vesicular transport of preassembled GABA_BRs and suggest an alternative mechanism of receptor targeting.     

Presynaptic Targeting of ��4��2 Nicotinic Acetylcholine Receptors Is Regulated by Neurexin-1��

The mechanisms involved in the targeting of neuronal nicotinic acetylcholine receptors (AChRs), critical for their functional organization at neuronal synapses, are not well understood. We have identified a novel functional association between α4β2 AChRs and the presynaptic cell adhesion molecule, neurexin-1β. In non-neuronal tsA 201 cells, recombinant neurexin-1β and mature α4β2 AChRs form complexes. α4β2 AChRs and neurexin-1β also coimmunoprecipitate from rat brain lysates. When exogenous α4β2 AChRs and neurexin-1β are coexpressed in hippocampal neurons, they are robustly targeted to hemi-synapses formed between these neurons and cocultured tsA 201 cells expressing neuroligin-1, a postsynaptic binding partner of neurexin-1β. The extent of synaptic targeting is significantly reduced in similar experiments using a mutant neurexin-1β lacking the extracellular domain. Additionally, when α4β2 AChRs, α7 AChRs, and neurexin-1β are coexpressed in the same neuron, only the α4β2 AChR colocalizes with neurexin-1β at presynaptic terminals. Collectively, these data suggest that neurexin-1β targets α4β2 AChRs to presynaptic terminals, which mature by trans-synaptic interactions between neurexins and neuroligins. Interestingly, human neurexin-1 gene dysfunctions have been implicated in nicotine dependence and in autism spectrum disorders. Our results provide novel insights as to possible mechanisms by which dysfunctional neurexins, through downstream effects on α4β2 AChRs, may contribute to the etiology of these neurological disorders.The clustering of ion channels or receptors and precise targeting to pre- and postsynaptic specializations in neurons is critical to efficiently regulate synaptic transmission. Within the central nervous system, neuronal nicotinic acetylcholine receptors (AChRs)⁵ regulate the release of neurotransmitters at presynaptic sites (1) and mediate fast synaptic transmission at postsynaptic sites of neurons (2). These receptors are part of a family of acetylcholine-gated ion channels that are assembled from various combinations of α2–α10 and β2–β4 subunits (3). AChRs participate in the regulation of locomotion, affect, reward, analgesia, anxiety, learning, and attention (4, 5).The α4β2 subtype is the most abundant AChR receptor expressed in the brain. Multiple lines of evidence support a major role for α4β2 AChRs in nicotine addiction. α4β2 AChRs show high affinity for nicotine (6) and are located on the dopaminergic projections of ventral tegmental area neurons to the medium spiny neurons of the nucleus accumbens (7, 8). Furthermore, β2 AChR subunit knock-out mice lose their sensitivity to nicotine in passive avoidance tasks (9) and show attenuated self-administration of nicotine (10). α4 AChR subunit knock-out mice also exhibit a loss of tonic control of striatal basal dopamine release (11). Finally, experiments with knock-in mice expressing α4β2 AChRs hypersensitive to nicotine demonstrate that α4β2 AChRs indeed mediate the essential features of nicotine addiction including reward, tolerance, and sensitization (12). High resolution ultrastructural studies show that α4 subunit-containing AChRs are clustered at dopaminergic axonal terminals (13), and a sequence motif has been identified within the α4 AChR subunit cytoplasmic domain that is essential for receptor trafficking to axons (14). However, the mechanisms underlying the targeting and clustering of α4β2 AChRs to presynaptic sites in neurons remain elusive.Recently, bi-directional interactions between neurexins and neuroligins have been shown to promote synapse assembly and maturation by fostering pre- and postsynaptic differentiation (reviewed in Refs. 15–17). The neurexins are encoded by three genes corresponding to neurexins I–III (18, 19), each encoding longer α-neurexins and shorter β-neurexins, because of differential promoter use. Neurexins recruit N- and P/Q-type calcium channels via scaffolding proteins, including calmodulin-associated serine/threonine kinase (20), to active zones of presynaptic terminals (21, 22). Recently, α-neurexins were shown to specifically induce GABAergic postsynaptic differentiation (23). Neuroligins, postsynaptic binding partners of neurexins, cluster N-methyl-d-aspartate receptors and GABA_A receptors by recruiting the scaffolding proteins PSD-95 (post-synaptic density 95) and gephyrin, respectively (24, 25). Interestingly, neurexins and neuroligins also modulate the postsynaptic clustering of α3-containing AChRs in chick ciliary ganglia (26, 27). In this study, using multiple experimental strategies, we provide evidence for the formation of complexes between neurexin-1β and α4β2 AChRs and a role for neurexin in the targeting of α4β2 AChRs to presynaptic terminals of neurons.     

Differential Effect of Membrane Cholesterol Removal on ��- and ��-Opioid Receptors: A PARALLEL COMPARISON OF ACUTE AND CHRONIC SIGNALING TO ADENYLYL CYCLASE*

According to the lipid raft theory, the plasma membrane contains small domains enriched in cholesterol and sphingolipid, which may serve as platforms to organize membrane proteins. Using methyl-β-cyclodextrin (MβCD) to deplete membrane cholesterol, many G protein-coupled receptors have been shown to depend on putative lipid rafts for proper signaling. Here we examine the hypothesis that treatment of HEK293 cells stably expressing FLAG-tagged μ-opioid receptors (HEK FLAG-μ) or δ-opioid receptors (HEK FLAG-δ) with MβCD will reduce opioid receptor signaling to adenylyl cyclase. The ability of the μ-opioid agonist [d-Ala²,N-Me-Phe⁴,Gly⁵-ol]enkephalin to acutely inhibit adenylyl cyclase or to cause sensitization of adenylyl cyclase following chronic treatment was attenuated with MβCD. These effects were due to removal of cholesterol, because replenishment of cholesterol restored [d-Ala²,N-Me-Phe⁴,Gly⁵-ol]enkephalin responses back to control values, and were confirmed in SH-SY5Y cells endogenously expressing μ-opioid receptors. The effects of MβCD may be due to uncoupling of the μ receptor from G proteins but were not because of decreases in receptor number and were not mimicked by cytoskeleton disruption. In contrast to the results in HEK FLAG-μ cells, MβCD treatment of HEK FLAG-δ cells had no effect on acute inhibition or sensitization of adenylyl cyclase by δ-opioid agonists. The differential responses of μ- and δ-opioid agonists to cholesterol depletion suggest that μ-opioid receptors are more dependent on cholesterol for efficient signaling than δ receptors and can be partly explained by localization of μ- but not δ-opioid receptors in cholesterol- and caveolin-enriched membrane domains.Membrane cholesterol can alter the function of integral proteins, such as G protein-coupled receptors, through cholesterol-protein interactions and by changes in membrane viscosity (1). In addition, cholesterol interacts with other lipids found in the bilayer, particularly sphingolipids (2), which allows for tight and organized packing that can precipitate the formation of specialized domains within the plasma membrane (3). These domains have become an area of intense research interest and have been termed lipid or membrane rafts (4). The study of membrane rafts in intact cells is controversial, due in part to the limitations of the current methods used to study rafts (5, 6). Regardless, the membrane environment formed in regions of high cholesterol and sphingolipids may be such that certain proteins have an affinity for these regions, especially proteins with a propensity to interact with cholesterol.Many G protein-coupled receptors and signaling proteins have been found to prefer cholesterol-enriched domains leading to the hypothesis that these domains can organize signaling molecules in the membrane to enhance or inhibit specific signaling events (7). This includes μ- (8, 9), δ- (10, 11), and κ-opioid receptors (12). In addition, Gα_i (12–17), Gα_o (16), and adenylyl cyclase isoforms 3 (18), 5/6 (9, 18, 19), and 8 (20) have been found to associate with cholesterol and/or the cholesterol-binding protein caveolin. Activated opioid receptors couple to Gα_i/o proteins and acutely inhibit the activity of adenylyl cyclase. Longer term exposure to opioid agonists causes sensitization of adenylyl cyclase and a rebound overshoot of cAMP production upon withdrawal of the agonist (21). Consequently, we sought to assess the role of cholesterol depletion on the ability of μ- and δ-opioid receptor agonists to inhibit and cause sensitization of adenylyl cyclase.There are conflicting data for the effect of changes in membrane cholesterol on opioid signaling. For example, an increase in plasma membrane microviscosity by addition of cholesteryl hemisuccinate to SH-SY5Y cell membranes increased μ-opioid receptor coupling to G proteins (22). Conversely, removal of membrane cholesterol from Chinese hamster ovary cells has been shown to either decrease (23) or increase (24) the coupling of μ-opioid receptors to G proteins, as measured by [³⁵S]GTPγS³ binding stimulated by the μ-opioid agonist DAMGO. Furthermore, the effect of cholesterol removal on δ-opioid agonist-stimulated [³⁵S]GTPγS binding varies by cell type (10, 25). In these previous studies, the variety of cell types utilized and the conflicting results make comparisons between opioid receptor types difficult. The objective of this study was to directly compare the role of membrane cholesterol in modulating acute and chronic μ- and δ-opioid signaling in the same cell systems using identical methods, including the following: 1) depletion of cholesterol by the cholesterol-sequestering agent methyl-β-cyclodextrin (MβCD); 2) separation of cholesterol-enriched membranes by sucrose gradient ultracentrifugation; and 3) clustering of lipid raft patches in whole cells with cholera toxin B subunit.In initial experiments using human embryonic kidney (HEK) cells heterologously expressing μ- or δ-opioid receptors, we found that δ-opioid receptors were located in caveolin-poor fractions following 1% Triton X-100 homogenization and sucrose gradient ultracentrifugation. This differs from studies using a detergent-free method to identify lipid raft fractions (10, 11). In contrast, we found that the μ-opioid receptor was found in both caveolin-poor and caveolin-rich fractions, in accordance with previous literature (8, 9). This differential localization of opioid receptors led us to test the hypothesis that, in contrast to the μ-opioid receptor, the δ-opioid receptor would not be dependent on cholesterol for signaling. The results show that μ- but not δ-opioid receptors have a dependence on cholesterol for signaling to adenylyl cyclase and that this effect is much more pronounced following chronic exposure to opioids.     

Anaesthetic impairment of immune function is mediated via GABA(A) receptors

Background

GABA_A receptors are members of the Cys-loop family of neurotransmitter receptors, proteins which are responsible for fast synaptic transmission, and are the site of action of wide range of drugs [1]. Recent work has shown that Cys-loop receptors are present on immune cells, but their physiological roles and the effects of drugs that modify their function in the innate immune system are currently unclear [2]. We are interested in how and why anaesthetics increase infections in intensive care patients; a serious problem as more than 50% of patients with severe sepsis will die [3]–[6]. As many anaesthetics act via GABA_A receptors [7], the aim of this study was to determine if these receptors are present on immune cells, and could play a role in immunocompromising patients.

Principal Findings

We demonstrate, using RT-PCR, that monocytes express GABA_A receptors constructed of α1, α4, β2, γ1 and/or δ subunits. Whole cell patch clamp electrophysiological studies show that GABA can activate these receptors, resulting in the opening of a chloride-selective channel; activation is inhibited by the GABA_A receptor antagonists bicuculline and picrotoxin, but not enhanced by the positive modulator diazepam. The anaesthetic drugs propofol and thiopental, which can act via GABA_A receptors, impaired monocyte function in classic immunological chemotaxis and phagocytosis assays, an effect reversed by bicuculline and picrotoxin.

Significance

Our results show that functional GABA_A receptors are present on monocytes with properties similar to CNS GABA_A receptors. The functional data provide a possible explanation as to why chronic propofol and thiopental administration can increase the risk of infection in critically ill patients: their action on GABA_A receptors inhibits normal monocyte behaviour. The data also suggest a potential solution: monocyte GABA_A receptors are insensitive to diazepam, thus the use of benzodiazepines as an alternative anesthetising agent may be advantageous where infection is a life threatening problem.     

Crystal Structure of the LG1-3 Region of the Laminin ��2 Chain

Laminins are large heterotrimeric glycoproteins with many essential functions in basement membrane assembly and function. Cell adhesion to laminins is mediated by a tandem of five laminin G-like (LG) domains at the C terminus of the α chain. Integrin binding requires an intact LG1-3 region, as well as contributions from the coiled coil formed by the α, β, and γ chains. We have determined the crystal structure at 2.8-Å resolution of the LG1-3 region of the laminin α2 chain (α2LG1-3). The three LG domains adopt typical β-sandwich folds, with canonical calcium binding sites in LG1 and LG2. LG2 and LG3 interact through a substantial interface, but LG1 is completely dissociated from the LG2-3 pair. We suggest that the missing γ chain tail may be required to stabilize the interaction between LG1 and LG2-3 in the biologically active conformation. A global analysis of N-linked glycosylation sites shows that the β-sandwich faces of LG1 are free of carbohydrate modifications in all five laminin α chains, suggesting that these surfaces may harbor the integrin binding site. The α2LG1-3 structure provides the first atomic view of the integrin binding region of laminins.The laminins constitute a major class of cell-adhesive glycoproteins that are intimately involved in basement membrane assembly and function. Their essential roles in embryo development and tissue function have been demonstrated by numerous genetic studies and the analysis of severe human diseases resulting from mutations in laminin genes (1–4). All laminins are heterotrimers composed of three different gene products, termed α, β, and γ chains. At present, 16 mouse and human laminins are known, assembled from five α, three β, and three γ chains. The different laminins have characteristic expression patterns and functions in the embryo and adult animal (1). Laminins are cross-shaped molecules: the three short arms are composed of one chain each, while the long arm is a coiled coil of all three chains, terminating in a tandem of five laminin G-like (LG)² domains, LG1-5, contributed by the α chain (2). Basement membrane assembly requires polymerization via the short arms and cell attachment via the LG1-5 region (5, 6).Cell adhesion to laminins is mediated by multiple receptors: integrins bind to the LG1-3 region, whereas α-dystroglycan, heparan sulfate proteoglycans, and sulfated glycolipids bind predominantly to sites in the LG4-5 pair (7). Integrins are heterodimers with a large extracellular domain consisting of one α and one β chain, which both span the cell membrane and engage in transmembrane signaling (8). Of the 24 mouse and human integrins, the major laminin binding integrins are α3β1, α6β1, α7β1, and α6β4, which have distinct affinities for the different laminin isoforms (9). Although some studies have reported integrin binding or integrin-mediated cell adhesion to isolated LG domains or tandems (10–12), there is strong evidence to suggest that the coiled coil region and an intact γ chain tail are required for full integrin binding to the laminin LG1-3 region (13–18). Compared with integrin binding to collagen and fibronectin, which is understood in atomic detail (19, 20), the laminin-integrin interaction remains poorly characterized in structural terms. We previously determined crystal structures of the LG4-5 region of the laminin α1 and α2 chains and defined their receptor binding sites (21–23). Here, we report the crystal structure of the remainder of the laminin α2 receptor binding region, LG1-3.     

Evidence for a Second, High Affinity G�¦� Binding Site on G��i1(GDP) Subunits

It is well known that Gα_i1(GDP) binds strongly to Gβγ subunits to form the Gα_i1(GDP)-Gβγ heterotrimer, and that activation to Gα_i1(GTP) results in conformational changes that reduces its affinity for Gβγ subunits. Previous studies of G protein subunit interactions have used stoichiometric amounts of the proteins. Here, we have found that Gα_i1(GDP) can bind a second Gβγ subunit with an affinity only 10-fold weaker than the primary site and close to the affinity between activated Gα_i1 and Gβγ subunits. Also, we find that phospholipase Cβ2, an effector of Gβγ, does not compete with the second binding site implying that effectors can be bound to the Gα_i1(GDP)-(Gβγ)₂ complex. Biophysical measurements and molecular docking studies suggest that this second site is distant from the primary one. A synthetic peptide having a sequence identical to the putative second binding site on Gα_i1 competes with binding of the second Gβγ subunit. Injection of this peptide into cultured cells expressing eYFP-Gα_i1(GDP) and eCFP-Gβγ reduces the overall association of the subunits suggesting this site is operative in cells. We propose that this second binding site serves to promote and stabilize G protein subunit interactions in the presence of competing cellular proteins.The plasma membranes of cells are organized as a series of protein-rich and lipid-rich domains (1–3). Many of the protein-rich domains, in particular those organized by caveolin proteins, are thought to be complexes of functionally related proteins that transduce extracellular signals (2). There is increasing evidence that heterotrimeric G proteins exist in pre-formed membrane complexes with their receptors and their intracellular effectors (4–8).The G protein signaling system is initiated when an extracellular agonist binds to its specific G protein-coupled receptor (for review see Refs. 9–12). The ligand-bound receptor will then catalyze the exchange of GTP for GDP on the Gα subunit in the G protein heterotrimer. In the basal state, Gα(GDP) binds strongly to Gβγ, but in the GTP-bound state this affinity is reduced, allowing Gα(GTP) and Gβγ subunits to individually bind to a host of specific intracellular enzymes and change their catalytic activity.Although the interactions between G protein subunits have been studied extensively in vitro, their behavior in cells may differ. For example, in pure or semi-pure systems, activation of Gα(GDP) sufficiently weakens its affinity for Gβγ resulting in dissociation (13). However, in cells separation of the heterotrimer is observed under some circumstances, but not others (7, 14–17). The reason for these differences in behavior is not clear. There are four families of Gα subunits that each contain several members, and, additionally, there are many subtypes of Gβγ subunits (18). It is possible that differences in dissociation behavior reflect differences in affinity between G protein subunit subtypes (19), the presence of various protein partners, and/or differences in post-synthetic modifications of the subunits (20).The mechanism that allows activated G proteins to remain bound is not apparent from the crystal structure (21, 22). If G protein subunits do not dissociate in cells, then their interaction must change in such a manner as to expose the effector interaction site(s). We have found that phospholipase Cβ1 (PLCβ1),⁴ an important effector of Gα_q (23), is bound to Gα_q prior to activation and throughout the activation cycle (6) implying that Gα_q(GDP) interacts with PLCβ1 in a non-functional manner.We have evidence that signaling complexes are stabilized by a series of secondary interactions. Using purified proteins and model membranes, we have found that membranes of the Gα_q-Gβγ/PLCβ1/RGS4 signaling system have secondary, weaker binding sites to members of this signaling system in addition to their high affinity site(s) to their functional partner(s). We speculate that secondary contacts allow for self-scaffolding of signaling proteins. To understand the nature of these secondary contacts, we have studied the ability of the Gα_i1(GDP)-Gβγ heterotrimer to remain complexed through the activation cycle (24). Here, we present evidence that Gα_i1(GDP) has two distinct Gβγ binding sites that only differ in affinity by an order of magnitude and may allow for continued association between the subunits upon activation. We also find that this site plays an important role in stabilizing G protein associations in cells and provides a mechanism of self-scaffolding.     

The Caenorhabditis elegans A��1�C42 Model of Alzheimer Disease Predominantly Expresses A��3�C42

Transgenic expression of human amyloid β (Aβ) peptide in body wall muscle cells of Caenorhabditis elegans has been used to better understand aspects of Alzheimer disease (AD). In human aging and AD, Aβ undergoes post-translational changes including covalent modifications, truncations, and oligomerization. Amino truncated Aβ is increasingly recognized as potentially contributing to AD pathogenesis. Here we describe surface-enhanced laser desorption ionization-time of flight mass spectrometry mass spectrometry of Aβ peptide in established transgenic C. elegans lines. Surprisingly, the Aβ being expressed is not full-length 1–42 (amino acids) as expected but rather a 3–42 truncation product. In vitro analysis demonstrates that Aβ_3–42 self-aggregates like Aβ_1–42, but more rapidly, and forms fibrillar structures. Similarly, Aβ_3–42 is also the more potent initiator of Aβ_1–40 aggregation. Seeded aggregation via Aβ_3–42 is further enhanced via co-incubation with the transition metal Cu(II). Although unexpected, the C. elegans model of Aβ expression can now be co-opted to study the proteotoxic effects and processing of Aβ_3–42.Numerous studies support a role for aggregating Aβ³ in mediating the toxicity that underlies AD (1, 2). However, several key questions remain central to understanding how AD and Aβ pathology are related. What is the connection between Aβ aggregation and toxicity? Is there a specific toxic Aβ conformation or species? How and why does aging impact on Aβ precipitation? Significant effort to address these questions has been invested in the use of vertebrate and simple invertebrate model organisms to simulate neurodegenerative diseases through transgenic expression of human Aβ (3). From these models, several novel insights into the proteotoxicity of Aβ have been gained (4–7).Human Aβ (e.g. in brain, cerebrospinal fluid, or plasma) is not found as a single species but rather as diverse mixtures of various modified, truncated, and cross-linked forms (8–10). Specific truncations, covalent modifications, and cross-linked oligomers of Aβ have potentially important roles in determining Aβ-associated neurotoxicity. For example, N-terminal truncations of Aβ have increased abundance in AD, rapidly aggregate, and are neurotoxic (9, 11). Furthermore, the N-terminal glutamic acid residue of Aβ_3–42 can be cyclized to pyroglutamate (Aβ_3(pE)-42) (12), which may be particularly important in AD pathogenesis (13, 14). Aβ_3(pE)-42 is a significant fraction of total Aβ in AD brain (15), accounting for more than 50% of Aβ accumulated in plaques (16). Aβ_3(pE)-42 seeds Aβ aggregation (17), confers proteolytic resistance, and is neurotoxic (13). Recently, glutaminyl cyclase (QC) has been proposed to catalyze, in vivo, pyroglutamate formation of Aβ_3(pE)-40/42 (14, 18). Aβ_1–42 itself cannot be cyclized by QC to Aβ_3(pE)-42 (19), unlike Aβ that commences with an N-terminal glutamic acid-residue (e.g. Aβ_3–42 and Aβ_11–42) (20). QC has broad expression in mammalian brain (21, 22), and its inhibition attenuates accumulation of Aβ_3(pE)-42 into plaques and improves cognition in a transgenic mouse model of AD that overexpresses human amyloid precursor protein (14). N-terminal truncations at position 3 have been reported in senile plaques (23, 24); however, the process that generates Aβ_3–42 is unknown. Currently there are no reported animal models of Aβ_3–42 expression.Advances in surface-enhanced laser desorption ionization-time of flight mass spectrometry (SELDI-TOF MS) analysis now facilitate accurate identification of particular Aβ species. Using this technology, we examined well characterized C. elegans transgenic models of AD that develop amyloid aggregates (25, 26) to see whether the human Aβ they express is post-translationally modified.     

Risk of serious adverse cardiovascular events associated with varenicline: a systematic review and meta-analysis

Background:

There have been postmarketing reports of adverse cardiovascular events associated with the use of varenicline, a widely used smoking cessation drug. We conducted a systematic review and meta-analysis of randomized controlled trials to ascertain the serious adverse cardiovascular effects of varenicline compared with placebo among tobacco users.

Methods:

We searched MEDLINE, EMBASE, the Cochrane Database of Systematic Reviews, websites of regulatory authorities and registries of clinical trials, with no date or language restrictions, through September 2010 (updated March 2011) for published and unpublished studies. We selected double-blind randomized controlled trials of at least one week’s duration involving smokers or people who used smokeless tobacco that reported on cardiovascular events (ischemia, arrhythmia, congestive heart failure, sudden death or cardiovascular-related death) as serious adverse events asociated with the use of varenicline.

Results:

We analyzed data from 14 double-blind randomized controlled trials involving 8216 participants. The trials ranged in duration from 7 to 52 weeks. Varenicline was associated with a significantly increased risk of serious adverse cardiovascular events compared with placebo (1.06% [52/4908] in varenicline group v. 0.82% [27/3308] in placebo group; Peto odds ratio [OR] 1.72, 95% confidence interval [CI] 1.09–2.71; I² = 0%). The results of various sensitivity analyses were consistent with those of the main analysis, and a funnel plot showed no publication bias. There were too few deaths to allow meaningful comparisons of mortality.

Interpretation:

Our meta-analysis raises safety concerns about the potential for an increased risk of serious adverse cardiovascular events associated with the use of varenicline among tobacco users.Varenicline is one of the most widely used drugs for smoking cessation. It is a partial agonist at the α₄–β₂ nicotinic acetylcholine receptors and a full agonist at the α₇ nicotinic acetylcholine receptor.^1,2 The drug modulates parasympathetic output from the brainstem to the heart because of activities of the α₇ receptor.³ Acute nicotine administration can induce thrombosis.⁴ Possible mechanisms by which varenicline may be associated with cardiovascular disease might include the action of varenicline at the α₇ receptor in the brainstem or, similar to nicotine, a prothrombotic effect.^2–4At the time of its priority safety review of varenicline in 2006, the US Food and Drug Administration (FDA) noted that “[t]he serious adverse event data suggest that varenicline may possibly increase the risk of cardiac events, both ischemic and arrhythmic, particularly over longer treatment period.”⁵ Subsequently, the product label was updated: “Post marketing reports of myocardial infarction and cerebrovascular accidents including ischemic and hemorrhagic events have been reported in patients taking Chantix.”⁶ There are published reports of cardiac arrest associated with varenicline.⁷Cardiovascular disease is an important cause of morbidity and mortality among tobacco users. The long-term cardiovascular benefits of smoking cessation are well established.⁸ Although one statistically underpowered trial reported a trend toward excess cardiovascular events associated with the use of varenicline,⁹ a systematic review of information on the cardiovascular effects of varenicline is unavailable to clinicians.We conducted a systematic review and meta-analysis of randomized controlled trials (RCTs) to ascertain the serious adverse cardiovascular effects of varenicline compared with placebo among tobacco users.     

Novel Eicosapentaenoic Acid-derived F3-isoprostanes as Biomarkers of Lipid Peroxidation

Isoprostanes (iPs) are prostaglandin (PG) isomers generated by free radical-catalyzed peroxidation of polyunsaturated fatty acids (PUFAs). Urinary F₂-iPs, PGF_2α isomers derived from arachidonic acid (AA) are used as indices of lipid peroxidation in vivo. We now report the characterization of two major F₃-iPs, 5-epi-8,12-iso-iPF_3α-VI and 8,12-iso-iPF_3α-VI, derived from the ω-3 fatty acid, eicosapentaenoic acid (EPA). Although the potential therapeutic benefits of EPA receive much attention, a shift toward a diet rich in ω-3 PUFAs may also predispose to enhanced lipid peroxidation. Urinary 5-epi-8,12-iso-iPF_3α-VI and 8,12-iso-iPF_3α-VI are highly correlated and unaltered by cyclooxygenase inhibition in humans. Fish oil dose-dependently elevates urinary F₃-iPs in mice and a shift in dietary ω-3/ω-6 PUFAs is reflected by an increasing slope [m] of the line relating urinary 8, 12-iso-iPF_3α-VI and 8,12-iso-iPF_2α-VI. Administration of bacterial lipopolysaccharide evokes a reversible increase in both urinary 8,12-iso-iPF_3α-VI and 8,12-iso-iPF_2α-VI in humans on an ad lib diet. However, while excretion of the iPs is highly correlated (R² median = 0.8), [m] varies by an order of magnitude, reflecting marked inter-individual variability in the relative peroxidation of ω-3 versus ω-6 substrates. Clustered analysis of F₂- and F₃-iPs refines assessment of the oxidant stress response to an inflammatory stimulus in vivo by integrating variability in dietary intake of ω-3/ω-6 PUFAs.Isoprostanes (iPs),² a family of prostaglandin isomers, are generated initially in situ by free radical attack on polyunsaturated fatty acids (PUFAs) in cell membranes. There, they can be immunodetected and quantified by mass spectrometry (1). They are then cleaved by phospholipases (2), circulate in plasma, and are excreted in urine (3). F₂-iPs, isomers of PGF_2α (3), derived from peroxidation of arachidonic acid (AA), are the most studied species. F₂-iPs can be quantified in normal animal and human biological fluids and tissues, implying ongoing lipid peroxidation under physiological conditions, despite replete and diversified endogenous antioxidant defense systems (4). The measurement of urinary F₂-isoprostanes has been used to reflect lipid peroxidation noninvasively in several human diseases (5–8). In addition to their utility as markers of oxidant stress (OS), high concentrations of some F₂-iPs also possess biological activity in vitro, including bronchoconstriction (9), vasoconstriction (10), platelet aggregation (11, 12), and adhesion (13). These effects result from iPs acting as incidental ligands at prostaglandin receptors. It is unknown whether this capacity of individual iPs to ligate prostanoid receptors has relevance to the concentration of the multiple endogenous iP species likely to be formed simultaneously under conditions of oxidant stress in vivo.iPs analogous to the F₂-iPs may be formed from other fatty acid substrates (14–19), including the fish oil constituent, eicosapentaenoic acid (EPA) (20). Potentially beneficial effects of EPA consumption have been supported by a variety of epidemiological and interventional studies. EPA competes with AA for access to the cyclooxygenase enzymes (21), reducing production of AA-derived PGs (22, 23). This effect and/or substituted formation of EPA-derived PGs may explain the anti-inflammatory and cardioprotective effects ascribed to fish oils.The relatively unsaturated EPA and docosahexaenoic acid (DHA) in fatty fish are likely to be more susceptible to lipid peroxidation than AA, although it is unknown whether this might constrain beneficial effects derived from a shift in substrate-dependent enzymatic product formation. While F₂-iPs and F₃-iPs are excreted into urine in their original form, the F₄-iPs formed from DHA (Fig. 1) are at least partly metabolized to F₃-iPs before excretion (20, 24). Given the potential utility of noninvasive biomarkers of EPA and DHA peroxidation and our previous work with F₂-iPs (25, 26), we sought to determine whether members of group VI (Fig. 1B), 5-epi-8,12-iso-iPF_3α-VI and 8,12-iso-iPF_3α-VI (Fig. 1C) might be detectable in urine.Open in a separate windowFIGURE 1.F₃-iPs derived from EPA. A, formation and metabolism of isoprostanes. AA is more abundant than EPA and DHA in cell membranes obtained from individuals consuming a Western diet. Isoprostanes are formed from the corresponding PUFA substrate in situ following free radical attack. F₂-iPs and F₃-iPs are excreted into urine in their original form, but F₄-iPs are at least partly metabolized to F₃-iPs before excretion. (F₂-iPs: F₂-isoprostanes; F₃-iPs: F₃-isoprostanes; F₄-iPs: F₄-isoprostanes). B, six types of F₃-iPs. C, 5-epi-8,12-iso-iPF_3α-VI and 8,12-iso-iPF_3α-VI.     

Purification and Functional Reconstitution of Monomeric ��-Opioid Receptors: ALLOSTERIC MODULATION OF AGONIST BINDING BY Gi2*

Despite extensive characterization of the μ-opioid receptor (MOR), the biochemical properties of the isolated receptor remain unclear. In light of recent reports, we proposed that the monomeric form of MOR can activate G proteins and be subject to allosteric regulation. A μ-opioid receptor fused to yellow fluorescent protein (YMOR) was constructed and expressed in insect cells. YMOR binds ligands with high affinity, displays agonist-stimulated [³⁵S]guanosine 5′-(γ-thio)triphosphate binding to Gα_i, and is allosterically regulated by coupled G_i protein heterotrimer both in insect cell membranes and as purified protein reconstituted into a phospholipid bilayer in the form of high density lipoprotein particles. Single-particle imaging of fluorescently labeled receptor indicates that the reconstituted YMOR is monomeric. Moreover, single-molecule imaging of a Cy3-labeled agonist, [Lys⁷, Cys⁸]dermorphin, illustrates a novel method for studying G protein-coupled receptor-ligand binding and suggests that one molecule of agonist binds per monomeric YMOR. Together these data support the notion that oligomerization of the μ-opioid receptor is not required for agonist and antagonist binding and that the monomeric receptor is the minimal functional unit in regard to G protein activation and strong allosteric regulation of agonist binding by G proteins.Opioid receptors are members of the G protein-coupled receptor (GPCR)² superfamily and are clinical mainstays for inducing analgesia. Three isoforms of opioid receptors, μ, δ, and κ, have been cloned and are known to couple to G_i/o proteins to regulate adenylyl cyclase and K⁺/Ca⁺ ion channels (1–3). An ever growing amount of data suggests that many GPCRs oligomerize (4, 5), and several studies have suggested that μ-opioid receptors (MORs) and δ-opioid receptors heterodimerize to form unique ligand binding and G protein-activating units (6–10). Although intriguing, these studies utilize cellular overexpression systems where it is difficult to know the exact nature of protein complexes formed between the receptors.To study the function of isolated GPCRs, our laboratory and others have utilized a novel phospholipid bilayer reconstitution method (11–16). In this approach purified GPCRs are reconstituted into the phospholipid bilayer of a high density lipoprotein (HDL) particle. The reconstituted HDL (rHDL) particles are monodispersed, uniform in size, and preferentially incorporate a GPCR monomer (14, 15). Previous work in our lab has shown that rhodopsin, a class A GPCR previously proposed to function as a dimer (17–19), is fully capable of activating its G protein when reconstituted as a monomer in the rHDL lipid bilayer (15). Moreover, we have demonstrated that agonist binding to a monomeric β₂-adrenergic receptor, another class A GPCR, can be allosterically regulated by G proteins (14). This led us to determine whether a monomer of MOR, a class A GPCR that endogenously binds peptide ligands, is the minimal functional unit required to activate coupled G proteins. We additionally investigated whether agonist binding to monomeric MOR is allosterically regulated by inhibitory G protein heterotrimer.To study the function of monomeric MOR we have purified a modified version of the receptor to near homogeneity. A yellow fluorescent protein was fused to the N terminus of MOR, and this construct (YMOR) was expressed in insect cells for purification. After reconstitution of purified YMOR into rHDL particles, single-molecule imaging of Cy3-labeled and Cy5-labeled YMOR determined that the rHDL particles contained one receptor. This monomeric YMOR sample binds ligands with affinities nearly equivalent to those observed in plasma membrane preparations. Monomeric YMOR efficiently stimulates GTPγS binding to G_i2 heterotrimeric G protein. G_i2 allosteric regulation of agonist binding to rHDL·YMOR was also observed. Single-particle imaging of binding of [Lys⁷, Cys⁸]dermorphin-Cy3, a fluorophore-labeled agonist, to rHDL·YMOR supports the notion that the rHDL particles contain a single YMOR. Taken together, these results suggest that a monomeric MOR is the minimal functional unit for ligand binding and G protein activation and illustrate a novel method for imaging ligand binding to opioid receptors.