The Pharmacology of the Cannabinoid System—A Question of Efficacy and Selectivity

Our knowledge of the function of the cannabinoid system in the body has been aided by the availability of pharmacological agents that affect its function. This has been achieved by the design of agents that either directly interact with the receptor (agonists and antagonist/inverse agonists) and agents that indirectly modulate the receptor output by changing the levels of the endogenous cannabinoids (endocannabinoids). In this review, examples of the most commonly used receptor agonists, antagonists/inverse agonists, and indirectly acting agents (anandamide uptake inhibitors, fatty acid amide hydrolase inhibitors, monoacylglycerol lipase inhibitors) are given, with particular focus upon their selectivity and, in the case of the directly acting compounds, efficacy. Finally, the links between the endocannabinoid and cyclooxygenase pathways are explored, in particular, with respect to agents whose primary function is to inhibit cyclooxygenase activity, but which also interact with the endocannabinoid system.     

Mutation in the M1 Domain of the Acetylcholine Receptor α Subunit Decreases the Rate of Agonist Dissociation

We describe the kinetic consequences of the mutation N217K in the M1 domain of the acetylcholine receptor (AChR) α subunit that causes a slow channel congenital myasthenic syndrome (SCCMS). We previously showed that receptors containing αN217K expressed in 293 HEK cells open in prolonged activation episodes strikingly similar to those observed at the SCCMS end plates. Here we use single channel kinetic analysis to show that the prolonged activation episodes result primarily from slowing of the rate of acetylcholine (ACh) dissociation from the binding site. Rate constants for channel opening and closing are also slowed but to much smaller extents. The rate constants derived from kinetic analysis also describe the concentration dependence of receptor activation, revealing a 20-fold shift in the EC₅₀ to lower agonist concentrations for αN217K. The apparent affinity of ACh binding, measured by competition against the rate of ¹²⁵I-α-bungarotoxin binding, is also enhanced 20-fold by αN217K. Both the slowing of ACh dissociation and enhanced apparent affinity are specific to the lysine substitution, as the glutamine and glutamate substitutions have no effect. Substituting lysine for the equivalent asparagine in the β, ε, or δ subunits does not affect the kinetics of receptor activation or apparent agonist affinity. The results show that a mutation in the amino-terminal portion of the M1 domain produces a localized perturbation that stabilizes agonist bound to the resting state of the AChR.     

β-Lactam Selectivity of Multidrug Transporters AcrB and AcrD Resides in the Proximal Binding Pocket

β-Lactams are mainstream antibiotics that are indicated for the prophylaxis and treatment of bacterial infections. The AcrA-AcrD-TolC multidrug efflux system confers much stronger resistance on Escherichia coli to clinically relevant anionic β-lactam antibiotics than the homologous AcrA-AcrB-TolC system. Using an extensive combination of chimeric analysis and site-directed mutagenesis, we searched for residues that determine the difference in β-lactam specificity between AcrB and AcrD. We identified three crucial residues at the “proximal” (or access) substrate binding pocket. The simultaneous replacement of these residues in AcrB by those in AcrD (Q569R, I626R, and E673G) transferred the β-lactam specificity of AcrD to AcrB. Our findings indicate for the first time that the difference in β-lactam specificity between AcrB and AcrD relates to interactions of the antibiotic with residues in the proximal binding pocket.     

Changes in Cationic Selectivity of the Nicotinic Channel at the Rat Ganglionic Synapse: A Role for Chloride Ions?

The permeability of the nicotinic channel (nAChR) at the ganglionic synapse has been examined, in the intact rat superior cervical ganglion in vitro, by fitting the Goldman current equation to the synaptic current (EPSC) I-V relationship. Subsynaptic nAChRs, activated by neurally-released acetylcholine (ACh), were thus analyzed in an intact environment as natively expressed by the mature sympathetic neuron. Postsynaptic neuron hyperpolarization (from -40 to -90 mV) resulted in a change of the synaptic potassium/sodium permeability ratio (P(K)/P(Na)) from 1.40 to 0.92, corresponding to a reversible shift of the apparent acetylcholine equilibrium potential, E(ACh), by about +10 mV. The effect was accompanied by a decrease of the peak synaptic conductance (g(syn)) and of the EPSC decay time constant. Reduction of [Cl(-)](o) to 18 mM resulted in a change of P(K)/P(Na) from 1.57 (control) to 2.26, associated with a reversible shift of E(ACh) by about -10 mV. Application of 200 nM αBgTx evoked P(K)/P(Na) and g(syn) modifications similar to those observed in reduced [Cl(-)](o). The two treatments were overlapping and complementary, as if the same site/mechanism were involved. The difference current before and after chloride reduction or toxin application exhibited a strongly positive equilibrium potential, which could not be explained by the block of a calcium component of the EPSC. Observations under current-clamp conditions suggest that the driving force modification of the EPSC due to P(K)/P(Na) changes represent an additional powerful integrative mechanism of neuron behavior. A possible role for chloride ions is suggested: the nAChR selectivity was actually reduced by increased chloride gradient (membrane hyperpolarization), while it was increased, moving towards a channel preferentially permeable for potassium, when the chloride gradient was reduced.     

Non-equivalent Ligand Selectivity of Agonist Sites in (α4β2)2α4 Nicotinic Acetylcholine Receptors: A KEY DETERMINANT OF AGONIST EFFICACY*

The α4β2 nicotinic acetylcholine receptor (nAChR) is the most abundant nAChR type in the brain, and this receptor type exists in alternate (α4β2)₂α4 and (α4β2)₂β2 forms, which are activated by agonists with strikingly differing efficacies. Recent breakthroughs have identified an additional operational agonist binding site in the (α4β2)₂α4 nAChR that is responsible for the signature sensitivity of this receptor to activation by agonists, yet the structural mechanisms determining agonist efficacy at this receptor type are not yet fully understood. In this study, we characterized the ligand selectivity of the individual agonist sites of the (α4β2)₂α4 nAChR to determine whether differences in agonist selectivity influence agonist efficacy. Applying the substituted cysteine accessibility method to individual agonist sites in concatenated (α4β2)₂α4 receptors, we determined the agonist selectivity of the agonist sites of the (α4β2)₂α4 receptor. We show that (a) accessibility of substituted cysteines to covalent modification by methanesulfonate reagent depends on the agonist site at which the modification occurs and (b) that agonists such as sazetidine-A and TC-2559 are excluded from the site at the α4/α4 interface. Given that additional binding to the agonist site in the α4/α4 interface increases acetylcholine efficacy and that agonists excluded from the agonist site at the α4/α4 interface behave as partial agonists, we conclude that the ability to engage all agonist sites in (α4β2)₂α4 nAChRs is a key determinant of agonist efficacy. The findings add another level of complexity to the structural mechanisms that govern agonist efficacy in heteromeric nAChRs and related ligand-gated ion channels.     

Agonist mediated internalization of M2 mAChR is β-arrestin-dependent

Background

Muscarinic acetylcholine receptors (mAChRs) undergo agonist-promoted internalization, but evidence suggesting that the mechanism of internalization is β-arrestin dependent has been contradictory and unclear. Previous studies using heterologous over-expression of wild type or dominant-negative forms of β-arrestins have reported that agonist-promoted internalization of M₂ mAChRs is a β-arrestin- and clathrin-independent phenomenon. In order to circumvent the complications associated with the presence of endogenous β-arrestin that may have existed in these earlier studies, we examined agonist-promoted internalization of the M₂ mAChR in mouse embryonic fibroblasts (MEFs) derived from β-arrestin knockout mice that lack expression of either one or both isoforms of β-arrestin (β-arrestin 1 and 2).

Results

In wild type MEF cells transiently expressing M₂ mAChRs, 40% of surface M₂ mAChRs underwent internalization and sorted into intracellular compartments following agonist stimulation. In contrast, M₂ mAChRs failed to undergo internalization and sorting into intracellular compartments in MEF β-arrestin double knockout cells following agonist stimulation. In double knockout cells, expression of either β-arrestin 1 or 2 isoforms resulted in rescue of agonist-promoted internalization. Stimulation of M₂ mAChRs led to a stable co-localization with GFP-tagged β-arrestin within endocytic structures in multiple cell lines; the compartment to which β-arrestin localized was determined to be the early endosome. Agonist-promoted internalization of M₂ mAChRs was moderately rescued in MEF β-arrestin 1 and 2 double knockout cells expressing exogenous arrestin mutants that were selectively defective in interactions with clathrin (β-arrestin 2 ΔLIELD), AP-2 (β-arrestin 2-F391A), or both clathrin/AP-2. Expression of a truncated carboxy-terminal region of β-arrestin 1 (319–418) completely abrogated agonist-promoted internalization of M₂ mAChRs in wild type MEF cells.

Conclusion

In summary, this study demonstrates that agonist-promoted internalization of M₂ mAChRs is β-arrestin- and clathrin-dependent, and that the receptor stably co-localizes with β-arrestin in early endosomal vesicles.     

Substrate Selectivity of the Acid-activated Glutamate/γ-Aminobutyric acid (GABA) Antiporter GadC from Escherichia coli

GadC, a central component of the Escherichia coli acid resistance system, is a Glu/GABA antiporter. A previous structural study and biochemical characterization showed that GadC exhibits a stringent pH dependence for substrate transport, with no detectable activity at pH values above 6.5. However, the substrate selectivity and the mechanism of pH-dependent transport activity of GadC remain enigmatic. In this study, we demonstrate that GadC selectively transports Glu with no net charge and GABA with a positive charge. A C-plug-truncated variant of GadC (residues 1–470) transported Gln (a mimic of Glu with no net charge), but not Glu, even at pH 8.0. The pH-dependent transport of Gln by this GadC variant was shifted ∼1 unit toward a higher pH compared with Glu transport. Taken together, the results identify the substrate selectivity for GadC and show that the protonation states of substrates are crucial for transport.     

Specificity and Selectivity of a Modified Radioligand Method for Estimating the Binding Activity of β1 Adrenergic Receptors in Human T Lymphocytes

Russian Journal of Bioorganic Chemistry - A radioligand-based method was proposed for quantifying the binding activity of β1-adrenergic receptors (ARs) on the surface of human T lymphocytes....     

Alteration of the Circadian Rhythm in Peak Expiratory Flow of Nocturnal Asthma Following Nighttime Transdermal β2‐adrenoceptor Agonist Tulobuterol Chronotherapy

We investigated the efficacy of nighttime transdermal tulobuterol (β₂‐adrenoceptor agonist) chronotherapy for nocturnal asthma by assessing changes both in the frequency of symptoms and features of the circadian rhythm in peak expiratory flow (PEF), a measure of airway caliber. Thirteen patients with nocturnal asthma were evaluated before and during tulobuterol patch chronotherapy, applied once daily in the evening for 6 consecutive days. Patients were asked to record their PEF every 4 h between 03:00 and 23:00 h for one day. Circadian rhythms in PEF were examined by group‐mean cosinor analysis. The group average PEF at 03:00 h, the time during the 24 h when PEF is generally the poorest, before the application of the chronotherapy, when asthma was unstable and nocturnal symptoms frequent, was 276±45 L/min. Application of the tulobuterol patch at nighttime significantly increased (p<0.001) the 03:00 h group average PEF to 363±67 L/min. Significant circadian rhythms in PEF were observed during the span of study when nocturnal symptoms were frequent as well as with the use of the tulobuterol patch. Before the initiation of tulobuterol chronotherapy, the bathyphase (trough time of the circadian rhythm) in PEF narrowed to around 04:00 h, and the group circadian amplitude was 28.8 L/min. In contrast, the group circadian amplitude significantly (p<0.01) decreased to 10.4 L/min, and the 24 h mean PEF increased significantly with tulobuterol patch chronotherapy. These changes indicate that tulobuterol chronotherapy significantly increased both the level and stability of airway function over the 24 h. The circadian rhythm in PEF varied with the severity and frequency of asthmatic symptoms with and without the nighttime application of the tulobuterol patch medication. We conclude that the parameters of the circadian rhythm of PEF proved useful both in determining the need for and effectiveness of tulobuterol chronotherapy for nocturnal asthma.     

Speeding the Recovery from Ultraslow Inactivation of Voltage-Gated Na+ Channels by Metal Ion Binding to the Selectivity Filter: A Foot-on-the-Door?

Slow inactivated states in voltage-gated ion channels can be modulated by binding molecules both to the outside and to the inside of the pore. Thus, external K⁺ inhibits C-type inactivation in Shaker K⁺ channels by a “foot-in-the-door” mechanism. Here, we explore the modulation of a very long-lived inactivated state, ultraslow inactivation (I_US), by ligand binding to the outer vestibule in voltage-gated Na⁺ channels. Blocking the outer vestibule by a mutant μ-conotoxin GIIIA substantially accelerated recovery from I_US. A similar effect was observed if Cd²⁺ was bound to a cysteine engineered to the selectivity filter (K1237C). In K1237C channels, exposed to 30 μM Cd²⁺, the time constant of recovery from I_US was decreased from 145.0 ± 10.2 s to 32.5 ± 3.3 s (P < 0.001). Recovery from I_US was only accelerated if Cd²⁺ was added to the bath solution during recovery (V = −120 mV) from I_US, but not when the channels were selectively exposed to Cd²⁺ during the development of I_US (−20 mV). These data could be explained by a kinetic model in which Cd²⁺ binds with high affinity to a slow inactivated state (I_S), which is transiently occupied during recovery from I_US. A total of 50 μM Cd²⁺ produced an ∼8 mV hyperpolarizing shift of the steady-state inactivation curve of I_S, supporting this kinetic model. Binding of lidocaine to the internal vestibule significantly reduced the number of channels entering I_US, suggesting that I_US is associated with a conformational change of the internal vestibule of the channel. We propose a molecular model in which slow inactivation (I_S) occurs by a closure of the outer vestibule, whereas I_US arises from a constriction of the internal vestibule produced by a widening of the selectivity filter region. Binding of Cd²⁺ to C1237 promotes the closure of the selectivity filter region, thereby hastening recovery from I_US. Thus, Cd²⁺ ions may act like a foot-on-the-door, kicking the I_S gate to close.     

Synthetic Peptide TPLVTLFK,a Selective Agonist of Nonopioid β-Endorphin Receptor,Reduces the Corticotropin and Corticosterone Response

The synthetic peptide octarphin (TPLVTLFK) corresponding to the sequence 12–19 of β-endorphin, a selective agonist of nonopioid β-endorphin receptor, was labeled with tritium to specific activity of 29 Ci/mmol. The analysis of the specific binding of [³H]octarphin to anterior pituitary membranes obtained from rats before and after the lipopolysaccharide (LPS)-injection showed that 2 h after LPS administration the value of maximal binding capacity of the membranes (B_max) was increased by 1.6 times (B_max 12.3 ± 0.8 and 20.0 ± 1.9 pmol/mg of protein, respectively), while the binding affinity was not changed (K _d 5.8 ± 0.3 and 5.5 ± 0.4 nM, respectively). At the same time, LPS did not have a significant effect on the characteristics of the labeled peptide binding to adrenal cortex membranes. Intranasal injection of octarphin at doses of 10–30 μg/rat was found to reduce the LPS-induced corticotropin and corticosterone response. The effect of the peptide was dose-dependent with a maximum at a dose 20 μg/rat. Aminoguanidine (AG 100 mg/kg i.p.), a selective inducible nitric oxide synthase (iNOS) inhibitor, completely abolished the inhibitory effect of the peptide on the LPS-induced corticotropin and corticosterone response. At the same time, octarphin in vitro stimulated in a time- and concentration-dependent manner the anterior pituitary iNOS expression of rats injected with LPS (1 mg/kg i.p.). The maximum level of the iNOS expression was observed at a peptide concentration of 10 nM after 2 h cultivation. These results indicate that the inhibitory effect of octarphin on LPS-induced secretion of corticotropin and corticosterone due to the ability of the peptide to stimulate the expression of iNOS in the anterior pituitary.     

Neurons and β-Cells of the Pancreas Express Connexin36, Forming Gap Junction Channels that Exhibit Strong Cationic Selectivity

We examined the permeability of connexin36 (Cx36) homotypic gap junction (GJ) channels, expressed in neurons and β-cells of the pancreas, to dyes differing in molecular mass and net charge. Experiments were performed in HeLa cells stably expressing Cx36 tagged with EGFP by combining a dual whole-cell voltage clamp and fluorescence imaging. To assess the permeability of the single GJ channel (P(γ)), we used a dual-mode excitation of fluorescent dyes that allowed us to measure cell-to-cell dye transfer at levels not resolvable using whole-field excitation solely. We demonstrate that P(γ) of Cx36 for cationic dyes (EAM-1? and EAM-2?) is ~10-fold higher than that for an anionic dye of the same net charge and similar molecular mass, Alexa fluor-350 (AFl-350?). In addition, P(γ) for Lucifer yellow (LY2?) is approximately fourfold smaller than that for AFl-350?, which suggests that the higher negativity of LY2? significantly reduces permeability. The P(γ) of Cx36 for AFl-350 is approximately 358, 138, 23 and four times smaller than the P(γ)s of Cx43, Cx40, Cx45, and Cx57, respectively. In contrast, it is 6.5-fold higher than the P(γ) of mCx30.2, which exhibits a smaller single-channel conductance. Thus, Cx36 GJs are highly cation-selective and should exhibit relatively low permeability to numerous vital negatively charged metabolites and high permeability to K?, a major charge carrier in cell-cell communication.     

Glutamine 57 at the Complementary Binding Site Face Is a Key Determinant of Morantel Selectivity for ��7 Nicotinic Receptors

Nicotinic receptors (AChRs) play key roles in synaptic transmission. We explored activation of neuronal α7 and mammalian muscle AChRs by morantel and oxantel. Our results revealed a novel action of morantel as a high efficacy and more potent agonist than ACh of α7 receptors. The EC₅₀ for activation by morantel of both α7 and α7-5HT_3A receptors is 7-fold lower than that determined for ACh. The minimum morantel concentration required to activate α7-5HT_3A channels is 6-fold lower than that of ACh, and activation episodes are more prolonged than in the presence of ACh. By contrast, oxantel is a weak agonist of α7 and α7-5HT_3A, and both drugs are very low efficacy agonists of muscle AChRs. The replacement of Gln⁵⁷ in α7 by glycine, which is found in the equivalent position of the muscle AChR, decreases the efficacy for activation and turns morantel into a partial agonist. The reverse mutation in the muscle AChR (ϵG57Q) increases 7-fold the efficacy of morantel. The mutations do not affect activation by ACh or oxantel, indicating that this position is selective for morantel. In silico studies show that the tetrahydropyrimidinyl group, common to both drugs, is close to Trp¹⁴⁹ of the principal face of the binding site, whereas the other cyclic group is proximal to Gln⁵⁷ of the complementary face in morantel but not in oxantel. Thus, position 57 at the complementary face is a key determinant of the high selectivity of morantel for α7. These results provide new information for further progress in drug design.Nicotinic acetylcholine receptors (AChRs),³ members of the Cys-loop receptor superfamily, are of fundamental importance in synaptic transmission throughout the nervous system in both vertebrates and invertebrates. They are implicated in a wide range of important pathologies and are targets of clinically relevant drugs. AChRs are pentameric proteins composed of highly homologous subunits (1, 2). Subunits are classified as either α, which contain a disulfide bridge formed by two adjacent cysteine residues important for acetylcholine (ACh) binding, or non-α subunits, which lack this motif (3).AChRs assemble from five identical α subunits, forming homomeric receptors, such as neuronal α7 receptors, or from different α and non-α subunits, forming heteromeric receptors, such as the muscle AChR. Human adult muscle AChRs are composed of two α1, one β, one ϵ, and one δ subunits. The five homologous subunits are arranged as barrel staves around a central ion-conducting pore (4). Approximately half of each subunit is extracellular with the remainder comprising transmembrane domains M1–M4 and a large cytoplasmic domain spanning M3 and M4 (4). The neurotransmitter binding sites are formed within the extracellular domain at interfaces between subunits (4, 5). One of the sides, called the principal face, is formed by three discontinuous loops of the α subunit, whereas the complementary face is formed by three discontinuous β-strands of the adjacent subunit. Key residues of the principal face are grouped in regions called loop A (Trp⁸⁶ and Tyr⁹³), loop B (Trp¹⁴⁹ and Gly¹⁵³), and loop C (Tyr¹⁹⁰, Cys¹⁹², Cys¹⁹³, and Tyr¹⁹⁸). The complementary face is formed by residues from α7 or δ or ϵ subunits in the adult muscle AChR. At this face of the muscle AChR, residues are clustered in loop D (Trp⁵⁵), E (Leu¹⁰⁹, Tyr¹¹¹, Tyr¹¹⁷, and Leu¹¹⁹), and F (Asp¹⁷⁴ and Glu¹⁷⁶) (2, 5, 6). Residues of the principal face are highly conserved between α7 and α1 subunits, whereas less conservation is found in residues located at the complementary face (5, 7).The anthelmintic agents levamisole, pyrantel, oxantel, and morantel are full agonists of nematode muscle AChRs, and exert their therapeutic actions by producing muscle paralysis (8). By contrast, levamisole and pyrantel have been shown to be low efficacy agonists of mammalian muscle AChRs (9). A few lines of experimental evidence suggest that these compounds also interact with some types of neuronal AChRs, but instead of acting as agonists, they act as modulators. Morantel and levamisole have been shown to allosterically potentiate responses of α3β2 and α3β4 receptors (10, 11). Thus, the actions of anthelmintic agents seem to be strongly dependent on the AChR subtype. Therefore, these compounds are useful tools for the identification of determinants of drug selectivity, which, in turn, is required for rational design of novel and more specific drugs.We have here determined that, similarly to pyrantel and levamisole (9), morantel and oxantel are low efficacy agonists of mammalian muscle AChRs. However, whereas oxantel is also a weak agonist of α7, morantel is more potent than ACh. By site-directed mutagenesis we determined that position 57, located at the complementary face of the binding site, is involved in the differential selectivity of morantel for α7 and mammalian muscle AChR.Neuronal α7 receptors may be involved in a range of neurological and psychiatric disorders that lead to cognitive impairment, including Alzheimer disease, attention deficit hyperactivity disorder, and schizophrenia (12). Given that its deficit is associated with cognitive impairment in these diseases, enhancement of its activity has recently emerged as a physiological and effective therapeutic strategy. Therefore, the characterization of the novel action of morantel as a potent agonist of α7 together with the identification of the structural basis of this high selectivity become of importance as they provide new information for further progress in drug design.     

Magnitude of a Conformational Change in the Glycine Receptor ��1-��2 Loop Is Correlated with Agonist Efficacy

The efficacy of agonists at Cys-loop ion channel receptors is determined by the rate they isomerize receptors to a pre-open flip state. Once the flip state is reached, the shut-open reaction is similar for low and high efficacy agonists. The present study sought to identify a conformational change associated with the closed-flip transition in the α1-glycine receptor. We employed voltage-clamp fluorometry to compare ligand-binding domain conformational changes induced by the following agonists, listed from highest to lowest affinity and efficacy: glycine > β-alanine > taurine. Voltage-clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements from ligand-induced fluorescence changes. Agonist affinity and efficacy correlated inversely with maximum fluorescence magnitudes at labeled residues in ligand-binding domain loops D and E, suggesting that large conformational changes in this region preclude efficacious gating. However, agonist affinity and efficacy correlated directly with maximum fluorescence magnitudes from a label attached to A52C in loop 2, near the transmembrane domain interface. Because glycine experiences the largest affinity increase between closed and flip states, we propose that the magnitude of this fluorescence signal is directly proportional to the agonist affinity increase. In contrast, labeled residues in loops C, F, and the pre-M1 domain yielded agonist-independent fluorescence responses. Our results support the conclusion that a closed-flip conformation change, with a magnitude proportional to the agonist affinity increase from closed to flip states, occurs in the microenvironment of Ala-52.Glycine receptors (GlyRs)³ are pentameric chloride-selective ion channels that mediate fast inhibitory neurotransmission (1). They are members of the Cys-loop receptor family that includes the prototypical nicotinic acetylcholine receptor (nAChR), the γ-aminobutyric acid type-A receptors (GABA_ARs), and serotonin type-3 receptors (5-HT₃Rs). Recent structural studies have provided a wealth of information on the structure and function of this receptor family (2–6). In Cys-loop receptors, the ligand-binding domain (LBD) preceding the four transmembrane helices consists of two twisted β-sheets. The inner (vestibule facing) β-sheet comprises seven β-strands, while the outer β-sheet is formed by three β-strands (3). The ligand binding site is located at the interface of adjacent subunits and is lined by six domains: three loops from the principal and the complementary sides, termed A-C and D-F, respectively (3).GlyRs are activated by endogenous amino acid agonists in the following order of efficacy: glycine > β-alanine > taurine (7, 8). As these amino acids share considerable structural similarity (Fig. 1A), they are likely to compete for the same binding site (9–11). A recent ground-breaking study on an intermediate pre-open state, the so-called “flip” state (12), has provided new insights into the mechanism of partial agonism in Cys-loop receptors (13). This study suggested that agonist efficacy depends on the ability of the agonist to convert the inert agonist-bound receptor to the pre-open flip state. Once the flip state is reached, the shut-open reaction is similar for high and low efficacy agonists. To date there is, however, very little information concerning the structural basis for the lower efficacies of partial agonists. To address this, the present study employed the voltage-clamp fluorometry (VCF) technique (14) to compare the conformational changes induced by glycine, β-alanine, and taurine at various positions in the GlyR LBD.Open in a separate windowFIGURE 1.A, structures of glycine, β-alanine, and taurine. B, model of the LBD, based on carbomylcholine-bound AChBP (PDB code 1uv6). The inner β-sheet is displayed in red, the outer β-sheet in blue. Connecting loops are shown in gray. Colored balls represent approximate locations of selected residues labeled in regions flanking the outer β-sheet (black, G181C in loop F; N203C in loop C; Q219C in the pre-M1 domain) and in the inner β-sheet (yellow, L127C in loop E; Q67C in loop D; A52C in loop 2).VCF involves tethering of an environmentally sensitive fluorophore to a cysteine engineered into a domain of interest. If ligand-binding and/or channel opening leads to a changed dielectric environment surrounding the fluorophore, a change in quantum yield or emission spectrum can be detected. VCF was first employed on voltage-gated potassium channels (15) and has since provided a wealth of information on Cys-loop receptor structure and function (16–23). Here we employ VCF to identify an agonist-specific conformational change that may control or reflect the rate at which the GlyR isomerizes to the flip state.     

Catalysis and Selectivity in Prebiotic Synthesis: Initiation of the Formation of Oligo(U)s on Montmorillonite Clay by Adenosine-5′-methylphosphate

Adenosine-5′-methylphosphate (MepA) initiates the oligomerization of the 5′-phosphorimidazolide of uridine (ImpU) in the presence of montmorillonite clay. Longer oligomers form because the 5′-phosphate is blocked with a methyl group that prevents the formation of cyclic- and pyrophosphate-containing compounds. The MepA initiates 69–84% of the 5–9 charge oligomers, respectively. The montmorillonite catalyst also provides selectivity in the oligomerization reactions so that the main reaction pathway is MepA → MepA³′pU → MepA³′pU²′pU → MepA³′pU²′pU³′pU. MepA did not enhance the oligomerization of ImpA. The relative rates of the reactions were determined from an investigation of the products in competitive reactions. Selectivity was observed in the reaction of ImpU with equimolar amounts of MepA³′pU and MepA²′pU where the relative reaction rates are 10.3:1, respectively. In the reaction of ImpA with MepA³′pA and MepA²′pA the ImpA reacts 5.2 times faster with MepA³′pA. In the competitive reaction of ImpU and ImpA with MepA³′pA and MepA³′pU the elongation proceeds on MepA³′pA 5.6 times more rapidly than with MepA³′pU. There is no correlation between the extent of binding to the montmorillonite and reaction rates in the formation of longer oligomers. The formation of more than two sequential 2′,5′-linkages in the oligomer chain proceeds more slowly than the addition to a single 2′,5′-link or a 3′,5′-link and either chain termination or elongation by a 3′,5′-linage occurs. The central role that catalysis may have had in the prebiotic formation of biopolymers is discussed. Note added in proof: There are errors in the high resolution mass spectral data given in Section 4.2.1. The high resolution mass spectrum found for the cyclic dimer of UpUp (C-UpUp) was 657.02260. C₁₈H₂₁N₄O₁₆P₂Na₂ requires 657.02232. The high resolution mass spectrum found for the cyclic dimer of ApAp (C-ApAp) was 725.05850. C₂₀H₂₂N₁₀O₁₂P₂Na₃ requires 725.05839.     

The AMPK Agonist AICAR Inhibits TGF-β1 Induced Activation of Kidney Myofibroblasts

Activation of interstitial myofibroblasts and excessive production of extracellular matrix proteins are common pathways that contribute to chronic kidney disease. In a number of tissues, AMP-activated kinase (AMPK) activation has been shown to inhibit fibrosis. Here, we examined the inhibitory effect of the AMPK activator, 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR), on renal fibrosis in vivo and TGF-β1-induced renal fibroblasts activation in vitro. A unilateral ureteral obstruction (UUO) model was induced in male BALB/c mice. Mice with UUO were administered AICAR (500 mg/Kg/day) or saline intraperitoneally 1 day before UUO surgery and daily thereafter. Both kidneys were harvested 7 days after surgery for further analysis. For the in vitro studies, NRK-49F rat fibroblasts were pre-incubated with AICAR before TGF-β1 stimulation. The inhibitory effects of AICAR on signaling pathways down-stream of TGF-β1 were analyzed. In UUO model mice, administration of AICAR attenuated extracellular matrix protein deposition and the expression of α-smooth muscle actin (α-SMA), type I collagen and fibronectin. Pre-incubation of NRK-49F cells with AICAR inhibited TGF-β1-induced myofibroblast activation. Silencing of AMPKα1 by siRNA or by blocking AMPK activation with Compound C diminished the inhibitory effect of AICAR. Moreover, the inhibitory effects of AICAR on TGF-β1-mediated myofibroblast activation were associated with down-regulation of ERK 1/2 and STAT3. Our results suggest that AICAR reduces tubulointerstitial fibrosis in UUO mice and inhibits TGF-β1-induced kidney myofibroblast activation. AMPK activation by AICAR may have therapeutic potential for the treatment of renal tubulointerstitial fibrosis.     

Effects of Peroxisome Proliferator-Activated Receptor-δ Agonist on Cardiac Healing after Myocardial Infarction

Peroxisome proliferator-activated receptor-delta (PPAR-δ)-dependent signaling is associated with rapid wound healing in the skin. Here, we investigated the therapeutic effects of PPAR-δ-agonist treatment on cardiac healing in post-myocardial infarction (MI) rats. Animals were assigned to the following groups: sham-operated control group, left anterior descending coronary artery ligation (MI) group, or MI with administration of the PPAR-δ agonist GW610742 group. GW610742 (1 mg/kg) was administrated intraperitoneally after the operation and repeated every 3 days. Echocardiographic data showed no differences between the two groups in terms of cardiac function and remodeling until 4 weeks. However, the degrees of angiogenesis and fibrosis after MI were significantly higher in the GW610742-treated rats than in the untreated MI rats at 1 week following MI, which changes were not different at 2 weeks after MI. Naturally, PPAR-δ expression in infarcted myocardium was highest increased in 3 day after MI and then disappeared in 14 day after MI. GW610742 increased myofibroblast differentiation and transforming growth factor-beta 2 expression in the infarct zone at 7 days after MI. GW610742 also increased bone marrow-derived mesenchymal stem cell (MSC) recruitment in whole myocardium, and increased serum platelet-derived growth factor B, stromal-derived factor-1 alpha, and matrix metallopeptidase 9 levels at day 3 after MI. PPAR-δ agonists treatment have the temporal effect on early fibrosis of infarcted myocardium, which might not sustain the functional and structural beneficial effect.