Fentanyl Structure as a Scaffold for Opioid/Non-Opioid Multitarget Analgesics

One of the strategies in the search for safe and effective analgesic drugs is the design of multitarget analgesics. Such compounds are intended to have high affinity and activity at more than one molecular target involved in pain modulation. In the present contribution we summarize the attempts in which fentanyl or its substructures were used as a μ-opioid receptor pharmacophoric fragment and a scaffold to which fragments related to non-opioid receptors were attached. The non-opioid ‘second’ targets included proteins as diverse as imidazoline I₂ binding sites, CB₁ cannabinoid receptor, NK₁ tachykinin receptor, D₂ dopamine receptor, cyclooxygenases, fatty acid amide hydrolase and monoacylglycerol lipase and σ₁ receptor. Reviewing the individual attempts, we outline the chemistry, the obtained pharmacological properties and structure-activity relationships. Finally, we discuss the possible directions for future work.     

TGF-β1 Protection against Aβ1–42-Induced Neuroinflammation and Neurodegeneration in Rats

Transforming growth factor (TGF)-β1, a cytokine that can be expressed in the brain, is a key regulator of the brain’s responses to injury and inflammation. Alzheimer’s disease (AD), the most common neurodegenerative disorder, involves inflammatory processes in the brain in addition to the hallmarks, amyloid-β (Aβ) plaques and neurofibrillary tangles. Recently, we have shown that T-helper (Th) 17 cells, a subpopulation of CD4⁺ T-cells with high proinflammation, also participate in the brain inflammatory process of AD. However, it is poorly known whether TGF-β1 ameliorates the lymphocyte-mediated neuroinflammation and, thereby, alleviates neurodegeneration in AD. Herein, we administered TGF-β1 via the intracerebroventricle (ICV) in AD model rats, by Aβ_1–42 injection in both sides of the hippocampus, to show the neuroprotection of TGF-β1. The TGF-β1 administration after the Aβ_1–42 injection ameliorated cognitive deficit and neuronal loss and apoptosis, reduced amyloid precursor protein (APP) expression, elevated protein phosphatase (PP)2A expression, attenuated glial activation and alleviated the imbalance of the pro-inflammatory/anti-inflammatory responses of T-lymphocytes, compared to the Aβ_1–42 injection alone. These findings demonstrate that TGF-β1 provides protection against AD neurodegeneration and suggest that the TGF-β1 neuroprotection is implemented by the alleviation of glial and T-cell-mediated neuroinflammation.     

State-Targeting Stabilization of Adenosine A2A Receptor by Fusing a Custom-Made De Novo Designed α-Helical Protein

G-protein coupled receptors (GPCRs) are known for their low stability and large conformational changes upon transitions between multiple states. A widely used method for stabilizing these receptors is to make chimeric receptors by fusing soluble proteins (i.e., fusion partner proteins) into the intracellular loop 3 (ICL3) connecting the transmembrane helices 5 and 6 (TM5 and TM6). However, this fusion approach requires experimental trial and error to identify appropriate soluble proteins, residue positions, and linker lengths for making the fusion. Moreover, this approach has not provided state-targeting stabilization of GPCRs. Here, to rationally stabilize a class A GPCR, adenosine A_2A receptor (A_2AR) in a target state, we carried out the custom-made de novo design of α-helical fusion partner proteins, which can fix the conformation of TM5 and TM6 to that in an inactive state of A_2AR through straight helical connections without any kinks or intervening loops. The chimeric A_2AR fused with one of the designs (FiX1) exhibited increased thermal stability. Moreover, compared with the wild type, the binding affinity of the chimera against the agonist NECA was significantly decreased, whereas that against the inverse agonist ZM241385 was similar, indicating that the inactive state was selectively stabilized. Our strategy contributes to the rational state-targeting stabilization of GPCRs.     

AmOctα2R: Functional Characterization of a Honeybee Octopamine Receptor Inhibiting Adenylyl Cyclase Activity

The catecholamines norepinephrine and epinephrine are important regulators of vertebrate physiology. Insects such as honeybees do not synthesize these neuroactive substances. Instead, they use the phenolamines tyramine and octopamine for similar physiological functions. These biogenic amines activate specific members of the large protein family of G protein-coupled receptors (GPCRs). Based on molecular and pharmacological data, insect octopamine receptors were classified as either α- or β-adrenergic-like octopamine receptors. Currently, one α- and four β-receptors have been molecularly and pharmacologically characterized in the honeybee. Recently, an α₂-adrenergic-like octopamine receptor was identified in Drosophila melanogaster (DmOctα2R). This receptor is activated by octopamine and other biogenic amines and causes a decrease in intracellular cAMP ([cAMP]_i). Here, we show that the orthologous receptor of the honeybee (AmOctα2R), phylogenetically groups in a clade closely related to human α₂-adrenergic receptors. When heterologously expressed in an eukaryotic cell line, AmOctα2R causes a decrease in [cAMP]_i. The receptor displays a pronounced preference for octopamine over tyramine. In contrast to DmOctα2R, the honeybee receptor is not activated by serotonin. Its activity can be blocked efficiently by 5-carboxamidotryptamine and phentolamine. The functional characterization of AmOctα2R now adds a sixth member to this subfamily of monoaminergic receptors in the honeybee and is an important step towards understanding the actions of octopamine in honeybee behavior and physiology.     

Guanosine-Mediated Anxiolytic-Like Effect: Interplay with Adenosine A1 and A2A Receptors

Acute or chronic administration of guanosine (GUO) induces anxiolytic-like effects, for which the adenosine (ADO) system involvement has been postulated yet without a direct experimental evidence. Thus, we aimed to investigate whether adenosine receptors (ARs) are involved in the GUO-mediated anxiolytic-like effect, evaluated by three anxiety-related paradigms in rats. First, we confirmed that acute treatment with GUO exerts an anxiolytic-like effect. Subsequently, we investigated the effects of pretreatment with ADO or A₁R (CPA, CCPA) or A_2AR (CGS21680) agonists 10 min prior to GUO on a GUO-induced anxiolytic-like effect. All the combined treatments blocked the GUO anxiolytic-like effect, whereas when administered alone, each compound was ineffective as compared to the control group. Interestingly, the pretreatment with nonselective antagonist caffeine or selective A₁R (DPCPX) or A_2AR (ZM241385) antagonists did not modify the GUO-induced anxiolytic-like effect. Finally, binding assay performed in hippocampal membranes showed that [³H]GUO binding became saturable at 100–300 nM, suggesting the existence of a putative GUO binding site. In competition experiments, ADO showed a potency order similar to GUO in displacing [³H]GUO binding, whereas AR selective agonists, CPA and CGS21680, partially displaced [³H]GUO binding, but the sum of the two effects was able to displace [³H]GUO binding to the same extent of ADO alone. Overall, our results strengthen previous data supporting GUO-mediated anxiolytic-like effects, add new evidence that these effects are blocked by A₁R and A_2AR agonists and pave, although they do not elucidate the mechanism of GUO and ADO receptor interaction, for a better characterization of GUO binding sites in ARs.     

Identification of the Ghrelin and Cannabinoid CB2 Receptor Heteromer Functionality and Marked Upregulation in Striatal Neurons from Offspring of Mice under a High-Fat Diet

Cannabinoids have been reported as orexigenic, i.e., as promoting food intake that, among others, is controlled by the so-called “hunger” hormone, ghrelin. The aim of this paper was to look for functional and/or molecular interactions between ghrelin GHSR1a and cannabinoid CB₂ receptors at the central nervous system (CNS) level. In a heterologous system we identified CB₂-GHSR1a receptor complexes with a particular heteromer print consisting of impairment of CB₂ receptor/G_i-mediated signaling. The blockade was due to allosteric interactions within the heteromeric complex as it was reverted by antagonists of the GHSR1a receptor. Cannabinoids acting on the CB₂ receptor did not affect cytosolic increases of calcium ions induced by ghrelin acting on the GHSR1a receptor. In situ proximity ligation imaging assays confirmed the expression of CB₂-GHSR1a receptor complexes in both heterologous cells and primary striatal neurons. We tested heteromer expression in neurons from offspring of high-fat-diet mouse mothers as they have more risk to be obese. Interestingly, there was a marked upregulation of those complexes in striatal neurons from siblings of pregnant female mice under a high-fat diet.     

Celastrol Attenuates Inflammatory and Neuropathic Pain Mediated by Cannabinoid Receptor Type 2

Celastrol, a major active ingredient of Chinese herb Tripterygium wilfordii Hook. f. (thunder god vine), has exhibited a broad spectrum of pharmacological activities, including anti-inflammation, anti-cancer and immunosuppression. In the present study, we used animal models of inflammatory pain and neuropathic pain, generated by carrageenan injection and spared nerve injury (SNI), respectively, to evaluate the effect of celastrol and to address the mechanisms underlying pain processing. Intraperitoneal (i.p.) injection of celastrol produced a dose-dependent inhibition of carrageenan-induced edema and allodynia. Real-time PCR analysis showed that celastrol (0.3 mg/kg, i.p.) significantly reduced mRNA expressions of inflammatory cytokines, TNF-α, IL-6, IL-1β, in carrageenan-injected mice. In SNI mice, pain behavior studies showed that celastrol (1 mg/kg, i.p.) effectively prevented the hypersensitivity of mechanical nociceptive response on the third day post-surgery and the seventh day post-surgery. Furthermore, the anti-hyperalgesic effects of celastrol in carrageenan-injected mice and SNI mice were reversed by SR144528 (1 mg/kg, i.p.), a specific cannabinoid receptor-2 (CB₂) receptor antagonist, but not by SR141716 (1 mg/kg, i.p.), a specific cannabinoid receptor-1 (CB₁) receptor antagonist. Taken together, our results demonstrate the analgesia effects of celastrol through CB₂ signaling and propose the potential of exploiting celastrol as a novel candidate for pain relief.     

Design and synthesis of novel dual-action compounds targeting the adenosine A(2A) receptor and adenosine transporter for neuroprotection

A novel compound, N⁶‐(4‐hydroxybenzyl)adenosine, isolated from Gastrodia elata and which has been shown to be a potential therapeutic agent for preventing and treating neurodegenerative disease, was found to target both the adenosine A_2A receptor (A_2AR) and the equilibrative nucleoside transporter 1 (ENT1). As A_2AR and ENT1 are proximal in the synaptic crevice of striatum, where the mutant huntingtin aggregate is located, the dual‐action compounds that concomitantly target these two membrane proteins may be beneficial for the therapy of Huntington’s disease. To design the desired dual‐action compounds, pharmacophore models of the A_2AR agonists and the ENT1 inhibitors were constructed. Accordingly, potentially active compounds were designed and synthesized by chemical modification of adenosine, particularly at the N⁶ and C^5’ positions, if the predicted activity was within an acceptable range. Indeed, some of the designed compounds exhibit significant dual‐action properties toward both A_2AR and ENT1. Both pharmacophore models exhibit good statistical correlation between predicted and measured activities. In agreement with competitive ligand binding assay results, these compounds also prevent apoptosis in serum‐deprived PC12 cells, rendering a crucial function in neuroprotection and potential utility in the treatment of neurodegenerative diseases.     

Neuroprotective Effect of Aurantio-Obtusin,a Putative Vasopressin V1A Receptor Antagonist,on Transient Forebrain Ischemia Mice Model

Traditional Chinese medicines (TCMs) have been a rich source of novel drug discovery, and Cassia seed is one of the common TCMs with numerous biological effects. Based on the existing reports on neuroprotection by Cassia seed extract, the present study aims to search possible pharmacological targets behind the neuroprotective effects of the Cassia seeds by evaluating the functional effect of specific Cassia compounds on various G-protein-coupled receptors. Among the four test compounds (cassiaside, rubrofusarin gentiobioside, aurantio-obtusin, and 2-hydroxyemodin 1-methylether), only aurantio-obtusin demonstrated a specific V_1AR antagonist effect (71.80 ± 6.0% inhibition at 100 µM) and yielded an IC₅₀ value of 67.70 ± 2.41 μM. A molecular docking study predicted an additional interaction of the hydroxyl group at C6 and a methoxy group at C7 of aurantio-obtusin with the Ser341 residue as functional for the observed antagonist effect. In the transient brain ischemia/reperfusion injury C57BL/6 mice model, aurantio-obtusin attenuated the latency time that was reduced in the bilateral common carotid artery occlusion (BCCAO) groups. Likewise, compared to neuronal damage in the BCCAO groups, treatment with aurantio-obtusin (10 mg/kg, p.o.) significantly reduced the severity of damage in medial cornu ammonis 1 (mCA1), dorsal CA1, and cortex regions. Overall, the findings of this study highlight V_1AR as a possible target of aurantio-obtusin for neuroprotection.     

Cannabis sativa L. as a Natural Drug Meeting the Criteria of a Multitarget Approach to Treatment

Cannabis sativa L. turned out to be a valuable source of chemical compounds of various structures, showing pharmacological activity. The most important groups of compounds include phytocannabinoids and terpenes. The pharmacological activity of Cannabis (in epilepsy, sclerosis multiplex (SM), vomiting and nausea, pain, appetite loss, inflammatory bowel diseases (IBDs), Parkinson’s disease, Tourette’s syndrome, schizophrenia, glaucoma, and coronavirus disease 2019 (COVID-19)), which has been proven so far, results from the affinity of these compounds predominantly for the receptors of the endocannabinoid system (the cannabinoid receptor type 1 (CB₁), type two (CB₂), and the G protein-coupled receptor 55 (GPR₅₅)) but, also, for peroxisome proliferator-activated receptor (PPAR), glycine receptors, serotonin receptors (5-HT), transient receptor potential channels (TRP), and GPR, opioid receptors. The synergism of action of phytochemicals present in Cannabis sp. raw material is also expressed in their increased bioavailability and penetration through the blood–brain barrier. This review provides an overview of phytochemistry and pharmacology of compounds present in Cannabis extracts in the context of the current knowledge about their synergistic actions and the implications of clinical use in the treatment of selected diseases.     

High-Dose Benzodiazepines Positively Modulate GABAA Receptors via a Flumazenil-Insensitive Mechanism

Benzodiazepines (BZDs) produce versatile pharmacological actions through positive modulation of GABA_A receptors (GABA_ARs). A previous study has demonstrated that high concentrations of diazepam potentiate GABA currents on the α₁β₂γ₂ and α₁β₂ GABA_ARs in a flumazenil-insensitive manner. In this study, the high-concentration effects of BZDs and their sensitivity to flumazenil were determined on synaptic (α₁β₂γ₂, α₂β₂γ₂, α₅β₂γ₂) and extra-synaptic (α₄β₂δ) GABA_ARs using the voltage-clamp electrophysiology technique. The in vivo evaluation of flumazenil-insensitive BZD effects was conducted in mice via the loss of righting reflex (LORR) test. Diazepam induced biphasic potentiation on the α₁β₂γ₂, α₂β₂γ₂ and α₅β₂γ₂ GABA_ARs, but did not affect the α₄β₂δ receptor. In contrast to the nanomolar component of potentiation, the second potentiation elicited by micromolar diazepam was insensitive to flumazenil. Midazolam, clonazepam, and lorazepam at 200 µM exhibited similar flumazenil-insensitive effects on the α₁β₂γ₂, α₂β₂γ₂ and α₅β₂γ₂ receptors, whereas the potentiation induced by 200 µM zolpidem or triazolam was abolished by flumazenil. Both the GABA_AR antagonist pentylenetetrazol and Fa173, a proposed transmembrane site antagonist, abolished the potentiation induced by 200 µM diazepam. Consistent with the in vitro results, flumazenil antagonized the zolpidem-induced LORR, but not that induced by diazepam or midazolam. Pentylenetetrazol and Fa173 antagonized the diazepam-induced LORR. These findings support the existence of non-classical BZD binding sites on certain GABA_AR subtypes and indicate that the flumazenil-insensitive effects depend on the chemical structures of BZD ligands.     

Adenosine and Cordycepin Accelerate Tissue Remodeling Process through Adenosine Receptor Mediated Wnt/β-Catenin Pathway Stimulation by Regulating GSK3b Activity

Adenosine is a cellular metabolite with diverse derivatives that possesses a wide range of physiological roles. We investigated the molecular mechanisms of adenosine and cordycepin for their promoting effects in wound-healing process. The mitochondrial energy metabolism and cell proliferation markers, cAMP responsive element binding protein 1 (CREB1) and Ki67, were enhanced by adenosine and cordycepin in cultured dermal fibroblasts. Adenosine and cordycepin stimulated adenosine receptor signaling via elevated cAMP. The phosphorylation of mitogen-activated protein kinase kinase (MEK) 1/2, mammalian target of rapamycin (mTOR) and glycogen synthase kinase 3 beta (Gsk3b) and Wnt target genes such as bone morphogenetic protein (BMP) 2/4 and lymphoid enhancer binding factor (Lef) 1 were activated. The enhanced gene expression by adenosine and cordycepin was abrogated by adenosine A_2A and A_2B receptor inhibitors, ZM241385 and PSH603, and protein kinase A (PKA) inhibitor H89, indicating the involvement of adenosine receptor A_2A, A_2B and PKA. As a result of Wnt/β-catenin pathway activation, the secretion of growth factors such as insulin-like growth factor (IGF)-1 and transforming growth factor beta (TGFβ) 3 was increased, previously reported to facilitate the wound healing process. In addition, in vitro fibroblast migration was also increased, demonstrating their possible roles in facilitating the wound healing process. In conclusion, our data strongly demonstrate that adenosine and cordycepin stimulate the Wnt/β-catenin signaling through the activation of adenosine receptor, possibly promoting the tissue remodeling process and suggest their therapeutic potential for treating skin wounds.     

Distinct Functional Alterations and Therapeutic Options of Two Pathological De Novo Variants of the T292 Residue of GABRA1 Identified in Children with Epileptic Encephalopathy and Neurodevelopmental Disorders

Mutations of GABA_AR have reportedly led to epileptic encephalopathy and neurodevelopmental disorders. We have identified a novel de novo T292S missense variant of GABRA1 from a pediatric patient with grievous global developmental delay but without obvious epileptic activity. This mutation coincidentally occurs at the same residue as that of a previously reported GABRA1 variant T292I identified from a pediatric patient with severe epilepsy. The distinct phenotypes of these two patients prompted us to compare the impacts of the two mutants on the receptor function and to search for suitable therapeutics. In this study, we used biochemical techniques and patch-clamp recordings in HEK293 cells overexpressing either wild-type or mutated rat recombinant GABA_ARs. We found that the α1T292S variant significantly increased GABA-evoked whole-cell currents, shifting the dose–response curve to the left without altering the maximal response. In contrast, the α1T292I variant significantly reduced GABA-evoked currents, shifting the dose–response curve to the right with a severely diminished maximum response. Single-channel recordings further revealed that the α1T292S variant increased, while the α1T292I variant decreased the GABA_AR single-channel open time and open probability. Importantly, we found that the T292S mutation-induced increase in GABA_AR function could be fully normalized by the negative GABA_AR modulator thiocolchicoside, whereas the T292I mutation-induced impairment of GABA_AR function was largely rescued with a combination of the GABA_AR positive modulators diazepam and verapamil. Our study demonstrated that α1T292 is a critical residue for controlling GABA_AR channel gating, and mutations at this residue may produce opposite impacts on the function of the receptors. Thus, the present work highlights the importance of functionally characterizing each individual GABA_AR mutation for ensuring precision medicine.     

Synthesis,Pharmacological Evaluation,and Docking Studies of Novel Pyridazinone‐Based Cannabinoid Receptor Type 2 Ligands

In recent years, cannabinoid type 2 receptors (CB₂R) have emerged as promising therapeutic targets in a wide variety of diseases. Selective ligands of CB₂R are devoid of the psychoactive effects typically observed for CB₁R ligands. Based on our recent studies on a class of pyridazinone 4‐carboxamides, further structural modifications of the pyridazinone core were made to better investigate the structure–activity relationships for this promising scaffold with the aim to develop potent CB₂R ligands. In binding assays, two of the new synthesized compounds [6‐(3,4‐dichlorophenyl)‐2‐(4‐fluorobenzyl)‐cis‐N‐(4‐methylcyclohexyl)‐3‐oxo‐2,3‐dihydropyridazine‐4‐carboxamide ( 2 ) and 6‐(4‐chloro‐3‐methylphenyl)‐cis‐N‐(4‐methylcyclohexyl)‐3‐oxo‐2‐pentyl‐2,3‐dihydropyridazine‐4‐carboxamide ( 22 )] showed high CB₂R affinity, with K_i values of 2.1 and 1.6 nm , respectively. In addition, functional assays of these compounds and other new active related derivatives revealed their pharmacological profiles as CB₂R inverse agonists. Compound 22 displayed the highest CB₂R selectivity and potency, presenting a favorable in silico pharmacokinetic profile. Furthermore, a molecular modeling study revealed how 22 produces inverse agonism through blocking the movement of the toggle‐switch residue, W6.48.     

Regulator of G Protein Signalling 4 (RGS4) as a Novel Target for the Treatment of Sensorineural Hearing Loss

We and others have previously identified signalling pathways associated with the adenosine A₁ receptor (A₁R) as important regulators of cellular responses to injury in the cochlea. We have shown that the “post-exposure” treatment with adenosine A₁R agonists confers partial protection against acoustic trauma and other forms of sensorineural hearing loss (SNHL). The aim of this study was to determine if increasing A₁R responsiveness to endogenous adenosine would have the same otoprotective effect. This was achieved by pharmacological targeting of the Regulator of G protein Signalling 4 (RGS4). RGS proteins inhibit signal transduction pathways initiated by G protein-coupled receptors (GPCR) by enhancing GPCR deactivation and receptor desensitisation. A molecular complex between RGS4 and neurabin, an intracellular scaffolding protein expressed in neural and cochlear tissues, is the key negative regulator of A₁R activity in the brain. In this study, Wistar rats (6–8 weeks) were exposed to traumatic noise (110 dBSPL, 8–16 kHz) for 2 h and a small molecule RGS4 inhibitor CCG-4986 was delivered intratympanically in a Poloxamer-407 gel formulation for sustained drug release 24 or 48 h after noise exposure. Intratympanic administration of CCG-4986 48 h after noise exposure attenuated noise-induced permanent auditory threshold shifts by up to 19 dB, whilst the earlier drug administration (24 h) led to even better preservation of auditory thresholds (up to 32 dB). Significant improvement of auditory thresholds and suprathreshold responses was linked to improved survival of sensorineural tissues and afferent synapses in the cochlea. Our studies thus demonstrate that intratympanic administration of CCG-4986 can rescue cochlear injury and hearing loss induced by acoustic overexposure. This research represents a novel paradigm for the treatment of various forms of SNHL based on regulation of GPCR.     

Effect of Linker Length and Composition on Heterobivalent Ligand‐Mediated Receptor Cross‐Talk between the A1 Adenosine and β2 Adrenergic Receptors

Heterobivalent ligands that possess pharmacophores designed to interact with both the A₁ adenosine receptor (A₁AR) and the β₂ adrenergic receptor (β₂AR) were prepared. More specifically, these ligands contain an adenosine moiety that is linked via its N⁶‐position to the amino group of the saligenin‐substituted ethanolamine moiety present in the well‐known β₂AR agonist, salbutamol. The affinities of these ligands were determined at both receptors and found to vary with linker length and composition. With all compounds, affinity and functional potencies were found to have selectivity for the A₁AR over the β₂AR. In all cases, cAMP accumulation (a β₂AR‐mediated response) was mainly observed when the A₁AR was blocked or its function decreased by pertussis toxin or chronic agonist treatment. This suggests that heterobivalent compounds for receptors that mediate opposite responses might be useful for elucidating the mechanisms of receptor cross‐talk and how this interaction, in terms of responsiveness, may change under pathophysiological conditions.     

Structure,Dynamics, and Ligand Recognition of Human-Specific CHRFAM7A (Dupα7) Nicotinic Receptor Linked to Neuropsychiatric Disorders

Cholinergic α7 nicotinic receptors encoded by the CHRNA7 gene are ligand-gated ion channels directly related to memory and immunomodulation. Exons 5–7 in CHRNA7 can be duplicated and fused to exons A-E of FAR7a, resulting in a hybrid gene known as CHRFAM7A, unique to humans. Its product, denoted herein as Dupα7, is a truncated subunit where the N-terminal 146 residues of the ligand binding domain of the α7 receptor have been replaced by 27 residues from FAM7. Dupα7 negatively affects the functioning of α7 receptors associated with neurological disorders, including Alzheimer’s diseases and schizophrenia. However, the stoichiometry for the α7 nicotinic receptor containing dupα7 monomers remains unknown. In this work, we developed computational models of all possible combinations of wild-type α7 and dupα7 pentamers and evaluated their stability via atomistic molecular dynamics and coarse-grain simulations. We assessed the effect of dupα7 subunits on the Ca²⁺ conductance using free energy calculations. We showed that receptors comprising of four or more dupα7 subunits are not stable enough to constitute a functional ion channel. We also showed that models with dupα7/α7 interfaces are more stable and are less detrimental for the ion conductance in comparison to dupα7/dupα7 interfaces. Based on these models, we used protein–protein docking to evaluate how such interfaces would interact with an antagonist, α-bungarotoxin, and amyloid Aβ₄₂. Our findings show that the optimal stoichiometry of dupα7/α7 functional pentamers should be no more than three dupα7 monomers, in favour of a dupα7/α7 interface in comparison to a homodimer dupα7/dupα7 interface. We also showed that receptors bearing dupα7 subunits are less sensitive to Aβ₄₂ effects, which may shed light on the translational gap reported for strategies focused on nicotinic receptors in ‘Alzheimer’s disease research.     

Living-Cell Diffracted X-ray Tracking Analysis Confirmed Internal Salt Bridge Is Critical for Ligand-Induced Twisting Motion of Serotonin Receptors

Serotonin receptors play important roles in neuronal excitation, emotion, platelet aggregation, and vasoconstriction. The serotonin receptor subtype 2A (5-HT_2AR) is a Gq-coupled GPCR, which activate phospholipase C. Although the structures and functions of 5-HT_2ARs have been well studied, little has been known about their real-time dynamics. In this study, we analyzed the intramolecular motion of the 5-HT_2AR in living cells using the diffracted X-ray tracking (DXT) technique. The DXT is a very precise single-molecular analytical technique, which tracks diffraction spots from the gold nanocrystals labeled on the protein surface. Trajectory analysis provides insight into protein dynamics. The 5-HT_2ARs were transiently expressed in HEK 293 cells, and the gold nanocrystals were attached to the N-terminal introduced FLAG-tag via anti-FLAG antibodies. The motions were recorded with a frame rate of 100 μs per frame. A lifetime filtering technique demonstrated that the unliganded receptors contain high mobility population with clockwise twisting. This rotation was, however, abolished by either a full agonist α-methylserotonin or an inverse agonist ketanserin. Mutation analysis revealed that the “ionic lock” between the DRY motif in the third transmembrane segment and a negatively charged residue of the sixth transmembrane segment is essential for the torsional motion at the N-terminus of the receptor.