The Minimum M3-M4 Loop Length of Neurotransmitter-activated Pentameric Receptors Is Critical for the Structural Integrity of Cytoplasmic Portals

The 5-HT₃A receptor homology model, based on the partial structure of the nicotinic acetylcholine receptor from Torpedo marmorata, reveals an asymmetric ion channel with five portals framed by adjacent helical amphipathic (HA) stretches within the 114-residue loop between the M3 and M4 membrane-spanning domains. The positive charge of Arg-436, located within the HA stretch, is a rate-limiting determinant of single channel conductance (γ). Further analysis reveals that positive charge and volume of residue 436 are determinants of 5-HT₃A receptor inward rectification, exposing an additional role for portals. A structurally unresolved stretch of 85 residues constitutes the bulk of the M3-M4 loop, leaving a >45-Å gap in the model between M3 and the HA stretch. There are no additional structural data for this loop, which is vestigial in bacterial pentameric ligand-gated ion channels and was largely removed for crystallization of the Caenorhabditis elegans glutamate-activated pentameric ligand-gated ion channels. We created 5-HT3A subunit loop truncation mutants, in which sequences framing the putative portals were retained, to determine the minimum number of residues required to maintain their functional integrity. Truncation to between 90 and 75 amino acids produced 5-HT₃A receptors with unaltered rectification. Truncation to 70 residues abolished rectification and increased γ. These findings reveal a critical M3-M4 loop length required for functions attributable to cytoplasmic portals. Examination of all 44 subunits of the human neurotransmitter-activated Cys-loop receptors reveals that, despite considerable variability in their sequences and lengths, all M3-M4 loops exceed 70 residues, suggesting a fundamental requirement for portal integrity.     

Molecular Basis of Cannabinoid CB1 Receptor Coupling to the G Protein Heterotrimer Gαiβγ: IDENTIFICATION OF KEY CB1 CONTACTS WITH THE C-TERMINAL HELIX α5 OF Gαi*

The cannabinoid (CB1) receptor is a member of the rhodopsin-like G protein-coupled receptor superfamily. The human CB1 receptor, which is among the most expressed receptors in the brain, has been implicated in several disease states, including drug addiction, anxiety, depression, obesity, and chronic pain. Different classes of CB1 agonists evoke signaling pathways through the activation of specific subtypes of G proteins. The molecular basis of CB1 receptor coupling to its cognate G protein is unknown. As a first step toward understanding CB1 receptor-mediated G protein signaling, we have constructed a ternary complex structural model of the CB1 receptor and G_i heterotrimer (CB1-G_i), guided by the x-ray structure of β₂-adrenergic receptor (β₂AR) in complex with G_s (β₂AR-G_s), through 824-ns duration molecular dynamics simulations in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer environment. We identified a group of residues at the juxtamembrane regions of the intracellular loops 2 and 3 (IC2 and IC3) of the CB1 receptor, including Ile-218^3.54, Tyr-224^IC2, Asp-338^6.30, Arg-340^6.32, Leu-341^6.33, and Thr-344^6.36, as potential key contacts with the extreme C-terminal helix α₅ of Gα_i. Ala mutations of these residues at the receptor-G_i interface resulted in little G protein coupling activity, consistent with the present model of the CB1-G_i complex, which suggests tight interactions between CB1 and the extreme C-terminal helix α₅ of Gα_i. The model also suggests that unique conformational changes in the extreme C-terminal helix α₅ of Gα play a crucial role in the receptor-mediated G protein activation.     

Subunit Symmetry at the Extracellular Domain-Transmembrane Domain Interface in Acetylcholine Receptor Channel Gating

Transmitter molecules bind to synaptic acetylcholine receptor channels (AChRs) to promote a global channel-opening conformational change. Although the detailed mechanism that links ligand binding and channel gating is uncertain, the energy changes caused by mutations appear to be more symmetrical between subunits in the transmembrane domain compared with the extracellular domain. The only covalent connection between these domains is the pre-M1 linker, a stretch of five amino acids that joins strand β10 with the M1 helix. In each subunit, this linker has a central Arg (Arg^3′), which only in the non-α-subunits is flanked by positively charged residues. Previous studies showed that mutations of Arg^3′ in the α-subunit alter the gating equilibrium constant and reduce channel expression. We recorded single-channel currents and estimated the gating rate and equilibrium constants of adult mouse AChRs with mutations at the pre-M1 linker and the nearby residue Glu⁴⁵ in non-α-subunits. In all subunits, mutations of Arg^3′ had similar effects as in the α-subunit. In the ϵ-subunit, mutations of the flanking residues and Glu⁴⁵ had only small effects, and there was no energy coupling between ϵGlu⁴⁵ and ϵArg^3′. The non-α-subunit Arg^3′ residues had Φ-values that were similar to those for the α-subunit. The results suggest that there is a general symmetry between the AChR subunits during gating isomerization in this linker and that the central Arg is involved in expression more so than gating. The energy transfer through the AChR during gating appears to mainly involve Glu⁴⁵, but only in the α-subunits.     

Defining the roles of Asn-128, Glu-129 and Phe-130 in loop A of the 5-HT3 receptor

The ligand binding pocket of Cys-loop receptors consists of a number of binding loops termed A–F. Here we examine the 5-HT₃ receptor loop A residues Asn-128, Glu-129 and Phe-130 using modelling, mutagenesis, radioligand binding and functional studies on HEK 293 cells. Replacement of Asn-128 results in receptors that have wild type [³H]granisetron binding characteristics but large changes (ranging from a five-fold decrease to a 1500-fold increase) in the 5-HT EC₅₀ when compared to wild type receptors. Phe-130 mutant receptors show both increases and decreases in K_d and EC₅₀ values, depending on the amino acid substituted. The most critical of these residues appears to be Glu-129; its replacement with a range of other amino acids results in non-binding and non-functional receptors. Lack of binding and function in some, but not all, of these receptors is due to poor membrane expression. These data suggest that Glu-129 is important primarily for receptor expression, although it may also play a role in ligand binding; Phe-130 is important for both ligand binding and receptor function, and Asn-128 plays a larger role in receptor function than ligand binding. In light of these results, we have created two new homology models of the 5-HT₃ receptor, with alternative positions of loop A. In our preferred model Glu-129 and Phe-130 contribute to the binding site, while the location of Asn-128 immediately behind the binding pocket could contribute to the conformation changes that result in receptor gating. This study provides a new model of the 5-HT₃ receptor binding pocket, and also highlights the importance of experimental data to support modelling studies.     

5-HT3 receptor ion size selectivity is a property of the transmembrane channel, not the cytoplasmic vestibule portals

5-HT3A receptors select among permeant ions based on size and charge. The membrane-associated (MA) helix lines the portals into the channel’s cytoplasmic vestibule in the 4-Å resolution structure of the homologous acetylcholine receptor. 5-HT3A MA helix residues are important determinants of single-channel conductance. It is unknown whether the portals into the cytoplasmic vestibule also determine the size selectivity of permeant ions. We sought to determine whether the portals form the size selectivity filter. Recently, we showed that channels functioned when the entire 5-HT3A M3–M4 loop was replaced by the heptapeptide M3–M4 loop sequence from GLIC, a bacterial Cys-loop neurotransmitter gated ion channel homologue from Gloebacter violaceus. We used homomeric 5-HT3A receptors with either a wild-type (WT) M3–M4 loop or the chimeric heptapeptide (5-HT3A–glvM3M4) loop, i.e., with or without portals. In Na⁺-containing buffer, the WT receptor current–voltage relationship was inwardly rectifying. In contrast, the 5-HT3A–glvM3M4 construct had a negative slope conductance region at voltages less than −80 mV. Glutamine substitution for the heptapeptide M3–M4 loop arginine eliminated the negative slope conductance region. We measured the relative permeabilities and conductances of a series of inorganic and organic cations ranging from 0.9 to 4.5 Å in radius (Li⁺, Na⁺, ammonium, methylammonium, ethanolammonium, 2-methylethanolammonium, dimethylammonium, diethanolammonium, tetramethylammonium, choline, tris [hydroxymethyl] aminomethane, and N-methyl-d-glucamine). Both constructs had measurable conductances with Li⁺, ammonium, and methylammonium (size range of 0.9–1.8-Å radius). Many of the organic cations >2.4 Å acted as competitive antagonists complicating measurement of conductance ratios. Analysis of the permeability ratios by excluded volume theory indicates that the minimal pore radius for 5-HT3A and 5-HT3–glvM3M4 receptors was similar, ∼5 Å. We infer that the 5-HT3A size selectivity filter is located in the transmembrane channel and not in the portals into the cytoplasmic vestibule. Thus, the determinants of size selectivity and conductance are located in physically distinct regions of the channel protein.     

Agonists and Antagonists Bind to an A-A Interface in the Heteromeric 5-HT3AB Receptor

The 5-HT₃ receptor is a member of the Cys-loop family of transmitter receptors. It can function as a homopentamer (5-HT3A-only subunits) or as a heteropentamer. The 5-HT₃AB receptor is the best characterized heteropentamer. This receptor differs from a homopentamer in its kinetics, voltage dependence, and single-channel conductance, but its pharmacology is similar. To understand the contribution of the 5-HT3B subunit to the binding site, we created homology models of 5-HT₃AB receptors and docked 5-HT and granisetron into AB, BA, and BB interfaces. To test whether ligands bind in any or all of these interfaces, we mutated amino acids that are important for agonist and antagonist binding in the 5-HT3A subunit to their corresponding residues in the 5-HT3B subunit and vice versa. Changes in [³H]granisetron binding affinity (K_d) and 5-HT EC₅₀ were determined using receptors expressed in HEK-293 cells and Xenopus oocytes, respectively. For all A-to-B mutant receptors, except T181N, antagonist binding was altered or eliminated. Functional studies revealed that either the receptors were nonfunctional or the EC₅₀ values were increased. In B-to-A mutant receptors there were no changes in K_d, although EC₅₀ values and Hill slopes, except for N170T mutant receptors, were similar to those for 5-HT₃A receptors. Thus, the experimental data do not support a contribution of the 5-HT3B subunit to the binding pocket, and we conclude that both 5-HT and granisetron bind to an AA binding site in the heteromeric 5-HT₃AB receptor.     

Glycosylation of β2 Subunits Regulates GABAA Receptor Biogenesis and Channel Gating

γ-Aminobutyric acid type A (GABA_A) receptors are heteropentameric glycoproteins. Based on consensus sequences, the GABA_A receptor β2 subunit contains three potential N-linked glycosylation sites, Asn-32, Asn-104, and Asn-173. Homology modeling indicates that Asn-32 and Asn-104 are located before the α1 helix and in loop L3, respectively, near the top of the subunit-subunit interface on the minus side, and that Asn-173 is located in the Cys-loop near the bottom of the subunit N-terminal domain. Using site-directed mutagenesis, we demonstrated that all predicted β2 subunit glycosylation sites were glycosylated in transfected HEK293T cells. Glycosylation of each site, however, produced specific changes in α1β2 receptor surface expression and function. Although glycosylation of Asn-173 in the Cys-loop was important for stability of β2 subunits when expressed alone, results obtained with flow cytometry, brefeldin A treatment, and endo-β-N-acetylglucosaminidase H digestion suggested that glycosylation of Asn-104 was required for efficient α1β2 receptor assembly and/or stability in the endoplasmic reticulum. Patch clamp recording revealed that mutation of each site to prevent glycosylation decreased peak α1β2 receptor current amplitudes and altered the gating properties of α1β2 receptor channels by reducing mean open time due to a reduction in the proportion of long open states. In addition to functional heterogeneity, endo-β-N-acetylglucosaminidase H digestion and glycomic profiling revealed that surface β2 subunit N-glycans at Asn-173 were high mannose forms that were different from those of Asn-32 and N104. Using a homology model of the pentameric extracellular domain of α1β2 channel, we propose mechanisms for regulation of GABA_A receptors by glycosylation.     

Identification of Three Residues Essential for 5-Hydroxytryptamine 2A-Metabotropic Glutamate 2 (5-HT2A·mGlu2) Receptor Heteromerization and Its Psychoactive Behavioral Function

Serotonin and glutamate G protein-coupled receptor (GPCR) neurotransmission affects cognition and perception in humans and rodents. GPCRs are capable of forming heteromeric complexes that differentially alter cell signaling, but the role of this structural arrangement in modulating behavior remains unknown. Here, we identified three residues located at the intracellular end of transmembrane domain four that are necessary for the metabotropic glutamate 2 (mGlu2) receptor to be assembled as a GPCR heteromer with the serotonin 5-hydroxytryptamine 2A (5-HT_2A) receptor in the mouse frontal cortex. Substitution of these residues (Ala-677^4.40, Ala-681^4.44, and Ala-685^4.48) leads to absence of 5-HT_2A·mGlu2 receptor complex formation, an effect that is associated with a decrease in their heteromeric ligand binding interaction. Disruption of heteromeric expression with mGlu2 attenuates the psychosis-like effects induced in mice by hallucinogenic 5-HT_2A agonists. Furthermore, the ligand binding interaction between the components of the 5-HT_2A·mGlu2 receptor heterocomplex is up-regulated in the frontal cortex of schizophrenic subjects as compared with controls. Together, these findings provide structural evidence for the unique behavioral function of a GPCR heteromer.     

A Dynamic View of Molecular Switch Behavior at Serotonin Receptors: Implications for Functional Selectivity

Functional selectivity is a property of G protein-coupled receptors that allows them to preferentially couple to particular signaling partners upon binding of biased agonists. Publication of the X-ray crystal structure of serotonergic 5-HT_1B and 5-HT_2B receptors in complex with ergotamine, a drug capable of activating G protein coupling and β-arrestin signaling at the 5-HT_1B receptor but clearly favoring β-arrestin over G protein coupling at the 5-HT_2B subtype, has recently provided structural insight into this phenomenon. In particular, these structures highlight the importance of specific residues, also called micro-switches, for differential receptor activation. In our work, we apply classical molecular dynamics simulations and enhanced sampling approaches to analyze the behavior of these micro-switches and their impact on the stabilization of particular receptor conformational states. Our analysis shows that differences in the conformational freedom of helix 6 between both receptors could explain their different G protein-coupling capacity. In particular, as compared to the 5-HT_1B receptor, helix 6 movement in the 5-HT_2B receptor can be constrained by two different mechanisms. On the one hand, an anchoring effect of ergotamine, which shows an increased capacity to interact with the extracellular part of helices 5 and 6 and stabilize them, hinders activation of a hydrophobic connector region at the center of the receptor. On the other hand, this connector region in an inactive conformation is further stabilized by unconserved contacts extending to the intracellular part of the 5-HT_2B receptor, which hamper opening of the G protein binding site. This work highlights the importance of considering receptor capacity to adopt different conformational states from a dynamic perspective in order to underpin the structural basis of functional selectivity.     

5-HT3 Receptors

5-Hydroxytryptamine type 3 (5-HT₃) receptors are cation-selective Cys loop receptors found in both the central and peripheral nervous systems. There are five 5-HT₃ receptor subunits (A–E), and all functional receptors require at least one A subunit. Regions from noncontiguous parts of the subunit sequence contribute to the agonist-binding site, and the roles of a range of amino acid residues that form the binding pocket have been identified. Drugs that selectively antagonize 5-HT₃ receptors (the “setrons”) are the current gold standard for treatment of chemotherapy-induced and postoperative nausea and vomiting and have potential for the treatment of a range of other conditions.     

Identification of Propofol Binding Sites in a Nicotinic Acetylcholine Receptor with a Photoreactive Propofol Analog

Propofol, a widely used intravenous general anesthetic, acts at anesthetic concentrations as a positive allosteric modulator of γ-aminobutyric acid type A receptors and at higher concentration as an inhibitor of nicotinic acetylcholine receptors (nAChRs). Here, we characterize propofol binding sites in a muscle-type nAChR by use of a photoreactive analog of propofol, 2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol (AziPm). Based upon radioligand binding assays, AziPm stabilized the Torpedo nAChR in the resting state, whereas propofol stabilized the desensitized state. nAChR-rich membranes were photolabeled with [³H]AziPm, and labeled amino acids were identified by Edman degradation. [³H]AziPm binds at three sites within the nAChR transmembrane domain: (i) an intrasubunit site in the δ subunit helix bundle, photolabeling in the nAChR desensitized state (+agonist) δM2-18′ and two residues in δM1 (δPhe-232 and δCys-236); (ii) in the ion channel, photolabeling in the nAChR resting, closed channel state (−agonist) amino acids in the M2 helices (αM2-6′, βM2-6′ and -13′, and δM2-13′) that line the channel lumen (with photolabeling reduced by >90% in the desensitized state); and (iii) at the γ-α interface, photolabeling αM2-10′. Propofol enhanced [³H]AziPm photolabeling at αM2-10′. Propofol inhibited [³H]AziPm photolabeling within the δ subunit helix bundle at lower concentrations (IC₅₀ = 40 μm) than it inhibited ion channel photolabeling (IC₅₀ = 125 μm). These results identify for the first time a single intrasubunit propofol binding site in the nAChR transmembrane domain and suggest that this is the functionally relevant inhibitory binding site.     

G-protein Receptor Kinase 5 Regulates the Cannabinoid Receptor 2-induced Up-regulation of Serotonin 2A Receptors

We have recently reported that cannabinoid agonists can up-regulate and enhance the activity of serotonin 2A (5-HT_2A) receptors in the prefrontal cortex (PFCx). Increased expression and activity of cortical 5-HT_2A receptors has been associated with neuropsychiatric disorders, such as anxiety and schizophrenia. Here we report that repeated CP55940 exposure selectively up-regulates GRK5 proteins in rat PFCx and in a neuronal cell culture model. We sought to examine the mechanism underlying the regulation of GRK5 and to identify the role of GRK5 in the cannabinoid agonist-induced up-regulation and enhanced activity of 5-HT_2A receptors. Interestingly, we found that cannabinoid agonist-induced up-regulation of GRK5 involves CB₂ receptors, β-arrestin 2, and ERK1/2 signaling because treatment with CB₂ shRNA lentiviral particles, β-arrestin 2 shRNA lentiviral particles, or ERK1/2 inhibitor prevented the cannabinoid agonist-induced up-regulation of GRK5. Most importantly, we found that GRK5 shRNA lentiviral particle treatment prevented the cannabinoid agonist-induced up-regulation and enhanced 5-HT_2A receptor-mediated calcium release. Repeated cannabinoid exposure was also associated with enhanced phosphorylation of CB₂ receptors and increased interaction between β-arrestin 2 and ERK1/2. These latter phenomena were also significantly inhibited by GRK5 shRNA lentiviral treatment. Our results suggest that sustained activation of CB₂ receptors, which up-regulates 5-HT_2A receptor signaling, enhances GRK5 expression; the phosphorylation of CB₂ receptors; and the β-arrestin 2/ERK interactions. These data could provide a rationale for some of the adverse effects associated with repeated cannabinoid agonist exposure.     

Common determinants of single channel conductance within the large cytoplasmic loop of 5-hydroxytryptamine type 3 and alpha4beta2 nicotinic acetylcholine receptors

Homomeric 5-hydroxytryptamine type 3A receptors (5-HT3ARs) have a single channel conductance (gamma) below the resolution of single channel recording (966 +/- 75 fS, estimated by variance analysis). By contrast, heteromeric 5-HT3A/B and nicotinic acetylcholine receptors (nAChRs) have picosiemen range gamma values. In this study, single channel recordings revealed that replacement of cytoplasmic membrane-associated (MA) helix arginine 432 (-4'), 436 (0'), and 440 (4') residues by 5-HT3B (-4'Gln, 0'Asp, and 4'Ala) residues increases gamma to 36.5 +/- 1.0 pS. The 0' residue makes the most substantial contribution to gamma of the 5-HT3AR. Replacement of 0'Arg by aspartate, glutamate (alpha7 nAChR subunit MA 0'), or glutamine (beta2 subunit MA 0') increases gamma to the resolvable range (>6 pS). By contrast, replacement of 0'Arg by phenylalanine (alpha4 subunit MA 0') reduced gamma to 416 +/- 107 fS. In reciprocal experiments with alpha4beta2 nAChRs (gamma = 31.3 +/- 0.8 pS), replacement of MA 0' residues by arginine in alpha4beta2(Q443R) and alpha4(F588R)beta2 reduced gamma slightly. By contrast, the gamma of double mutant alpha4(F588R)beta2(Q443R) was halved. The MA -4' and 4' residues also influenced gamma of 5-HT3ARs. Replacement of nAChR alpha4 or beta2 MA 4' residues by arginine made current density negligible. By contrast, replacement of both -4' residues by arginine produced functional nAChRs with substantially reduced gamma (11.4 +/- 0.5 pS). Homology models of the 5-HT3A and alpha4beta2 nAChRs against Torpedo nAChR revealed MA -4', 0', and 4' residues within five intracellular portals. This locus may be a common determinant of ion conduction throughout the Cys loop receptor family.     

Molecular and Pharmacological Characterization of Serotonin 5-HT2α and 5-HT7 Receptors in the Salivary Glands of the Blowfly Calliphora vicina

Secretion in blowfly (Calliphora vicina) salivary glands is stimulated by the biogenic amine serotonin (5-hydroxytryptamine, 5-HT), which activates both inositol 1,4,5-trisphosphate (InsP₃)/Ca²⁺ and cyclic adenosine 3′,5′-monophosphate (cAMP) signalling pathways in the secretory cells. In order to characterize the signal-inducing 5-HT receptors, we cloned two cDNAs (Cv5-ht2α, Cv5-ht7) that share high similarity with mammalian 5-HT₂ and 5-HT₇ receptor genes, respectively. RT-PCR demonstrated that both receptors are expressed in the salivary glands and brain. Stimulation of Cv5-ht2α-transfected mammalian cells with 5-HT elevates cytosolic [Ca²⁺] in a dose-dependent manner (EC₅₀ = 24 nM). In Cv5-ht7-transfected cells, 5-HT produces a dose-dependent increase in [cAMP]_i (EC₅₀ = 4 nM). We studied the pharmacological profile for both receptors. Substances that appear to act as specific ligands of either Cv5-HT_2α or Cv5-HT₇ in the heterologous expression system were also tested in intact blowfly salivary gland preparations. We observed that 5-methoxytryptamine (100 nM) activates only the Cv5-HT_2α receptor, 5-carboxamidotryptamine (300 nM) activates only the Cv5-HT₇ receptor, and clozapine (1 µM) antagonizes the effects of 5-HT via Cv5-HT₇ in blowfly salivary glands, providing means for the selective activation of each of the two 5-HT receptor subtypes. This study represents the first comprehensive molecular and pharmacological characterization of two 5-HT receptors in the blowfly and permits the analysis of the physiological role of these receptors, even when co-expressed in cells, and of the modes of interaction between the Ca²⁺- and cAMP-signalling cascades.     

Physical interaction of calmodulin with the 5-hydroxytryptamine2C receptor C-terminus is essential for G protein-independent, arrestin-dependent receptor signaling

The serotonin (5-hydroxytryptamine; 5-HT)_2C receptor is a G protein-coupled receptor (GPCR) exclusively expressed in CNS that has been implicated in numerous brain disorders, including anxio-depressive states. Like many GPCRs, 5-HT_2C receptors physically interact with a variety of intracellular proteins in addition to G proteins. Here, we show that calmodulin (CaM) binds to a prototypic Ca²⁺-dependent “1-10” CaM-binding motif located in the proximal region of the 5-HT_2C receptor C-terminus upon receptor activation by 5-HT. Mutation of this motif inhibited both β-arrestin recruitment by 5-HT_2C receptor and receptor-operated extracellular signal-regulated kinase (ERK) 1,2 signaling in human embryonic kidney-293 cells, which was independent of G proteins and dependent on β-arrestins. A similar inhibition was observed in cells expressing a dominant-negative CaM or depleted of CaM by RNA interference. Expression of the CaM mutant also prevented receptor-mediated ERK1,2 phosphorylation in cultured cortical neurons and choroid plexus epithelial cells that endogenously express 5-HT_2C receptors. Collectively, these findings demonstrate that physical interaction of CaM with recombinant and native 5-HT_2C receptors is critical for G protein-independent, arrestin-dependent receptor signaling. This signaling pathway might be involved in neurogenesis induced by chronic treatment with 5-HT_2C receptor agonists and their antidepressant-like activity.     

Serotonin 5-HT3 Receptor-Mediated Vomiting Occurs via the Activation of Ca2+/CaMKII-Dependent ERK1/2 Signaling in the Least Shrew (Cryptotis parva)

Stimulation of 5-HT₃ receptors (5-HT₃Rs) by 2-methylserotonin (2-Me-5-HT), a selective 5-HT₃ receptor agonist, can induce vomiting. However, downstream signaling pathways for the induced emesis remain unknown. The 5-HT₃R channel has high permeability to extracellular calcium (Ca²⁺) and upon stimulation allows increased Ca²⁺ influx. We examined the contribution of Ca²⁺/calmodulin-dependent protein kinase IIα (Ca²⁺/CaMKIIα), interaction of 5-HT₃R with calmodulin, and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling to 2-Me-5-HT-induced emesis in the least shrew. Using fluo-4 AM dye, we found that 2-Me-5-HT augments intracellular Ca²⁺ levels in brainstem slices and that the selective 5-HT₃R antagonist palonosetron, can abolish the induced Ca²⁺ signaling. Pre-treatment of shrews with either: i) amlodipine, an antagonist of L-type Ca²⁺ channels present on the cell membrane; ii) dantrolene, an inhibitor of ryanodine receptors (RyRs) Ca²⁺-release channels located on the endoplasmic reticulum (ER); iii) a combination of their less-effective doses; or iv) inhibitors of CaMKII (KN93) and ERK1/2 (PD98059); dose-dependently suppressed emesis caused by 2-Me-5-HT. Administration of 2-Me-5-HT also significantly: i) enhanced the interaction of 5-HT₃R with calmodulin in the brainstem as revealed by immunoprecipitation, as well as their colocalization in the area postrema (brainstem) and small intestine by immunohistochemistry; and ii) activated CaMKIIα in brainstem and in isolated enterochromaffin cells of the small intestine as shown by Western blot and immunocytochemistry. These effects were suppressed by palonosetron. 2-Me-5-HT also activated ERK1/2 in brainstem, which was abrogated by palonosetron, KN93, PD98059, amlodipine, dantrolene, or a combination of amlodipine plus dantrolene. However, blockade of ER inositol-1, 4, 5-triphosphate receptors by 2-APB, had no significant effect on the discussed behavioral and biochemical parameters. This study demonstrates that Ca²⁺ mobilization via extracellular Ca²⁺ influx through 5-HT₃Rs/L-type Ca²⁺ channels, and intracellular Ca²⁺ release via RyRs on ER, initiate Ca²⁺-dependent sequential activation of CaMKIIα and ERK1/2, which contribute to the 5-HT₃R-mediated, 2-Me-5-HT-evoked emesis.     

Influence of Cholesterol and Its Stereoisomers on Members of the Serotonin Receptor Family

Despite the ubiquity of cholesterol within the cell membrane, the mechanism by which it influences embedded proteins remains elusive. Numerous G-protein coupled receptors exhibit dramatic responses to membrane cholesterol with regard to the ligand-binding affinity and functional properties, including the 5-HT receptor family. Here, we use over 25 μs of unbiased atomistic molecular dynamics simulations to identify cholesterol interaction sites in the 5-HT_1B and 5-HT_2B receptors and evaluate their impact on receptor structure. Susceptibility to membrane cholesterol is shown to be subtype dependent and determined by the quality of interactions between the extracellular loops. Charged residues are essential for maintaining the arrangement of the extracellular surface in 5-HT_2B; in the absence of such interactions, the extracellular surface of the 5-HT_1B is malleable, populating a number of distinct conformations. Elevated cholesterol density near transmembrane helix 4 is considered to be conducive to the conformation of extracellular loop 2. Occupation of this site is also shown to be stereospecific, illustrated by differential behavior of nat-cholesterol isomers, ent- and epi-cholesterol. In simulations containing the endogenous agonist, serotonin, cholesterol binding at transmembrane helix 4 biases bound serotonin molecules toward an unexpected binding mode in the extended binding pocket. The results highlight the capability of membrane cholesterol to influence the mobility of the extracellular surface in the 5-HT₁ receptor family and manipulate the architecture of the extracellular ligand-binding pocket.     

A Residue in Loop 9 of the ��2-Subunit Stabilizes the Closed State of the GABAA Receptor

In γ-aminobutyric acid type A (GABA_A) receptors, the structural elements that couple ligand binding to channel opening remain poorly defined. Here, site-directed mutagenesis was used to determine if Loop 9 on the non-GABA binding site interface of the β2-subunit may be involved in GABA_A receptor activation. Specifically, residues Gly¹⁷⁰-Gln¹⁸⁵ of the β2-subunit were mutated to alanine, co-expressed with wild-type α1- and γ2S-subunits in human embryonic kidney (HEK) 293 cells and assayed for their activation by GABA, the intravenous anesthetic propofol and the endogenous neurosteroid pregnanolone using whole cell macroscopic recordings. Three mutants, G170A, V175A, and G177A, produced 2.5-, 6.7-, and 5.6-fold increases in GABA EC₅₀ whereas one mutant, Q185A, produced a 5.2-fold decrease in GABA EC₅₀. None of the mutations affected the ability of propofol or pregnanolone to potentiate a submaximal GABA response, but the Q185A mutant exhibited 8.3- and 3.5-fold increases in the percent direct activation by propofol and pregnanolone, respectively. Mutant Q185A receptors also had an increased leak current that was sensitive to picrotoxin, indicating an increased gating efficiency. Further Q185E, Q185L, and Q185W substitutions revealed a strong correlation between the hydropathy of the amino acid at this position and the GABA EC₅₀. Taken together, these results indicate that β2 Loop 9 is involved in receptor activation by GABA, propofol, and pregnanolone and that β2(Q185) participates in hydrophilic interactions that are important for stabilizing the closed state of the GABA_A receptor.     

Biophysical Investigations of Complement Receptor 2 (CD21 and CR2)-Ligand Interactions Reveal Amino Acid Contacts Unique to Each Receptor-Ligand Pair

Human complement receptor type 2 (CR2 and CD21) is a cell membrane receptor, with 15 or 16 extracellular short consensus repeats (SCRs), that promotes B lymphocyte responses and bridges innate and acquired immunity. The most distally located SCRs, SCR1–2, mediate the interaction of CR2 with its four known ligands (C3d, EBV gp350, IFNα, and CD23). To ascertain specific interacting residues on CR2, we utilized NMR studies wherein gp350 and IFNα were titrated into ¹⁵N-labeled SCR1–2, and chemical shift changes indicative of specific inter-molecular interactions were identified. With backbone assignments made, the chemical shift changes were mapped onto the crystal structure of SCR1–2. With regard to gp350, the binding region of CR2 is primarily focused on SCR1 and the inter-SCR linker, specifically residues Asn¹¹, Arg¹³, Ala²², Arg²⁸, Ser³², Arg³⁶, Lys⁴¹, Lys⁵⁷, Tyr⁶⁴, Lys⁶⁷, Tyr⁶⁸, Arg⁸³, Gly⁸⁴, and Arg⁸⁹. With regard to IFNα, the binding is similar to the CR2-C3d interaction with specific residues being Arg¹³, Tyr¹⁶, Arg²⁸, Ser⁴², Lys⁴⁸, Lys⁵⁰, Tyr⁶⁸, Arg⁸³, Gly⁸⁴, and Arg⁸⁹. We also report thermodynamic properties of each ligand-receptor pair determined using isothermal titration calorimetry. The CR2-C3d interaction was characterized as a two-mode binding interaction with K_d values of 0.13 and 160 μm, whereas the CR2-gp350 and CR2-IFNα interactions were characterized as single site binding events with affinities of 0.014 and 0.035 μm, respectively. The compilation of chemical binding maps suggests specific residues on CR2 that are uniquely important in each of these three binding interactions.