Affinity labeling of cysteine-mutants evidences contact residues in modeled receptor binding sites

To investigate the topology of binding sites in two ionotropic receptors, we have initiated a strategy combining affinity labeling with cysteine-scanning mutagenesis. For the GABAA receptor we have used reactive derivatives of non-competitive blockers (NCBs) to explore interacting positions in its channel. The polypeptide positions of the M2 segment of the alpha1 subunit which we mutated into cysteine were selected for their established accessibility, as determined by the substituted-cysteine accessibility method (SCAM). Using the Xenopus oocyte expression system, we show that receptors containing mutations V257C and S272C are inactivated by several reactive NCBs. These position-selective inactivations lead to an analysis of NCB binding in the channel. For the NMDA receptor glycine-binding site, the prototype antagonist L-701,324 was derivatized at different positions with different reactive groups. The receptor positions to mutate into cysteine were selected after a 3-D homology model. The observed receptor inactivations are mutant- and probe-selective, leading to an unambiguous chemical docking of the antagonist pharmacophore and supporting the model. The site-specificity of the inactivating reactions is assessed by protection experiments and by mutant to wild-type (WT) comparisons. The scope and limitations of the method are briefly discussed.     

Structural model of the N-methyl-D-aspartate receptor glycine site probed by site-directed chemical coupling

The N-methyl-d-aspartate (NMDA) receptor is a ligand-gated ion channel that requires both glutamate and glycine for efficient activation. Here, a strategy combining cysteine scanning mutagenesis and affinity labeling was used to investigate the glycine binding site located on the NR1 subunit. Based on homology modeling to the crystal structure of the glutamate binding site of the 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)-propionic acid receptor GluR2, cysteines were introduced into the NR1 subunit as chemical sensors for three thiol-reactive derivatives of the competitive antagonist L-701324. After coexpressing the mutant NR1 with wild-type NR2B subunits in Xenopus oocytes, agonist-induced currents were recorded to monitor irreversible receptor inactivation by the reactive antagonists. For each derivative, glycine site-specific inactivations were observed with a distinct subset of cysteine-substituted receptors. Together these inactivating substitutions identified seven NR1 residues (Ile-385, Gln-387, Glu-388, Thr-500, Asn-502, Ala-696, and Val-717) that undergo proximity-induced covalent coupling with specific regions of the bound antagonist and disclose its mode of docking in the glycine binding pocket of the NMDA receptor. Our approach may help to unravel the structural basis of distinct NMDA receptor subtype pharmacologies.     

Mutational and Cysteine Scanning Analysis of the Glucagon Receptor N-terminal Domain

The glucagon receptor belongs to the B family of G-protein coupled receptors. Little structural information is available about this receptor and its association with glucagon. We used the substituted cysteine accessibility method and three-dimensional molecular modeling based on the gastrointestinal insulinotropic peptide and glucagon-like peptide 1 receptor structures to study the N-terminal domain of this receptor, a central element for ligand binding and specificity. Our results showed that Asp⁶³, Arg¹¹⁶, and Lys⁹⁸ are essential for the receptor structure and/or ligand binding because mutations of these three residues completely disrupted or markedly impaired the receptor function. In agreement with these data, our models revealed that Asp⁶³ and Arg¹¹⁶ form a salt bridge, whereas Lys⁹⁸ is engaged in cation-π interactions with the conserved tryptophans 68 and 106. The native receptor could not be labeled by hydrophilic cysteine biotinylation reagents, but treatment of intact cells with [2-(trimethylammonium)ethyl]methanethiosulfonate increased the glucagon binding site density. This result suggested that an unidentified protein with at least one free cysteine associated with the receptor prevented glucagon recognition and that [2-(trimethylammonium)ethyl]methanethiosulfonate treatment relieved this inhibition. The substituted cysteine accessibility method was also performed on 15 residues selected using the three-dimensional models. Several receptor mutants, despite a relatively high predicted cysteine accessibility, could not be labeled by specific reagents. The three-dimensional models show that these mutated residues are located on one face of the protein. This could be part of the interface between the receptor and the unidentified inhibitory protein, making these residues inaccessible to biotinylation compounds.     

Probing bulky ligand entry in engineered archaeal ferritins

BackgroundA set of engineered ferritin mutants from Archaeoglobus fulgidus (Af-Ft) and Pyrococcus furiosus (Pf-Ft) bearing cysteine thiols in selected topological positions inside or outside the ferritin shell have been obtained. The two apo-proteins were taken as model systems for ferritin internal cavity accessibility in that Af-Ft is characterized by the presence of a 45 Å wide aperture on the protein surface whereas Pf-Ft displays canonical (threefold) channels.MethodsThiol reactivity has been probed in kinetic experiments in order to assess the protein matrix permeation properties towards the bulky thiol reactive DTNB (5,5′-dithiobis-2-nitrobenzoic acid) molecule.ResultsReaction of DTNB with thiols was observed in all ferritin mutants, including those bearing free cysteine thiols inside the ferritin cavity. As expected, a ferritin mutant from Pf-Ft, in which the cysteine thiol is on the outer surface displays the fastest binding kinetics. In turn, also the Pf-Ft mutant in which the cysteine thiol is placed within the internal cavity, is still capable of full stoichiometric DTNB binding albeit with an almost 200-fold slower rate. The behaviour of Af-Ft bearing a cysteine thiol in a topologically equivalent position in the internal cavity was intermediate among the two Pf-Ft mutants.Conclusions and general significanceThe data thus obtained indicate clearly that the protein matrix in archaea ferritins does not provide a significant barrier against bulky, negatively charged ligands such as DTNB, a finding of relevance in view of the multiple biotechnological applications of these ferritins that envisage ligand encapsulation within the internal cavity.     

Site-selective labeling and electron paramagnetic resonance studies of human cannabinoid receptor CB2

Integral membrane G protein-coupled receptors (GPCR) regulate multiple physiological processes by transmitting signals from extracellular milieu to intracellular proteins and are major targets of pharmaceutical drug development. Since GPCR are inherently flexible proteins, their conformational dynamics can be studied by spectroscopic techniques such as electron paramagnetic resonance (EPR) which requires selective chemical labeling of the protein. Here, we developed protocols for selective chemical labeling of the recombinant human cannabinoid receptor CB₂ by judiciously replacing naturally occurring reactive cysteine residues and introducing a new single cysteine residue in selected positions. The majority of the 47 newly generated single cysteine constructs expressed well in E. coli cells, and more than half of them retained high functional activity. The reactivity of newly introduced cysteine residues was assessed by incorporating nitroxide spin label and EPR measurement. The conformational transition of the receptor between the inactive and activated form were studied by EPR of selectively labeled constructs in the presence of either a full agonist CP-55,940 or an inverse agonist SR-144,528. We observed evidence for higher mobility of labels in the center of internal loop 3 and a structural change between agonist vs. inverse agonist-bound CB₂ in the extracellular tip of transmembrane helix 6. Our results demonstrate the utility of EPR for studies of conformational dynamics of CB₂.     

The Fifth Transmembrane Domain of Angiotensin II Type 1 Receptor Participates in the Formation of the Ligand-binding Pocket and Undergoes a Counterclockwise Rotation upon Receptor Activation

The octapeptide hormone angiotensin II exerts a wide variety of cardiovascular effects through the activation of the angiotensin II Type 1 (AT₁) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein- coupled receptors, the AT₁ receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. The role of the fifth transmembrane domain (TMD5) was investigated using the substituted cysteine accessibility method. All of the residues within Thr-190 to Leu-217 region were mutated one at a time to cysteine, and after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of L197C-AT₁, N200C-AT₁, I201C-AT₁, G203C-AT₁, and F204C-AT₁ mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT₁ receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD5 reporter cysteines engineered in a constitutively active N111G-AT₁ receptor background. Indeed, mutant I201C-N111G-AT₁ became more sensitive to MTSEA, whereas mutant G203C-N111G-AT₁ lost some sensitivity. Our results suggest that constitutive activation of AT₁ receptor causes an apparent counterclockwise rotation of TMD5 as viewed from the extracellular side.     

Combination therapy with bone marrow stromal cells and FK506 enhanced amelioration of ischemic brain damage in rats

AimsWe previously reported that cysteinyl leukotriene receptor 2 (CysLT₂) mediates ischemic astrocyte injury, and leukotriene D₄-activated CysLT₂ receptor up-regulates the water channel aquaporin 4 (AQP4). Here we investigated the mechanism underlying CysLT₂ receptor-mediated ischemic astrocyte injury induced by 4-h oxygen-glucose deprivation and 24-h recovery (OGD/R).Main methodsPrimary cultures of rat astrocytes were treated by OGD/R to construct the cell injury model. AQP4 expression was inhibited by small interfering RNA (siRNA). The expressions of AQP4 and CysLTs receptors, and the MAPK signaling pathway were determined.Key findingsOGD/R induced astrocyte injury, and increased expression of the CysLT₂ (but not CysLT₁) receptor and AQP4. OGD/R-induced cell injury and AQP4 up-regulation were inhibited by a CysLT₂ receptor antagonist (Bay cysLT2) and a non-selective CysLT receptor antagonist (Bay u9773), but not by a CysLT₁ receptor antagonist (montelukast). Knockdown of AQP4 by siRNA attenuated OGD/R injury. Furthermore, OGD/R increased phosphorylation of ERK1/2 and p38, whose inhibitors relieved the cell injury and AQP4 up-regulation.SignificanceThe CysLT₂ receptor mediates AQP4 up-regulation in astrocytes, and up-regulated AQP4 leads to OGD/R-induced injury, which results from activation of the ERK1/2 and p38 MAPK pathways.     

Reactive affinity probes for the mapping of the glycine-binding site of the NMDA receptor NR1 subunit

The glycine co-agonist binding site of the NMDA receptor is a target for the prevention and treatment of neurotoxic and neurodegenerative conditions. Until now, the interactions taking place at this site, and its structure, have been investigated by ligand structure-activity relationships and by site-directed mutagenesis. On the basis of a structural model which is currently proposed for this site, we have designed and synthesized six affinity markers by substituting electrophilic reactive groups in the 4, the 7 and the 3' positions of L 701,324, a high-affinity glycine site antagonist. These compounds compete with 3H-DCKA binding to rat brain membranes at equilibrium with nanomolar to low-micromolar affinities, and antagonize glycine-evoked currents in oocytes transfected with wild-type NR1-NR2B. However, they do not induce a time-shift in binding equilibria, and do not inactivate irreversibly the glycine evoked currents. Since they react only with cysteine at physiological pH, we conclude that there is no such residue in the site, in agreement with the model. Our affinity markers therefore represent potential topological probes for NMDA receptors with sequence positions related to the glycine-binding site mutated into cysteine.     

Mapping the rho1 GABA(C) receptor agonist binding pocket. Constructing a complete model

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian brain. The GABA receptor type C (GABA(C)) is a ligand-gated ion channel with pharmacological properties distinct from the GABA(A) receptor. To date, only three binding domains in the recombinant rho1 GABA(C) receptor have been recognized among six potential regions. In this report, using the substituted cysteine accessibility method, we scanned three potential regions previously unexplored in the rho1 GABA(C) receptor, corresponding to the binding loops A, E, and F in the structural model for ligand-gated ion channels. The cysteine accessibility scanning and agonist/antagonist protection tests have resulted in the identification of residues in loops A and E, but not F, involved in forming the GABA(C) receptor agonist binding pocket. Three of these newly identified residues are in a novel region corresponding to the extended stretch of loop E. In addition, the cysteine accessibility pattern suggests that part of loop A and part of loop E have a beta-strand structure, whereas loop F is a random coil. Finally, when all of the identified ligand binding residues are mapped onto a three-dimensional homology model of the amino-terminal domain of the rho1 GABA(C) receptor, they are facing toward the putative binding pocket. Combined with previous findings, a complete model of the GABA(C) receptor binding pocket was proposed and discussed in comparison with the GABA(A) receptor binding pocket.     

Multiple tyrosine residues at the GABA binding pocket influence surface expression and mediate kinetics of the GABAA receptor

The prevalence of aromatic residues in the ligand binding site of the GABA_A receptor, as with other cys‐loop ligand‐gated ion channels, is undoubtedly important for the ability of neurotransmitters to bind and trigger channel opening. Here, we have examined three conserved tyrosine residues at the GABA binding pocket (β₂Tyr97, β₂Tyr157, and β₂Tyr205), making mutations to alanine and phenylalanine. We fully characterized the effects each mutation had on receptor function using heterologous expression in HEK‐293 cells, which included examining surface expression, kinetics of macroscopic currents, microscopic binding and unbinding rates for an antagonist, and microscopic binding rates for an agonist. The assembly or trafficking of GABA_A receptors was disrupted when tyrosine mutants were expressed as αβ receptors, but interestingly not when expressed as αβγ receptors. Mutation of each tyrosine accelerated deactivation and slowed GABA binding. This provides strong evidence that these residues influence the binding of GABA. Qualitatively, mutation of each tyrosine has a very similar effect on receptor function; however, mutations at β₂Tyr157 and β₂Tyr205 are more detrimental than β₂Tyr97 mutations, particularly to the GABA binding rate. Overall, the results suggest that interactions involving multiple tyrosine residues are likely during the binding process.     

Analysis of the glucagon receptor first extracellular loop by the substituted cysteine accessibility method

Glucagon is an important hormone for the prevention of hypoglycemia, and contributes to the hyperglycemia observed in diabetic patients, yet very little is known about its receptor structure and the receptor-glucagon interaction. In related receptors, the first extracellular loop, ECL1, is highly variable in length and sequence, suggesting that it might participate in ligand recognition. We applied a variant of the SCAM (Substituted Cysteine Accessibility Method) to the glucagon receptor ECL1 and sequentially mutated positions 197 to 223 to cysteine. Most of the mutations (15/27) affected the glucagon potency, due either to a modification of the glucagon binding site, or to the destabilization of the active receptor conformation. We reasoned that side chains accessible to glucagon must also be accessible to large, hydrophilic cysteine reagents. We therefore evaluated the accessibility of the introduced cysteines to maleimide-PEO₂-biotin ((+)-biotinyl-3-maleimido-propionamidyl-3,6-dioxa-octanediamine), and tested the effect of pretreatment of intact cells with a large cationic cysteine reagent, MTSET ([2-(trimethylammonium)ethyl]methanethiosulfonate bromide), on glucagon potency. Our results suggest that the second and third transmembrane helices (TM2 and TM3) are extended to position 202 and from position 215, respectively, and separated by a short β stretch (positions 203-209). Glucagon binding induced a conformational change close to TM2: L198C was accessible to the biotin reagent only in the presence of glucagon. Most other mutations affected the receptor activation rather than glucagon recognition, but S217 and D218 (at the top of TM3) were good candidates for glucagon recognition and V221 was very close to the binding site.     

Mass Ligand Binding Screening for Receptor Antagonists: Prototype New Drugs and Blind Alleys

Abstract

The ligand binding assay is a powerful tool in the search for antagonists for novel receptors, and for identification of novel classes of antagonists for well-known receptors. Ligand binding mass screening can be adapted for very high throughput. In order for mass screening to be useful, it is necessary to strictly define the binding characteristics for a compound to be considered a putative receptor antagonist. In practice, we have found that synthetic pursuit of a compound with a K_i of ± 1 uM is likely to lead down a blind alley unless very good evidence for specificity is available. Even potent competitors for binding should be thoroughly evaluated in assays of biological activity before a synthetic program is initiated in earnest.     

Alpha1 Adrenergic Receptors in the Porcine Pituitary Neurointermediate Lobe: Detection with [3H]Ketanserin

Abstract

[³H]Ketanserin, a serotonin receptor antagonist, labelled high affinity, saturable sites in homogenates of porcine neurointermediate lobe tissue. Cinanserin, a potent and selective serotonin receptor antagonist, inhibited the specific binding of 5 × 10^-10M [³H]ketanserin with a high affinity component representing 20% of the total binding. Prazosin, a potent and selective alpha₁ adrenergic antagonist, inhibited [³H]ketanserin binding with a high affinity component representing 60% of total binding. The prazosin-specific component was demonstrated to be distinct from the cinanserin-specific component. 10^-7M cinanserin was co-incubated with [³H]ketanserin to eliminate the serotonergic component of the binding and allow pharmacological characterization of the remaining prazosin-specific component. The prazosin-specific binding of [³H]ketanserin binding closely resembled the results of experiments using [³H]prazosin to label alpha₁ receptors in neurointermediate lobe tissue homogenates. Ketanserin was found to be sevenfold more potent in inhibiting [³H]prazosin binding to alpha₁ adrenergic receptors in the neurointermediate lobe tissue than in brain tissue. This observation explains why low concentrations of [³H]ketanserin can selectively label serotonin receptors in the brain but will label both adrenergic and serotonin receptors in the neurointermediate lobe.     

Pharmacological Characterization of Mammalian D1 and D2 Dopamine Receptors Expressed in Drosophila Schneider‐2 Cells

Abstract

Mammalian D₁ and D₂ dopamine receptors were stably expressed in Drosophila Schneider‐2 (S2) cells and screened for their pharmacological properties. Saturable, dose‐dependent, high affinity binding of the D₁‐selective antagonist [³H]SCH‐23390 was detected only in membranes from S2 cells induced to express rat dopamine D₁ receptors, while saturable, dose‐dependent, high affinity binding of the D₂‐selective antagonist [³H]methylspiperone was detected only in membranes from S2 cells induced to express rat dopamine D₂ receptors. No specific binding of either radioligand could be detected in membranes isolated from uninduced or untransfected S2 cells. Both dopamine D₁ and D₂ receptor subtypes displayed the appropriate stereoselective binding of enantiomers of the nonselective antagonist butaclamol. Each receptor subtype also displayed the appropriate agonist stereoselectivities. The dopamine D₁ receptor bound the (+)‐enantiomer of the D₁‐selective agonist SKF38393 with higher affinity than the (?)‐enantiomer, while the dopamine D₂ receptor bound the (?)‐enantiomer of the D₂‐selective agonist norpropylapomorphine with higher affinity than the (+)‐enantiomer. At both receptor subtypes, dopamine binding was best characterized as occurring to a single low affinity site. In addition, the low affinity dopamine binding was also found to be insensitive to GTPγS and magnesium ions. Overall, the pharmacological profiles of mammalian dopamine D₁ and D₂ receptors expressed in Drosophila S2 cells is comparable to those observed for these same receptors when they are expressed in mammalian cell lines. A notable distinction is that there is no evidence for the coupling of insect G proteins to mammalian dopamine receptors. These results suggest that the S2 cell insect G system may provide a convenient source of pharmacologically active mammalian D₁ and D₂ dopamine receptors free of promiscuous G protein contaminants.     

Role of Disulfides and Sulfhydryl Groups in Agonist and Antagonist Binding in Serotonin1A Receptors from Bovine Hippocampus

SUMMARY 1. The serotonin_1A (5-HT_1A) receptors are members of a superfamily of seven-transmembrane-domain receptors that couple to G-proteins. They appear to be involved in various behavioral and cognitive functions. Mutagenesis and modeling studies point out that the ligand-binding sites in serotonin receptors are located in the transmembrane domain. However, these binding sites are not very well characterized. Since disulfide bonds and sulfhydryl groups have been shown to play vital roles in the assembly, organization, and function of various G-protein-coupled receptors, we report here the effect of disulfide and sulfhydryl group modifications on the agonist and antagonist binding activity of 5-HT_1A receptors from bovine hippocampus.2. DTT or NEM treatment caused a concentration-dependent reduction in specific binding of the agonist and antagonist in 5-HT_1A receptors from bovine hippocampal native and solubilized membranes. This is supported by a concomitant reduction in binding affinity.3. Pretreatment of the receptor with unlabeled ligands prior to chemical modifications indicate that the majority of disulfides or sulfhydryl groups that undergo modification giving rise to inhibition in binding activity could be at the vicinity of the ligand-binding sites.4. In addition, ligand-binding studies in presence of GTP--S, a nonhydrolyzable analogue of GTP, indicate that sulfhydryl groups (and disulfide bonds to a lesser extent) are vital for efficient coupling between the 5-HT_1A receptor and the G-protein.5. Our results point out that disulfide bonds and sulfhydryl groups could play an important role in ligand binding in 5-HT_1A receptors.     

An NMDA Receptor Gating Mechanism Developed from MD Simulations Reveals Molecular Details Underlying Subunit-Specific Contributions

N-methyl-D-aspartate (NMDA) receptors are obligate heterotetrameric ligand-gated ion channels that play critical roles in learning and memory. Here, using targeted molecular dynamics simulations, we developed an atomistic model for the gating of the GluN1/GluN2A NMDA receptor. Upon agonist binding, lobe closure of the ligand-binding domain produced outward pulling of the M3-D2 linkers, leading to outward movements of the C-termini of the pore-lining M3 helices and opening of the channel. The GluN2A subunits, similar to the distal (B/D) subunits in the homotetrameric GluA2 α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate receptor, had greater M3 outward movements and thus contributed more to channel gating than the GluN1 subunits. Our gating model is validated by functional studies, including cysteine modification data indicating wider accessibility to the GluN1 M3 helices than to the GluN2A M3 helices from the lumen of the open channel, and reveals why the Lurcher mutation in GluN1 has a stronger ability in maintaining channel opening than the counterpart in GluN2A. The resulting structural model for the open state provides an explanation for the Ca²⁺ permeability of NMDA receptors, and the structural differences between the closed and open states form the basis for drug design.     

Electrostatic and aromatic microdomains within the binding-site crevice of the D2 receptor: contributions of the second membrane-spanning segment.

The binding-site of the dopamine D2 receptor, like that of other homologous G protein-coupled receptors, is contained within a water-accessible crevice formed among its seven membrane-spanning segments. Using the substituted cysteine accessibility method (SCAM), we previously mapped the residues in the third, fifth, sixth, and seventh membrane-spanning segments that contribute to the surface of this binding-site crevice. We have now mutated to cysteine, one at a time, 22 consecutive residues in the second membrane-spanning segment (M2) and expressed the mutant receptors in HEK 293 cells. Eleven of these mutants reacted with charged, hydrophilic, lipophobic, sulfhydryl-specific reagents, added extracellularly, and 9 of these 11 were protected from reaction by a reversible dopamine antagonist, sulpiride. We infer that the side chains of the residues at the 11 reactive loci (D80, L81, V83, V87, P89, W90, V91, V92, L94, E95, V96) are on the water-accessible surface of the binding-site crevice and that 9 of these are occluded by bound antagonist. The pattern of accessibility suggests an alpha-helical conformation. A broadening of the angle of accessibility near the binding site is consistent with the presence of a kink at Pro89. On the basis of the enhanced rates of reaction of positively charged sulfhydryl reagents, we infer the presence of an electrostatic microdomain composed of three acidic residues in M2 and the adjacent M3 that could attract and orient cationic ligands. Furthermore, based on the enhanced reactivity of the hydrophobic cation-containing reagent, we infer the presence of an aromatic microdomain formed between M2, M3, and M7.     

Membrane topology of loop 13-14 of the Na/glucose cotransporter (SGLT1): A SCAM and fluorescent labelling study

The accessibility of the hydrophilic loop between putative transmembrane segments XIII and XIV of the Na⁺/glucose cotransporter (SGLT1) was studied in Xenopus oocytes, using the substituted cysteine accessibility method (SCAM) and fluorescent labelling. Fifteen cysteine mutants between positions 565 and 664 yielded cotransport currents of similar amplitude than the wild-type SGLT1 (wtSGLT1). Extracellular, membrane-impermeant MTSES⁽⁻⁾ and MTSET⁽⁺⁾ had no effect on either cotransport or Na⁺ leak currents of wtSGLT1 but 9 mutants were affected by MTSES and/or MTSET. We also performed fluorescent labelling on SGLT1 mutants, using tetramethylrhodamine-5-maleimide and showed that positions 586, 588 and 624 were accessible. As amino acids 604 to 610 in SGLT1 have been proposed to form part of a phlorizin (Pz) binding site, we measured the K_i^Pz and K_m^αMG for wtSGLT1 and for cysteine mutants at positions 588, 605-608 and 625. Although mutants A605C, Y606C and D607C had slightly higher K_i^Pz values than wtSGLT1 with minimal changes in K_m^αMG, the effects were modest and do not support the original hypothesis. We conclude that the large, hydrophilic loop near the carboxyl terminus of SGLT1 is thus accessible to the external solution but does not appear to play a major part in the binding of phlorizin.     

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.     

Receptor binding profile of R 41 468, a novel antagonist at 5-HT2 receptors

For a new antiserotonergic agent, R 41 468 and 13 reference compounds with alleged antiserotonergic activity, the receptor binding profile is reported, comprising K_i-values measured in ten different receptor binding models. R 41 468 appeared to be a particularly selective agent with respect to differentiation between two 5-hydroxytryptamine (5-HT) receptor models; it primarily displayed high binding affinity for 5-HT₂ receptors and was inactive at 5-HT₁ receptors. Besides showing a moderate binding affinity for histamine₁ and α₁ adrenergic receptors, the compound was very weakly active at dopamine receptors and inactive at the remaining receptors. Receptor binding profiles of the reference compounds differed widely. Apart from R 41 468 no other compound showed a similar selectivity towards 5-HT₂ receptors. Reference compounds either poorly differentiated between 5-HT₂ and 5-HT₁ receptors, showed other primary effects, or were only moderately active. In the 5-HT₂ and 5-HT₁ receptor binding models the ‘D-receptor’ antagonist phenoxybenzamine was weakly active and the ‘M-receptor’ antagonist morphine was inactive. It is concluded that R 41 468 will be a particularly suitable tool to antagonize 5-HT action mediated by 5-HT₂ receptors.