Influence of GABA(A) Receptor α Subunit Isoforms on the Benzodiazepine Binding Site

Classical benzodiazepines, such as diazepam, interact with α_xβ₂γ₂ GABA_A receptors, x = 1, 2, 3, 5 and modulate their function. Modulation of different receptor isoforms probably results in selective behavioural effects as sedation and anxiolysis. Knowledge of differences in the structure of the binding pocket in different receptor isoforms is of interest for the generation of isoform-specific ligands. We studied here the interaction of the covalently reacting diazepam analogue 3-NCS with α₁S204Cβ₂γ₂, α₁S205Cβ₂γ₂ and α₁T206Cβ₂γ₂ and with receptors containing the homologous mutations in α₂β₂γ₂, α₃β₂γ₂, α₅β_1/2γ₂ and α₆β₂γ₂. The interaction was studied using radioactive ligand binding and at the functional level using electrophysiological techniques. Both strategies gave overlapping results. Our data allow conclusions about the relative apposition of α₁S204Cβ₂γ₂, α₁S205Cβ₂γ₂ and α₁T206Cβ₂γ₂ and homologous positions in α₂, α₃, α₅ and α₆ with C-atom adjacent to the keto-group in diazepam. Together with similar data on the C-atom carrying Cl in diazepam, they indicate that the architecture of the binding site for benzodiazepines differs in each GABA_A receptor isoform α₁β₂γ₂, α₂β₂γ₂, α₃β₂γ₂, α₅β_1/2γ₂ and α₆β₂γ₂.     

The Effects of Transmembrane Sequence and Dimerization on Cleavage of the p75 Neurotrophin Receptor by γ-Secretase

Cleavage of transmembrane receptors by γ-secretase is the final step in the process of regulated intramembrane proteolysis (RIP) and has a significant impact on receptor function. Although relatively little is known about the molecular mechanism of γ-secretase enzymatic activity, it is becoming clear that substrate dimerization and/or the α-helical structure of the substrate can regulate the site and rate of γ-secretase activity. Here we show that the transmembrane domain of the pan-neurotrophin receptor p75^NTR, best known for regulating neuronal death, is sufficient for its homodimerization. Although the p75^NTR ligands NGF and pro-NGF do not induce homerdimerization or RIP, homodimers of p75^NTR are γ-secretase substrates. However, dimerization is not a requirement for p75^NTR cleavage, suggesting that γ-secretase has the ability to recognize and cleave each receptor molecule independently. The transmembrane cysteine 257, which mediates covalent p75^NTR interactions, is not crucial for homodimerization, but this residue is required for normal rates of γ-secretase cleavage. Similarly, mutation of the residues alanine 262 and glycine 266 of an AXXXG dimerization motif flanking the γ-secretase cleavage site within the p75^NTR transmembrane domain alters the orientation of the domain and inhibits γ-secretase cleavage of p75^NTR. Nonetheless, heteromer interactions of p75^NTR with TrkA increase full-length p75^NTR homodimerization, which in turn potentiates the rate of γ-cleavage following TrkA activation independently of rates of α-cleavage. These results provide support for the idea that the helical structure of the p75^NTR transmembrane domain, which may be affected by co-receptor interactions, is a key element in γ-secretase-catalyzed cleavage.     

The α-Subunit Regulates Stability of the Metal Ion at the Ligand-associated Metal Ion-binding Site in β3 Integrins

The aspartate in the prototypical integrin-binding motif Arg-Gly-Asp binds the integrin βA domain of the β-subunit through a divalent cation at the metal ion-dependent adhesion site (MIDAS). An auxiliary metal ion at a ligand-associated metal ion-binding site (LIMBS) stabilizes the metal ion at MIDAS. LIMBS contacts distinct residues in the α-subunits of the two β₃ integrins α_IIbβ₃ and α_Vβ₃, but a potential role of this interaction on stability of the metal ion at LIMBS in β₃ integrins has not been explored. Equilibrium molecular dynamics simulations of fully hydrated β₃ integrin ectodomains revealed strikingly different conformations of LIMBS in unliganded α_IIbβ₃ versus α_Vβ₃, the result of stronger interactions of LIMBS with α_V, which reduce stability of the LIMBS metal ion in α_Vβ₃. Replacing the α_IIb-LIMBS interface residue Phe¹⁹¹ in α_IIb (equivalent to Trp¹⁷⁹ in α_V) with Trp strengthened this interface and destabilized the metal ion at LIMBS in α_IIbβ₃; a Trp¹⁷⁹ to Phe mutation in α_V produced the opposite but weaker effect. Consistently, an F191/W substitution in cellular α_IIbβ₃ and a W179/F substitution in α_Vβ₃ reduced and increased, respectively, the apparent affinity of Mn²⁺ to the integrin. These findings offer an explanation for the variable occupancy of the metal ion at LIMBS in α_Vβ₃ structures in the absence of ligand and provide new insights into the mechanisms of integrin regulation.     

The Role of Cysteine 116 in the Active Site of the Antitumor Enzyme L-Methionine γ-Lyase from Pseudomonas putida

The cysteinyl residue at the active site of L-methionine γ-lyase from Pseudomonas putida (MGL_Pp) is highly conserved among the heterologous MGLs. To determine the role of Cys116, we constructed 19 variants of C116X MGL_Pp by saturation mutagenesis. The Cys116 mutants possessed little catalytic activity, while their affinity for each substrate was almost the same as that of the wild type. Especially, the C116S, C116A, and C116H variants composed active site catalytic function as measured by the kinetic parameter k _cat toward L-methionine. Furthermore, the mutagenesis of Cys116 also affected the substrate specificity of MGL_Pp at the active center. Substitution of Cys116 for His led to a marked increase in activity toward L-cysteine and a decrease in that toward L-methionine. Propargylglycine inactivated the WT MGL, C116S, and C116A mutants. Based on these results, we postulate that Cys116 plays an important role in the γ-elimination reaction of L-methionine and in substrate recognition in the MGLs.     

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.     

The Novel Recognition Site in the C-Terminal Heparin-Binding Domain of Fibronectin by Integrin α4β1 Receptor on HL-60 Cells

The hematopoietic cell recognition sites of human fibronectin (FN) are the Arg–Gly–Asp–Ser (RGDS) sequence recognized by widely distributed integrin receptor α5β1 and the type III connecting segment (III CS) containing two cell-binding sites, designated CS1 and CS5, that are recognized by the α4β1 receptor. The C-terminal heparin-binding domain of FN (Hep II) has recently been demonstrated to support adhesion of α4β1-dependent melanoma cells [A. P. Mould and M. J. Humphries (1991)EMBO J.10, 4089–4095]. Previously we demonstrated that this region of FN mediated binding of FN to HL-60 cells (acute promyelocytic leukemia cell line) by direct interaction independently of RGD and CS1 [H. Fujitaet al.,(1995)Exp. Cell Res.217, 484–488]. In this study we have characterized a novel site in the Hep II region for binding to HL-60 cells. α4β1 and α5β1 were expressed on HL-60 cells, while α2β1 and α3β1 were not present, as shown by flow cytometry using monoclonal antibodies specific for the different integrins. Anti-α4β1 (P4C2) and anti-β1 (JB1a) antibodies inhibited binding of a 29-kDa dispase-digestive fragment of FN to HL-60 cells. This fragment contains the C-terminal heparin-binding domain of FN but lacks CS1 and CS5. Only the peptide representing the sequence from Val¹⁸⁶⁶to Arg¹⁸⁸⁰, designated E1, inhibited the binding of the 29-kDa fragment to HL-60 cells. The active region of this peptide was a sequence of Thr–Asp–Ile–Asp–Ala–Pro–Ser (TAI- DAPS), which is homologous to Leu–Asp–Val–Pro–Ser (LDVPS) derived from the active site of CS1. Furthermore, labeled E1 peptide directly bound to HL-60 cells. The anti-α4β1 antibody (P4C2) inhibited this interaction. These results indicate that the site of binding to hematopoietic cells is present in the Hep II region of FN and the definition of the chemical structure of FN clarifies a fundamental mechanism of cell invasion of the extracellular matrix.     

The Oxygen Receptor of the Teleost Gill?

The gills of the Atlantic cod, Gadus morhua, were studied using immunohistochemical techniques. Primary antibodies directed against serotonin (5-hydroxytryptamine, 5-HT) and acetylated α-tubulin were used to visualise cells containing serotonin and nerve fibres, respectively. Three morphologically different 5-HT immunoreactive cell types were distinguished: (I) Neuroepithelial cells (NECs), which were abundant along the distal half of the efferent filamental arteries (EFAs), and particularly formed distinct clusters at the individual filamental tips, (II) bipolar neurones running next to the EFAs and (III) multipolar neurones innervating the proximal parts of the EFA. In addition, the study revealed a well-developed system of nerve fibres, some of which form plexuses in association with the NECs. A relatively rich innervation of the proximal part of the EFAs, in conjunction with the EFA sphincters was also observed. Delicate varicose terminals surround the bases of the efferent lamellar arterioles. The localisation of distinct clusters of NECs at the individual filamental tips and the close connection with nerve terminals suggests a function as external branchial oxygen receptors.     

The effects of γ-irradiation on the priming by deoxyribonucleohistone of ribonucleic acid polymerase

1. The effect of gamma-irradiation of solutions of DNA and deoxyribonucleohistone (DNH) on their ability to prime the synthesis of RNA by DNA-dependent RNA polymerase has been studied. 2. The priming ability of both DNA and DNH decreased continuously with increasing radiation dose, but more rapidly with DNH. 3. These decreases have been compared with decreases in molecular weight and with the breakdown of the specific hydrogen-bonded structure of DNA. 4. It is concluded that a process was occurring during gamma-irradiation of DNH that, although involving a decrease in molecular weight, did not diminish and even enhanced its priming ability. This is consistent with previous physicochemical evidence that gamma-irradiation causes dissociation of histone from DNH.     

Is the Redox State of the ci Heme of the Cytochrome b6f Complex Dependent on the Occupation and Structure of the Qi Site and Vice Versa?

Oxidoreductases of the cytochrome bc₁/b₆f family transfer electrons from a liposoluble quinol to a soluble acceptor protein and contribute to the formation of a transmembrane electrochemical potential. The crystal structure of cyt b₆f has revealed the presence in the Q_i site of an atypical c-type heme, heme c_i. Surprisingly, the protein does not provide any axial ligand to the iron of this heme, and its surrounding structure suggests it can be accessed by exogenous ligand. In this work we describe a mutagenesis approach aimed at characterizing the c_i heme and its interaction with the Q_i site environment. We engineered a mutant of Chlamydomonas reinhardtii in which Phe⁴⁰ from subunit IV was substituted by a tyrosine. This results in a dramatic slowing down of the reoxidation of the b hemes under single flash excitation, suggesting hindered accessibility of the heme to its quinone substrate. This modified accessibility likely originates from the ligation of the heme iron by the phenol(ate) side chain introduced by the mutation. Indeed, it also results in a marked downshift of the c_i heme midpoint potential (from +100 mV to −200 mV at pH 7). Yet the overall turnover rate of the mutant cytochrome b₆f complex under continuous illumination was found similar to the wild type one, both in vitro and in vivo. We propose that, in the mutant, a change in the ligation state of the heme upon its reduction could act as a redox switch that would control the accessibility of the substrate to the heme and trigger the catalysis.The cytochrome b₆f complex of oxygenic photosynthesis is the integral membrane protein, the quinol:plastocyanin oxido-reductase activity of which allows the linear electron flow between the two photosystems (PSI and PSII).⁴ The turnover of the cytochrome b₆f complex depends on the steady state of its redox partners, the liposoluble plastoquinol (PQH₂ reduced and protonated plastoquinone PQ) formed by the PSII, and the hydrosoluble plastocyanin oxidized by the PSI. In the Q_o site, exposed to the lumenal side, the quinol is oxidized, and this oxidation is coupled to the release of two protons into the lumen. The two electrons provided by the quinol are transferred along two bifurcated pathways, the high and low potential chains. The high potential chain involves two lumenal redox partners, the membrane-anchored flexible Rieske [2Fe-2S] cluster and the cytochrome f, which ultimately interacts with the soluble plastocyanin. In the low potential chain, electrons are transferred to the stroma via the low and high potential b hemes (b_L and b_H) of the transmembrane b₆ subunit. Two turnovers of the cyt b₆f complex lead to the reduction of the low potential chain, thereby allowing the reduction of a quinone molecule in the stromal Q_i pocket. This mechanism, which recycles reducing equivalents, is referred to as the “Q cycle,” initially described by Mitchell (1) and modified later by Crofts et al. and others (2, 3).Although this quinol:cytochrome oxidoreductase activity is involved in both the respiratory and photosynthetic electron transfer chains, recent x-ray data (4–6) have evidenced major structural differences between the b₆f complex and its mitochondrial counterpart the bc₁ complex (for reviews see Refs. 7–10). Indeed an additional heme localized in close contact with heme b_H stands as another putative electron carrier as proposed earlier by Lavergne (11). Since it was brought to light by the x-ray studies, knowledge of the basic properties of this heme, named c_i in reference to the Q_i site (5) or c_n in reference to its proximity to the negatively charged side of the membrane (4), has significantly improved. The proteins involved in the assembly machinery of the heme have been identified in Chlamydomonas reinhardtii and Arabidopsis thaliana (12, 13). Consistent with the structure, according to which the only axial ligand could be a water molecule interacting with the proponiate chain of the b_H heme, the spectroscopic properties of this heme are those of a high spin heme (14, 15). Evidences for a high spin heme covalently bound to the cytochrome b subunit were also found in Heliobacterium modesticaldum and Heliobacillus mobilis (16).In the b₆f complex from the oxygenic photosynthetic chain, EPR (15) and structural data (17) have shown that NQNO (2-n-nony l-4-hydroxyquinoline N-oxide), an inhibitor of the Q_i pocket (18, 19), can act as an axial ligand to c_i. This ligation is accompanied by a significant change in the redox properties of c_i, because, in the presence of NQNO, at least two titrations waves were observed (13, 14), one with a midpoint potential (E_m) similar to that observed in the absence of NQNO and the other with a midpoint potential downshifted by ∼250 mV. This, together with the widespread range of redox potential found for heme c_i (11, 14, 15, 20), points to a structural plasticity of the c_i ligand network.This plasticity may arise from the unusual coordination properties of the heme c_i. As a matter of fact, the x-ray models of the complex from C. reinhardtii and Mastigocladus laminosus evidenced a water or hydroxyl molecule as a fifth ligand. The sixth position of coordination is directed toward the Q_i pocket and appears as free. Nevertheless, the side chain of Phe⁴⁰ of subunit IV protrudes above the heme plane, leaving little space for any axial ligand to the heme c_i. Besides, modeling a quinone analogue in the electron density found in the Q_i pocket of structures obtained in presence of Tridecyl- Stigmatellin or NQNO implies a steric clash with the native position of the Phe⁴⁰ aromatic ring.⁵ The Phe⁴⁰ residue of subunit IV therefore stands as a key residue for the plasticity of the site, making it an ideal mutagenesis target when attempting to alter the possible interactions between c_i and the quinone or quinol (4, 5) (Fig. 1). Here we present the consequences of the substitution of Phe⁴⁰ by a tyrosine on the properties and function of the c_i heme.Open in a separate windowFIGURE 1.A view of the Q_i site from the dimer interface. The coordinates are from the Protein Data Bank 1Q90 model. The Van der Waal''s surface of the peptide chains was drawn with Pymol. Phe⁴⁰ is in van der Waal''s contact with the plane of the c_i, heme.     

Gating-induced Conformational Rearrangement of the γ-Aminobutyric Acid Type A Receptor β-α Subunit Interface in the Membrane-spanning Domain

GABA(A) receptors mediate fast inhibitory synaptic transmission. The transmembrane ion channel is lined by a ring of five α helices, M2 segments, one from each subunit. An outer ring of helices comprising the alternating M1, M3, and M4 segments from each subunit surrounds the inner ring and forms the interface with the lipid bilayer. The structural rearrangements that follow agonist binding and culminate in opening of the ion pore remain incompletely characterized. Propofol and other intravenous general anesthetics bind at the βM3-αM1 subunit interface. We sought to determine whether this region undergoes conformational changes during GABA activation. We measured the reaction rate of p-chloromercuribenzenesulfonate (pCMBS) with cysteines substituted in the GABA(A) receptor α1M1 and β2M3 segments. In the presence of GABA, the pCMBS reaction rate increased significantly in a cluster of residues in the extracellular third of the α1M1 segment facing the β2M3 segment. Mutation of the β2M2 segment 19' position, R269Q, altered the pCMBS reaction rate with several α1M1 Cys, some only in the resting state and others only in the GABA-activated state. Thus, β2R269 is charged in both states. GABA activation induced disulfide bond formation between β2R269C and α1I228C. The experiments demonstrate that α1M1 moves in relationship to β2M2R269 during gating. Thus, channel gating does not involve rigid body movements of the entire transmembrane domain. Channel gating causes changes in the relative position of transmembrane segments both within a single subunit and relative to the neighboring subunits.     

The β1-Subunit of the MaxiK Channel Associates with the Thromboxane A2 Receptor and Reduces Thromboxane A2 Functional Effects

The large conductance voltage- and Ca²⁺-activated K⁺ channel (MaxiK, BK_Ca, BK) is composed of four pore-forming α-subunits and can be associated with regulatory β-subunits. One of the functional roles of MaxiK is to regulate vascular tone. We recently found that the MaxiK channel from coronary smooth muscle is trans-inhibited by activation of the vasoconstricting thromboxane A₂ prostanoid receptor (TP), a mechanism supported by MaxiK α-subunit (MaxiKα)-TP physical interaction. Here, we examined the role of the MaxiK β1-subunit in TP-MaxiK association. We found that the β1-subunit can by itself interact with TP and that this association can occur independently of MaxiKα. Subcellular localization analysis revealed that β1 and TP are closely associated at the cell periphery. The molecular mechanism of β1-TP interaction involves predominantly the β1 extracellular loop. As reported previously, TP activation by the thromboxane A₂ analog U46619 caused inhibition of MaxiKα macroscopic conductance or fractional open probability (FP_o) as a function of voltage. However, the positive shift of the FP_o versus voltage curve by U46619 relative to the control was less prominent when β1 was coexpressed with TP and MaxiKα proteins (20 ± 6 mV, n = 7) than in cells expressing TP and MaxiKα alone (51 ± 7 mV, n = 7). Finally, β1 gene ablation reduced the EC₅₀ of the U46619 agonist in mediating aortic contraction from 18 ± 1 nm (n = 12) to 9 ± 1 nm (n = 12). The results indicate that the β1-subunit can form a tripartite complex with TP and MaxiKα, has the ability to associate with each protein independently, and diminishes U46619-induced MaxiK channel trans-inhibition as well as vasoconstriction.     

Characterization of the ��-d-Glucopyranoside Binding Site of the Human Bitter Taste Receptor hTAS2R16

G-protein-coupled receptors mediate the senses of taste, smell, and vision in mammals. Humans recognize thousands of compounds as bitter, and this response is mediated by the hTAS2R family, which is one of the G-protein-coupled receptors composed of only 25 receptors. However, structural information on these receptors is limited. To address the molecular basis of bitter tastant discrimination by the hTAS2Rs, we performed ligand docking simulation and functional analysis using a series of point mutants of hTAS2R16 to identify its binding sites. The docking simulation predicted two candidate binding structures for a salicin-hTAS2R16 complex, and at least seven amino acid residues in transmembrane 3 (TM3), TM5, and TM6 were shown to be involved in ligand recognition. We also identified the probable salicin-hTAS2R16 binding mode using a mutated receptor experiment. This study characterizes the molecular interaction between hTAS2R16 and β-d-glucopyranoside and will also facilitate rational design of bitter blockers.     

Association of Renal Na,K-ATPase α-Subunit with the β- and γ-Subunits Based on Cryoelectron Microscopy

Na,K-ATPase transports Na(+) and K(+) across cell membranes and consists of alpha- and beta-subunits. Na,K-ATPase also associates with small FXYD proteins that regulate the activity of the pump. We have used cryoelectron microscopy of two-dimensional crystals including data to 8 A resolution to determine the three-dimensional (3-D) structure of renal Na,K-ATPase containing FXYD2, the gamma-subunit. A homology model for the alpha-subunit was calculated from a Ca(2+)-ATPase structure and used to locate the additional beta- and gamma-subunits present in the 3-D map of Na,K-ATPase. Based on the 3-D map, the beta-subunit is located close to transmembrane helices M8 and M10 and the gamma-subunit is adjacent to helices M2 and M9 of the alpha-subunit.     

The Role of N53Q Mutation on the Rat μ-Opioid Receptor Function

Glycosylation of the μ-opioid receptor may play an important role on its function. Using nested PCR, N53Q mutation was prepared in the N-terminal region of the rat μ-opioid receptor cDNA and cloned into the pcDNA3 vector. The plasmids containing the wild-type and mutated receptor cDNA were transfected into the COS-7 cells. Intracellular cAMP was measured in the morphine-treated and untreated transfected cells using an ELISA kit. Plasmid expression was evaluated using X-gal staining. Intracellular concentration of cAMP in the N53Q-mutated cells was not significantly different from the wild-type. The expression of the transfected plasmids was confirmed. Therefore, based on these results, it seems that glycosylation at the N53 site of the rat μ-opioid receptor does not influence the function of this receptor significantly.     

The Production of a Stably Transformed Insect Cell Line Expressing An Invertebrate GABAA Receptor β-Subunit

Abstract

We have produced a stable insect cell line derived from Spodoptera frugiperda (Sf9) cells expressing a cDNA encoding a β-subunit of the Lymnaea stagnalis GABA_A receptor. The cDNA was randomly integrated into the insect cell genome under the control of a baculovirus immediate early gene (IE-1) promoter. Stable cell lines were established by transformation of Sf9 cells with the expression vector pIEK1.LGβ1 together with a plasmid encoding a selectable marker which confers neomycin (G418) resistance. Following growth in the presence of G418, neomycin resistant clones were selected, amplified and analysed for the presence of functional GABA-gated chloride channels. Electrophysiological analysis of one cell line showed the presence of a picrotoxin-sensitive chloride channel not present in control Sf9 cells. These channels were also sensitive to GABA, albeit at relatively high (mM) concentrations.     

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

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