ABSTRACT
BACKGROUND AND PURPOSE: It is assumed that ATP induces closure of the binding jaw of ligand-gated P2X receptors, which eventually results in the opening of the membrane channel and the flux of cations. Immobilization by cysteine mutagenesis of the binding jaw inhibited ATP-induced current responses, but did not allow discrimination between disturbances of binding, gating, subunit assembly or trafficking to the plasma membrane. EXPERIMENTAL APPROACH: A molecular model of the pain-relevant human (h)P2X3 receptor was used to identify amino acid pairs, which were located at the lips of the binding jaw and did not participate in agonist binding but strongly approached each other even in the absence of ATP. KEY RESULTS: A series of cysteine double mutant hP2X3 receptors, expressed in HEK293 cells or Xenopus laevis oocytes, exhibited depressed current responses to α,ß-methylene ATP (α,ß-meATP) due to the formation of spontaneous inter-subunit disulfide bonds. Reducing these bonds with dithiothreitol reversed the blockade of the α,ß-meATP transmembrane current. Amino-reactive fluorescence labelling of the His-tagged hP2X3 receptor and its mutants expressed in HEK293 or X. laevis oocytes demonstrated the formation of inter-subunit cross links in cysteine double mutants and, in addition, confirmed their correct trimeric assembly and cell surface expression. CONCLUSIONS AND IMPLICATIONS: In conclusion, spontaneous tightening of the binding jaw of the hP2X3 receptor by inter-subunit cross-linking of cysteine residues substituted at positions not directly involved in agonist binding inhibited agonist-evoked currents without interfering with binding, subunit assembly or trafficking.
Subject(s)
Adenosine Triphosphate/analogs & derivatives , Models, Molecular , Purinergic P2X Receptor Agonists/pharmacology , Receptors, Purinergic P2X3 , Adenosine Triphosphate/pharmacology , Animals , HEK293 Cells , Humans , Ion Channel Gating , Mutation , Oocytes , Protein Conformation , Receptors, Purinergic P2X3/chemistry , Receptors, Purinergic P2X3/genetics , Receptors, Purinergic P2X3/physiology , Xenopus laevisABSTRACT
BACKGROUND AND PURPOSE: In mammalian cells, the anti-parasitic drug ivermectin is known as a positive allosteric modulator of the ATP-activated ion channel P2X4 and is used to discriminate between P2X4- and P2X7-mediated cellular responses. In this paper we provide evidence that the reported isoform selectivity of ivermectin is a species-specific phenomenon. EXPERIMENTAL APPROACH: Complementary electrophysiological and fluorometric methods were applied to evaluate the effect of ivermectin on recombinantly expressed and on native P2X7 receptors. A biophysical characterization of ionic currents and of the pore dilation properties is provided. KEY RESULTS: Unexpectedly, ivermectin potentiated currents in human monocyte-derived macrophages that endogenously express hP2X7 receptors. Likewise, currents and [Ca(2+) ](i) influx through recombinant human (hP2X7) receptors were potently enhanced by ivermectin at submaximal or saturating ATP concentrations. Since intracellular ivermectin did not mimic or prevent its activity when applied to the bath solution, the binding site of ivermectin on hP2X7 receptors appears to be accessible from the extracellular side. In contrast to currents through P2X4 receptors, ivermectin did not cause a delay in hP2X7 current decay upon ATP removal. Interestingly, NMDG(+) permeability and Yo-Pro-1 uptake were not affected by ivermectin. On rat or mouse P2X7 receptors, ivermectin was only poorly effective, suggesting a species-specific mode of action. CONCLUSIONS AND IMPLICATIONS: The data indicate a previously unrecognized species-specific modulation of human P2X7 receptors by ivermectin that should be considered when using this cell-biological tool in human cells and tissues.
Subject(s)
Antiparasitic Agents/pharmacology , Ivermectin/pharmacology , Receptors, Purinergic P2X7/physiology , Adenosine Triphosphate/pharmacology , Allosteric Regulation/drug effects , Animals , Astrocytes/drug effects , Astrocytes/physiology , Benzoxazoles/metabolism , Calcium/physiology , Cells, Cultured , Fluorescent Dyes/metabolism , HEK293 Cells , Humans , Macrophages/drug effects , Macrophages/physiology , Mice , Mice, Inbred C57BL , Monocytes/cytology , Quinolinium Compounds/metabolism , Rats , Rats, Wistar , Receptors, Purinergic P2X4/physiology , Species SpecificityABSTRACT
Human P2X7 receptors (hP2X7Rs) belong to the P2X family, which opens an intrinsic cation channel when challenged by extracellular ATP. hP2X7Rs are expressed in cells of the inflammatory and immune system. During inflammation, ATP and protons are secreted into the interstitial fluid. Therefore, we investigated the effect of protons on the activation of hP2X7Rs. hP2X7Rs were expressed in Xenopus laevis oocytes and activated by the agonists ATP or benzoyl-benzoyl-ATP (BzATP) at different pH values. The protons reduced the hP2X7R-dependent cation current amplitude and slowed the current deactivation depending on the type and concentration of the agonist used. These effects can be explained by (i) the protonation of ATP, which reduces the effective concentration of the agonist ATP(4-) at the high- and low-affinity ATP activation site of the hP2XR, and (ii) direct allosteric inhibition of the hP2X7R channel opening that follows ATP(4-) binding to the low-affinity activation site. Due to the hampered activation via the low-affinity activation site, a low pH (as observed in inflamed tissues) leads to a relative increase in the contribution of the high-affinity activation site for hP2X7R channel opening.
Subject(s)
Ion Channel Gating/physiology , Protons , Receptors, Purinergic P2/metabolism , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Allosteric Regulation , Animals , Cations/metabolism , Female , Humans , Hydrogen-Ion Concentration , Oocytes/cytology , Oocytes/metabolism , Patch-Clamp Techniques , Receptors, Purinergic P2X7 , Xenopus laevisABSTRACT
Using the patch-clamp method, we studied the influence of external alkali and organic monovalent cations on the single-channel properties of the adenosine triphosphate (ATP)-activated recombinant human P2X(7) receptor. The slope conductance of the hP2X(7) channel decreased and the reversal potential was shifted to more negative values as the ionic diameter of the organic test cations increased. From the relationship between single-channel conductance and the dimensions of the inward current carrier, the narrowest portion of the pore was estimated to have a mean diameter of approximately 8.5 A. Single-channel kinetics and permeation properties remained unchanged during receptor activation by up to 1 mM ATP(4-) for >1 min, arguing against a molecular correlate of pore dilation at the single P2X(7) channel level. Substitution of extracellular Na(+) by any other alkali or organic cation drastically increased the open probability of the channels by prolonging the mean open time. This effect seems to be mediated allosterically through an extracellular voltage-dependent Na(+) binding site with a K(d) of approximately 5 mM Na(+) at a membrane potential of -120 mV. The modulation of the ATP-induced hP2X(7) receptor gating by extracellular Na(+) could be well described by altering the rate constant from the open to the neighboring closed state in a C-C-C-O kinetic receptor model. We suggest that P2X(7) receptor-induced depolarization and associated K(+)-efflux may reduce Na(+) occupancy of the regulatory Na(+) binding site and thus increase the efficacy of ATP(4-) in a feed-forward manner in P2X(7) receptor-expressing cells.
Subject(s)
Cations, Monovalent/pharmacology , Ion Channel Gating/physiology , Ion Channels/physiology , Receptors, Purinergic P2/physiology , Adenosine Triphosphate/metabolism , Adenosine Triphosphate/pharmacology , Animals , Calcium Chloride/pharmacology , Cell Membrane/drug effects , Cell Membrane/physiology , Female , Humans , Ion Channel Gating/drug effects , Ion Channels/drug effects , Multigene Family , Oocytes/drug effects , Oocytes/physiology , Patch-Clamp Techniques , Potassium/pharmacology , Receptors, Purinergic P2/drug effects , Receptors, Purinergic P2/genetics , Receptors, Purinergic P2X7 , Recombinant Proteins/drug effects , Recombinant Proteins/metabolism , Xenopus laevisABSTRACT
Human P2X7 receptors were expressed in Xenopus laevis oocytes and single channels were recorded using the patch-clamp technique in the outside-out configuration. ATP4- evoked two types of P2X7 receptor-mediated single channel currents characterized by short-lived and long-lived openings. The short- and long-lasting open states had mean open times of approximately 5 and approximately 20 ms and slope conductances near -60 mV of 9 and 13 pS, respectively. The open probabilities of the short and long openings were strongly [ATP4-]-dependent with EC50 values of approximately 0.3 mM and approximately 0.1 mM ATP4-, respectively. The channel kinetics did not change significantly during sustained P2X7 receptor activation for several minutes, as was also observed in recordings in the cell-attached patch-clamp configuration. Activation and deactivation of the short openings followed exponential time courses with time constants in the range of 20 ms, and displayed a shallow [ATP4-] dependence of the activation process. The kinetics of the short channel openings at negative membrane potentials fitted well to a linear C-C-C-O model with two ATP4- binding steps at equal binding sites with a dissociation constant Kd of 139 microM.
Subject(s)
Adenosine Triphosphate/metabolism , Ion Channel Gating/physiology , Models, Biological , Models, Chemical , Oocytes/physiology , Receptors, Purinergic P2/chemistry , Receptors, Purinergic P2/physiology , Adenosine Triphosphate/chemistry , Animals , Cells, Cultured , Computer Simulation , Kinetics , Receptors, Purinergic P2X7 , Xenopus laevisABSTRACT
Bordetella dermonecrotic toxin (DNT) catalyzes the transglutamination of glutamine-63/61 of Rho GTPases, thereby constitutively activating Rho proteins. Here we identified second substrates for transglutamination of RhoA by DNT. The enzymatically active fragment of DNT (residues 1136 to 1451, DeltaDNT) induced the incorporation of L-[(14)C]lysine in RhoA in a concentration-dependent manner. Also, Rac and Cdc42, but not Ras, were transglutaminated with lysine by DeltaDNT. Transglutamination of the GTPase with L-lysine inhibited intrinsic and Rho-GAP-stimulated GTP hydrolysis of RhoA. In contrast to lysine, treatment of RhoA with alanine, arginine, and glutamine were not able to substitute for lysine in the transglutamination reaction. DNT increased the incorporation of L-[(14)C]lysine into embryonic bovine lung cells. Microinjection of GST-RhoA together with the enzymatically active DNT fragment into Xenopus oocytes, subsequent affinity purification of modified GST-RhoA, and mass spectrometry identified attachment of putrescine or spermidine at glutamine-63 of RhoA. A comparison of putrescine, spermidine, and lysine as substrates for DNT-induced transglutamination of RhoA revealed that lysine is a preferred second substrate at least in vitro.
Subject(s)
Bacterial Toxins/metabolism , Bordetella/enzymology , Transglutaminases/metabolism , Virulence Factors, Bordetella , rho GTP-Binding Proteins/metabolism , Animals , Cross-Linking Reagents , Lysine/metabolism , Microinjections , Oocytes , Polyamines/metabolism , Putrescine/metabolism , Recombinant Fusion Proteins/metabolism , Spermidine/metabolism , Substrate Specificity , Xenopus , rho GTP-Binding Proteins/antagonists & inhibitors , rho GTP-Binding Proteins/genetics , rhoA GTP-Binding Protein/antagonists & inhibitors , rhoA GTP-Binding Protein/genetics , rhoA GTP-Binding Protein/metabolismABSTRACT
The inhibitory glycine receptor (GlyR) in developing spinal neurones is internalized efficiently upon antagonist inhibition. Here we used surface labeling combined with affinity purification to show that homopentameric alpha1 GlyRs generated in Xenopus oocytes are proteolytically nicked into fragments of 35 and 13 kDa upon prolonged incubation. Nicked GlyRs do not exist at the cell surface, indicating that proteolysis occurs exclusively in the endocytotic pathway. Consistent with this interpretation, elevation of the lysosomal pH, but not the proteasome inhibitor lactacystin, prevents GlyR cleavage. Prior to internalization, alpha1 GlyRs are conjugated extensively with ubiquitin in the plasma membrane. Our results are consistent with ubiquitination regulating the endocytosis and subsequent proteolysis of GlyRs residing in the plasma membrane. Ubiquitin-conjugating enzymes thus may have a crucial role in synaptic plasticity by determining postsynaptic receptor numbers.
Subject(s)
Acetylcysteine/analogs & derivatives , Cell Membrane/metabolism , Glycine/metabolism , Macrolides , Ubiquitin/metabolism , Acetylcysteine/metabolism , Acetylcysteine/pharmacology , Animals , Anti-Bacterial Agents/pharmacology , Cysteine Endopeptidases , DNA, Complementary/metabolism , Electrophoresis, Gel, Two-Dimensional , Electrophoresis, Polyacrylamide Gel , Endoplasmic Reticulum/metabolism , Enzyme Inhibitors/pharmacology , Histidine/chemistry , Hydrogen-Ion Concentration , Lysine/chemistry , Lysosomes/metabolism , Models, Biological , Multienzyme Complexes/antagonists & inhibitors , Mutagenesis, Site-Directed , Oocytes/metabolism , Phenylmethylsulfonyl Fluoride/pharmacology , Protease Inhibitors/pharmacology , Proteasome Endopeptidase Complex , Protein Binding , RNA, Complementary/metabolism , XenopusABSTRACT
Antagonistic effects of the novel suramin analogue 4,4',4",4"'-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid (NF449) were studied on contractions of the rat vas deferens elicited by alpha,beta-methylene ATP (alphabetameATP; mediated by P2X1 receptors), contractions of the guinea-pig ileal longitudinal smooth muscle elicited by alphabetameATP (mediated by P2X3 receptors) or adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS; mediated by P2Y1 receptors), ATP-induced increases of [Ca2+]i in human embryonic kidney (HEK) 293 cells (mediated by P2Y2 receptors), inward currents evoked by ATP in follicle cell-free Xenopus laevis oocytes expressing rP2X1 or rP2X3 receptors and degradation of ATP by ecto-nucleotidases in folliculated Xenopus laevis oocytes. In addition, NF449 was examined for its P2 receptor specificity in rat vas deferens (alpha1A-adrenoceptors) and guinea-pig ileum (histamine H1 and muscarinic M3 receptors). At native (pIC50=7.15) and recombinant (pIC50=9.54) P2X1 receptors, NF449 was a highly potent antagonist. The P2X3 receptors present in guinea-pig ileum (pIC50=5.04) or expressed in oocytes (pIC50 approximately 5.6) were much less sensitive for NF449. It also was a very weak antagonist at P2Y1 receptors in guinea-pig ileum (pIC50=4.85) and P2Y2 receptors in HEK 293 cells (pIC50=3.86), and showed very low inhibitory potency on ecto-nucleotidases (pIC50<3.5). NF449 (100 microM) did not interact with alpha1A-adrenoceptors or histamine H1 and muscarinic M3 receptors. Thus, the antagonism by NF449 is highly specific for P2 receptors. In conclusion, the subnanomolar potency at rP2X1 receptors and the rank order of potency, P2X1 >> P2X3 > P2Y1 > P2Y2 > ecto-nucleotidases, make NF449 unique among the P2 receptor antagonists reported to date. NF449 may fill the long-standing need for a P2X1-selective radioligand.
Subject(s)
Benzenesulfonates/pharmacology , Purinergic P2 Receptor Antagonists , Suramin/analogs & derivatives , Animals , Guinea Pigs , Ileum/drug effects , Ileum/metabolism , Male , Muscle Contraction/drug effects , Muscle, Smooth/drug effects , Rats , Receptors, Purinergic P2X , Suramin/pharmacology , Vas Deferens/drug effects , Vas Deferens/metabolism , Xenopus laevisABSTRACT
1. The effect of the agonist ATP on whole cell currents of Xenopus oocytes expressing either the wild-type human P2X(7) receptor (hP2X(7)), an N-terminally hexahistidyl-tagged hP2X(7) receptor (His-hP2X(7)), or a truncated His-hP2X(7) receptor (His-hP2X(7)DeltaC) lacking the C-terminal 156 amino acids was investigated using the two-microelectrode voltage clamp technique. 2. The activation time course of the wild-type hP2X(7) receptor can be described as the sum of an exponentially growing and an additional almost linearly activating current component. 3. The amplitude of the exponentially activating current component of the wild-type hP2X(7) receptor displayed a biphasic dependence on the agonist concentration, which could be best approximated by a model of two equal high-sensitivity and two equal low-sensitivity non-cooperative activation sites with apparent dissociation constants of about 4 and 200 microM free ATP(4-), respectively. 4. The linearly activating current was monophasically dependent on the agonist concentration with an apparent dissociation constant of about 200 microM. 5. The contribution of the low-sensitivity sites to current kinetics was reduced or almost abolished in oocytes expressing His-hP2X(7) or His-hP2X(7)DeltaC. 6. Our data indicate that the hP2X(7) receptor possesses at least two types of activation sites, which differ in ATP(4-) sensitivity by a factor of 50. The degree of occupation of these two sites influences both activation and deactivation kinetics. Both N- and C-terminal domains appear to be important determinants of the current elicited by activation of the sites with low ATP sensitivity, but not for that mediated by the highly ATP-sensitive sites.
Subject(s)
Adenosine Triphosphate/pharmacology , Receptors, Purinergic P2/drug effects , Receptors, Purinergic P2/physiology , Animals , Cations, Divalent/pharmacology , Dose-Response Relationship, Drug , Drug Synergism , Electric Conductivity , Extracellular Space/metabolism , Female , Humans , Kinetics , Magnesium/physiology , Oocytes , Osmolar Concentration , Peptide Fragments/pharmacology , Purinergic P2 Receptor Agonists , Reaction Time , Receptors, Purinergic P2/chemistry , Receptors, Purinergic P2X7 , Sequence Tagged Sites , Time Factors , Xenopus laevisABSTRACT
Na(+)/Cl(-)-dependent neurotransmitter transporters form a superfamily of transmembrane proteins that share 12 membrane-spanning regions. To gain information about the quaternary structure of these transporter proteins, we heterologously expressed the glial glycine transporter GlyT1 and its neuronal homolog GlyT2 in Xenopus oocytes. By using metabolic labeling with [(35)S]methionine or surface labeling with a plasma membrane impermeable reagent followed by affinity purification, we separately analyzed the total cellular pools of newly synthesized GlyTs and its functional plasma membrane-bound fractions. Upon blue native gel electrophoresis, the surface-localized transporter proteins were found to exist exclusively in complex-glycosylated monomeric form, whereas a significant fraction of the intracellular GlyT1 and GlyT2 was core-glycosylated and oligomeric. In contrast, even after treatment with the crosslinker glutaraldehyde, surface GlyTs failed to migrate as oligomeric proteins. These results indicate that plasma membrane-bound GlyT1 and GlyT2 are monomeric proteins. Thus, Na(+)/Cl(-)-dependent neurotransmitter transporters do not require oligomerization for substrate translocation.
Subject(s)
Amino Acid Transport Systems, Neutral , Carrier Proteins/metabolism , Glycine/metabolism , Membrane Proteins/metabolism , Animals , Carrier Proteins/genetics , Cell Membrane/metabolism , Glycine Plasma Membrane Transport Proteins , Membrane Proteins/genetics , Oligopeptides/genetics , Oligopeptides/metabolism , Oocytes/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Xenopus laevisABSTRACT
Clostridium botulinum C3 is the prototype of the family of the C3-like transferases that ADP-ribosylate exclusively RhoA, -B and -C. The ADP-ribose at Asn-41 results in functional inactivation of Rho reflected by disaggregation of the actin cytoskeleton. We report on a new C3-like transferase produced by a pathogenic Staphylococcus aureus strain. The transferase designated C3(Stau) was cloned from the genomic DNA. At the amino acid level, C3(Stau) revealed an identity of 35% to C3 from C. botulinum and Clostridium limosum exoenzyme, respectively, and of 78% to EDIN from S. aureus. In addition to RhoA, which is the target of the other C3-like transferases, C3(Stau) modified RhoE and Rnd3. RhoE was ADP-ribosylated at Asn-44, which is equivalent to Asn-41 of RhoA. RhoE and Rnd3 are members of the Rho subfamily, which are deficient in intrinsic GTPase activity and possess a RhoA antagonistic cell function. The protein substrate specificity found with recombinant Rho proteins was corroborated by expression of RhoE in Xenopus laevis oocytes showing that RhoE was also modified in vivo by C3(Stau) but not by C3 from C. botulinum. The poor cell accessibility of C3(Stau) was overcome by generation of a chimeric toxin recruiting the cell entry machinery of C. botulinum C2 toxin. The chimeric C3(Stau) caused the same morphological and cytoskeletal changes as the chimeric C. botulinum C3. C3(Stau) is a new member of the family of the C3-like transferases but is also the prototype of a subfamily of RhoE/Rnd modifying transferases.
Subject(s)
ADP Ribose Transferases/metabolism , Botulinum Toxins , GTPase-Activating Proteins/metabolism , Poly(ADP-ribose) Polymerases/metabolism , Staphylococcus aureus/enzymology , Amino Acid Sequence , Base Sequence , DNA, Bacterial , Molecular Sequence Data , Sequence Homology, Amino AcidABSTRACT
Human B lymphocytes express an ATP-gated ion channel (P2Z receptor), which shares similarities with the recently identified P2X7 receptor. Using gene specific primers, we have now isolated P2X7 cDNA from the total RNA of human B lymphocytes. This hP2X7 receptor subtype was expressed in Xenopus oocytes and electrophysiologically characterized. The hP2X7 receptor is similar to, but does not completely match, P2Z of human B cells. The hP2X7 receptors resemble the P2Z receptors with regard to the ATP concentration of half maximal activation, reproducibility, permeation characteristics and lack of desensitization of the ATP-evoked currents. However, in contrast to the native lymphocytic P2Z receptor, the time course of activation of hP2X7 displayed an additional linearly increasing current component. Furthermore, a second, small and slowly deactivating current component exists only in hP2X7 expressed in oocytes. The activation and deactivation kinetics as well as permeation characteristics of hP2X7 are different from rat P2X7 recently expressed in oocytes. Unlike in mammalian cells, hP2X7 expressed in Xenopus oocytes is not sufficient to induce large non-selective pores.
Subject(s)
B-Lymphocytes/metabolism , Receptors, Purinergic P2/metabolism , Adenosine Triphosphate/pharmacology , Animals , Base Sequence , DNA Primers/genetics , Female , Humans , In Vitro Techniques , Ion Channel Gating , Ion Channels/drug effects , Ion Channels/genetics , Ion Channels/metabolism , Kinetics , Membrane Potentials/drug effects , Oocytes/metabolism , Phenotype , Rats , Receptors, Purinergic P2/drug effects , Receptors, Purinergic P2/genetics , Receptors, Purinergic P2X7 , Recombinant Proteins/drug effects , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Species Specificity , Xenopus laevisABSTRACT
The suramin analogue 8,8'-(carbonylbis(imino-4, 1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)) bis(1,3,5-naphthalenetrisul fonic acid) (NF279) was analysed with respect to its potency and P2X receptor subtype selectivity. Two-electrode voltage-clamp measurements were performed with Xenopus laevis oocytes expressing homomultimeric rat P2X(1), P2X(2), P2X(3) and human P2X(4) receptors. For the fast desensitising P2X(1) and P2X(3) receptors, IC(50) values strongly depended on whether oocytes were pre-incubated with NF279 prior to ATP superfusion or exposed to NF279 simultaneously with ATP. With a 10 s pre-incubation period of NF279, IC(50) values of 19 nM and 1.62 microM were obtained for rat P2X(1) and P2X(3), respectively. Without pre-incubation, IC(50) values amounted to 2 microM and 85.5 microM for P2X(1) and P2X(3), respectively. For the non-desensitising rat P2X(2) receptor NF279 appeared to act as a competitive antagonist with an IC(50) value of 0.76 microM and a K(B) value of 0.36 microM, as derived from Schild analysis. P2X(4) receptors were the least sensitive subtypes for NF279 (IC(50)>300 microM). The antagonism was fully reversible at all P2X subtypes analysed. Our results indicate that NF279 is a potent P2X(1) receptor-selective and reversible antagonist.
Subject(s)
Purinergic P2 Receptor Antagonists , Suramin/analogs & derivatives , Adenosine Triphosphate/pharmacology , Animals , Female , Humans , Purinergic Antagonists , Rats , Receptors, Purinergic/physiology , Receptors, Purinergic P2/physiology , Receptors, Purinergic P2X , Receptors, Purinergic P2X3 , Receptors, Purinergic P2X4 , Suramin/pharmacology , Xenopus laevisABSTRACT
P2X(1) receptor subunits assemble in the ER of Xenopus oocytes to homotrimers that appear as ATP-gated cation channels at the cell surface. Here we address the extent to which N-glycosylation contributes to assembly, surface appearance, and ligand recognition of P2X(1) receptors. SDS-polyacrylamide gel electrophoresis (PAGE) analysis of glycan minus mutants carrying Gln instead of Asn at five individual NXT/S sequons reveals that Asn(284) remains unused because of a proline in the +4 position. The four other sites (Asn(153), Asn(184), Asn(210), and Asn(300)) carry N-glycans, but solely Asn(300) located only eight residues upstream of the predicted reentry loop of P2X(1) acquires complex-type carbohydrates. Like parent P2X(1), glycan minus mutants migrate as homotrimers when resolved by blue native PAGE. Recording of ATP-gated currents reveals that elimination of Asn(153) or Asn(210) diminishes or increases functional expression levels, respectively. In addition, elimination of Asn(210) causes a 3-fold reduction of the potency for ATP. If three or all four N-glycosylation sites are simultaneously eliminated, formation of P2X(1) receptors is severely impaired or abolished, respectively. We conclude that at least one N-glycan per subunit of either position is absolutely required for the formation of P2X(1) receptors and that individual N-glycans possess marked positional effects on expression levels (Asn(154), Asn(210)) and ATP potency (Asn(210)).
Subject(s)
Adenosine Triphosphate/metabolism , Polysaccharides/metabolism , Receptors, Purinergic P2/chemistry , Amino Acid Sequence , Animals , Glycosylation , Molecular Sequence Data , Polysaccharides/chemistry , Protein Subunits , Rats , Receptors, Purinergic P2/metabolism , Structure-Activity Relationship , Xenopus laevisABSTRACT
The effect of the suramin analogue 8,8'-(carbonylbis(imino-4, 1-phenylenecarbonylimino-4,1-phenylenecarbonylimino))bis(1,3 , 5-naphthalenetrisulfonic acid) (NF279) was analyzed on human P2X(1) and P2X(7) receptor subtypes (human P2X(1) and human P2X(7)) heterologously expressed in Xenopus oocytes using the two-microelectrode voltage-clamp technique. At activating ATP concentrations of 1 microM (human P2X(1)) and 10 microM ATP (human P2X(7)), IC(50) values of 0.05 microM and 2.8 microM were found for human P2X(1) and human P2X(7) receptors, respectively. An increase in the activating [ATP] shifted the NF279 concentration-inhibition curve rightwards for both receptors. NF279 slowed the activation of both human P2X(1) and human P2X(7) as well as the desensitization of human P2X(1). The data support a model in which desensitization of P2X(1) is dependent on preceding activation of these P2X receptors. It is concluded that NF279 acts as a competitive antagonist with much higher potency at human P2X(1) than at P2X(7) receptors. NF279 may hence be suited to discriminate between both receptors in native tissues.
Subject(s)
Purinergic P2 Receptor Antagonists , Suramin/analogs & derivatives , Animals , Female , Humans , Suramin/pharmacology , Xenopus laevisABSTRACT
Pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4',8'-disulfonat e) (PPNDS) potently antagonized P2X(1) receptor-mediated responses in rat vas deferens (pK(B)=7.43) and Xenopus laevis oocytes (pIC(50)=7. 84). It showed lower (up to 20,000-fold) inhibitory potency on ecto-nucleotidase in Xenopus oocytes and on P2Y(1) receptors in guinea-pig ileum (pA(2)=6.13). PPNDS did not interact with alpha(1A)-adrenoceptors, adenosine A(1) and A(2B), histamine H(1) and muscarinic M(3) receptors. Thus, PPNDS is a novel, specific P2 receptor antagonist and represents the pyridoxal-5'-phosphate derivative with the highest potency at P2X(1) receptors.
Subject(s)
Purinergic P2 Receptor Antagonists , Pyridoxal Phosphate/analogs & derivatives , Sulfonic Acids/pharmacology , Animals , Female , Guinea Pigs , In Vitro Techniques , Male , Pyridoxal Phosphate/pharmacology , Rats , XenopusSubject(s)
Ion Channels/chemistry , Ion Channels/physiology , Receptors, Purinergic P2/physiology , Amino Acid Sequence , Animals , Humans , Ion Channel Gating , Ligands , Molecular Sequence Data , Protein Structure, Quaternary , Receptors, Purinergic P2/chemistry , Receptors, Purinergic P2/genetics , Sequence AlignmentABSTRACT
Among suramin analogues, the properties of P2 receptor subtype blockade and ecto-nucleotidase inhibition appear to be controlled by different structural parameters (Fig. 1 and 2, Table 1; Van Rhee et al., 1994; Beukers et al., 1995; Bültmann et al., 1996; Damer et al., 1998a, 1998b; and this study): the molecular size of the compounds, the position of the sulfonic acid residues in the naphthalene rings, the substitution pattern of the benzoyl moieties and the 3'- or 4'-aminobenzoyl-linkages of the phenyl rings "1" and "2". As a result, compounds with different receptor selectivity profiles were obtained. A maximum in potency at and selectivity for P2X1 receptors is reached in NF279, which is a specific P2 receptor antagonist and the compound with the highest P2X1 vs. P2Y receptor and ecto-nucleotidase selectivity presently available.
Subject(s)
Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/pharmacology , Receptors, Purinergic P2/physiology , Suramin/analogs & derivatives , Suramin/pharmacology , Adenosine Triphosphate/pharmacokinetics , Animals , Isometric Contraction/drug effects , Ligands , Male , Purinergic P2 Receptor Agonists , Purinergic P2 Receptor Antagonists , Rats , Vas Deferens/drug effects , Vas Deferens/innervation , Vas Deferens/physiologySubject(s)
Adenosine Triphosphate/pharmacology , Adjuvants, Immunologic/pharmacology , B-Lymphocytes/physiology , Receptors, Purinergic/physiology , Cell Line, Transformed , Cells, Cultured , Herpesvirus 4, Human , Humans , Membrane Potentials/drug effects , Membrane Potentials/physiology , Palatine Tonsil/immunology , Receptors, Purinergic/drug effectsABSTRACT
The inhibitory glycine receptor (GlyR) is a pentameric transmembrane protein composed of homologous alpha and beta subunits. Single expression of alpha subunits generates functional homo-oligomeric GlyRs, whereas the beta subunit requires a co-expressed alpha subunit to assemble into hetero-oligomeric channels of invariant stoichiometry (alpha(3)beta(2)). Here, we identified eight amino acid residues within the N-terminal region of the alpha1 subunit that are required for the formation of homo-oligomeric GlyR channels. We show that oligomerization and N-glycosylation of the alpha1 subunit are required for transit from the endoplasmic reticulum to the Golgi apparatus and later compartments, and that addition of simple carbohydrate side chains occurs prior to GlyR subunit assembly. Our data are consistent with both intersubunit surface and conformational differences determining the different assembly behaviour of GlyR alpha and beta subunits.