Involvement of the cysteine-rich head domain in activation and desensitization of the P2X1 receptor.
ABSTRACT: P2X receptors (P2XRs) are ligand-gated ion channels activated by extracellular ATP. Although the crystal structure of the zebrafish P2X4R has been solved, the exact mode of ATP binding and the conformational changes governing channel opening and desensitization remain unknown. Here, we used voltage clamp fluorometry to investigate movements in the cysteine-rich head domain of the rat P2X1R (A118-I125) that projects over the proposed ATP binding site. On substitution with cysteine residues, six of these residues (N120-I125) were specifically labeled by tetramethyl-rhodamine-maleimide and showed significant changes in the emission of the fluorescence probe on application of the agonists ATP and benzoyl-benzoyl-ATP. Mutants N120C and G123C showed fast fluorescence decreases with similar kinetics as the current increases. In contrast, mutants P121C and I125C showed slow fluorescence increases that seemed to correlate with the current decline during desensitization. Mutant E122C showed a slow fluorescence increase and fast decrease with ATP and benzoyl-benzoyl-ATP, respectively. Application of the competitive antagonist 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) resulted in large fluorescence changes with the N120C, E122C, and G123C mutants and minor or no changes with the other mutants. Likewise, TNP-ATP-induced changes in control mutants distant from the proposed ATP binding site were comparably small or absent. Combined with molecular modeling studies, our data confirm the proposed ATP binding site and provide evidence that ATP orients in its binding site with the ribose moiety facing the solution. We also conclude that P2XR activation and desensitization involve movements of the cysteine-rich head domain.
Project description:The Pgp (P-glycoprotein) multidrug transporter couples ATP hydrolysis at two cytoplasmic NBDs (nucleotide-binding domains) to the transport of hydrophobic compounds. Orthovanadate (V(i)) and fluoroaluminate (AlF(x)) trap nucleotide in one NBD by forming stable catalytically inactive complexes (Pgp-M2+-ADP-X), which are proposed to resemble the catalytic transition state, whereas the complex formed by beryllium fluoride (BeF(x)) is proposed to resemble the ground state. We studied the trapped complexes formed via incubation of Pgp with ATP (catalytically forward) or ADP (reverse) and V(i), BeF(x) or AlF(x) using Mg2+ or Co2+ as the bivalent cation. Quenching of intrinsic Pgp tryptophan fluorescence by acrylamide, iodide and caesium indicated that conformational changes took place upon formation of the trapped complexes. Trapping with V(i) and ATP led to a 6-fold increase in the acrylamide quenching constant, K(SV), suggesting that large conformational changes take place in the Pgp transmembrane regions on trapping in the forward direction. Trapping with V(i) and ADP gave only a small change in quenching, indicating that the forward- and reverse-trapped complexes are different. TNP (trinitrophenyl)-ATP/TNP-ADP interacted with all of the trapped complexes, however, the fluorescence enhancement differed for the trapped states, suggesting a change in polarity in the nucleotide-binding sites. The nucleotide-binding site of the BeF(x)-trapped complex was much more polar than that of the V(i) and AlF(x) complexes. Functionally, all the trapped complexes were able to bind drugs and TNP-nucleotides with unchanged affinity compared with native Pgp.
Project description:Adenylyl cyclases (ACs) catalyze the conversion of ATP into the second messenger cAMP and play a key role in signal transduction. In a recent study (Mol Pharmacol 70:878-886, 2006), we reported that 2',3'-O-(2,4,6-trinitrophenyl)-substituted nucleoside 5'-triphosphates (TNP-NTPs) are potent inhibitors (K(i) values in the 10 nM range) of the purified catalytic subunits VC1 and IIC2 of membranous AC (mAC). The crystal structure of VC1:IIC2 in complex with TNP-ATP revealed that the nucleotide binds to the catalytic site with the TNP-group projecting into a hydrophobic pocket. The aims of this study were to analyze the interaction of TNP-nucleotides with VC1:IIC2 by fluorescence spectroscopy and to analyze inhibition of mAC isoforms, soluble AC (sAC), soluble guanylyl cyclase (sGC), and G-proteins by TNP-nucleotides. Interaction of VC1:IIC2 with TNP-NDPs and TNP-NTPs resulted in large fluorescence increases that were differentially reduced by a water-soluble forskolin analog. TNP-ATP turned out to be the most potent inhibitor for ACV (K(i), 3.7 nM) and sGC (K(i), 7.3 nM). TNP-UTP was identified as the most potent inhibitor for ACI (K(i), 7.1 nM) and ACII (K(i), 24 nM). TNP-NTPs inhibited sAC and GTP hydrolysis by G(s)- and G(i)-proteins only with low potencies. Molecular modeling revealed that TNP-GTP and TNP-ATP interact very similarly, but not identically, with VC1:IIC2. Collectively, our data show that TNP-nucleotides are useful fluorescent probes to monitor conformational changes in VC1:IIC2 and that TNP-NTPs are a promising starting point to develop isoform-selective AC and sGC inhibitors. TNP-ATP is the most potent sGC inhibitor known so far.
Project description:P2X receptors are trimeric eukaryotic ATP-gated cation channels. Extracellular ATP-their physiological ligand-is released as a neurotransmitter and in conditions of cell damage such as inflammation, and substantial evidence implicates P2X receptors in diseases including neuropathic pain, cancer, and arthritis. In 2009, the first P2X crystal structure, Danio rerio P2X4 in the apo- state, was published, and this was followed in 2012 by the ATP-bound structure. These structures transformed our understanding of the conformational changes induced by ATP binding and the mechanism of ligand specificity, and enabled homology modeling of mammalian P2X receptors for ligand docking and rational design of receptor modulators. P2X receptors are attractive drug targets, and a wide array of potent, subtype-selective modulators (mostly antagonists) have been developed. In 2016, crystal structures of human P2X3 in complex with the competitive antagonists TNP-ATP and A-317491, and Ailuropoda melanoleuca P2X7 in complex with a series of allosteric antagonists were published, giving fascinating insights into the mechanism of channel antagonism. In this article we not only summarize current understanding of small-molecule modulator binding at P2X receptors, but also use this information in combination with previously published structure-function data and molecular docking experiments, to hypothesize a role for the dorsal fin loop region in differential ATP potency, and describe novel, testable binding conformations for both the semi-selective synthetic P2X7 agonist 2'-(3')-O-(4-benzoyl)benzoyl ATP (BzATP), and the P2X4-selective positive allosteric modulator ivermectin. We find that the distal benzoyl group of BzATP lies in close proximity to Lys-127, a residue previously implicated in BzATP binding to P2X7, potentially explaining the increased potency of BzATP at rat P2X7 receptors. We also present molecular docking of ivermectin to rat P2X4 receptors, illustrating a plausible binding conformation between the first and second transmembrane domains which not only tallies with previous mutagenesis studies, but would also likely have the effect of stabilizing the open channel structure, consistent with the mode of action of this positive allosteric modulator. From our docking simulations and analysis of sequence homology we propose a series of mutations likely to confer ivermectin sensitivity to human P2X1.
Project description:The analogue of ATP, 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP), binds tightly to pig muscle 3-phosphoglycerate kinase. A dissociation constant Kd of 0.0095 +/- 0.0015 mM was determined by fluorimetric titration on the basis of 1:1 stoichiometry. TNP-ATP is a strong competitive inhibitor towards MgATP and MgADP with a Ki of 0.008 +/- 0.001 mM for both substrates. It is also a mixed-type inhibitor towards 3-phosphoglycerate with similar inhibition constants. Binding of TNP-ATP to 3-phosphoglycerate kinase is accompanied by a tenfold intensity increase and a blue shift of about 20 nm in its fluorescence emission spectrum and a shift of the pK of its trinitrophenyl group towards a more acidic pH. These findings suggest that the negatively charged trinitrophenyl group of TNP-ATP significantly contributes to the binding of the analogue. By stepwise replacement of the fluorescent TNP-ATP, the dissociation constants (Kd) for ADP and MgADP binding were determined and found to be 0.78 +/- 0.08 and 0.048 +/- 0.006 mM respectively, which are consistent with the values previously determined by equilibrium dialysis [Molnár and Vas (1993) Biochem J. 293, 595-599]. In similar competitive-titration experiments, ATP and MgATP did not completely substitute for TNP-ATP. For the fraction of the analogue that could be substituted, the dissociation constants for MgATP and ATP were estimated to be 0.27 +/- 0.09 and 0.33 +/- 0.15 mM respectively, close to the values determined by equilibrium dialysis. Using the same method, a significant weakening of binding of both (Mg)ADP and (Mg)ATP could be detected in the presence of 3-phosphoglycerate: their respective Kd values became 0.34 +/- 0.04 and 0.51 +/- 0.22 mM. The reciprocal effect, i.e. weakening of 3-phosphoglycerate binding in the presence of the nucleotide substrates, has been observed previously [Vas and Batke (1984) Eur. J. Biochem. 139, 115-123]. Similarly, a much weaker binding of (Mg)ATP could be observed in the presence of 1,3-bisphosphoglycerate (Kd = 2.30 +/- 0.68 mM). The possible reason for the mutual weakening of substrate binding is discussed in the light of the available structural data.
Project description:Calcium-dependent domain movements of the actuator (A) and nucleotide (N) domains of the SERCA2a isoform of the Ca-ATPase were assessed using constructs containing engineered tetracysteine binding motifs, which were expressed in insect High-Five cells and subsequently labeled with the biarsenical fluorophore 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH-EDT(2)). Maximum catalytic function is retained in microsomes isolated from High-Five cells and labeled with FlAsH-EDT(2). Distance measurements using the nucleotide analog 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP), which acts as a fluorescence resonance energy transfer (FRET) acceptor from FlAsH, identify a 2.4 A increase in the spatial separation between the N- and A-domains induced by high-affinity calcium binding; this structural change is comparable to that observed in crystal structures. No significant distance changes occur across the N-domain between FlAsH and TNP-ATP, indicating that calcium activation induces rigid body domain movements rather than intradomain conformational changes. Calcium-dependent decreases in the fluorescence of FlAsH bound, respectively, to either the N- or A-domains indicate coordinated and noncooperative domain movements, where both A- and N-domains display virtually identical calcium dependencies (i.e., K(d) = 4.8 +/- 0.4 microM). We suggest that occupancy of a single high-affinity calcium binding site induces the rearrangement of the A- and N-domains of the Ca-ATPase to form an intermediate state, which facilitates phosphoenzyme formation from ATP upon occupancy of the second high-affinity calcium site.
Project description:Structural studies of P2X receptors show a novel U shaped ATP orientation following binding. We used voltage clamp fluorometry (VCF) and molecular dynamics (MD) simulations to investigate agonist action. For VCF the P2X1 receptor (P2X1R) K190C mutant (adjacent to the agonist binding pocket) was labelled with the fluorophore MTS-TAMRA and changes in fluorescence on agonist treatment provided a real time measure of conformational changes. Studies with heteromeric channels incorporating a key lysine mutation (K68A) in the ATP binding site demonstrate that normally three molecules of ATP activate the receptor. The time-course of VCF responses to ATP, 2'-deoxy ATP, 3'-deoxy ATP, Ap5A and ??meATP were agonist dependent. Comparing the properties of the deoxy forms of ATP demonstrated the importance of the 2' hydroxyl group on the ribose ring in determining agonist efficacy consistent with MD simulations showing that it forms a hydrogen bond with the ?-phosphate oxygen stabilizing the U-shaped conformation. Comparison of the recovery of fluorescence on agonist washout, with channel activation to a second agonist application for the partial agonists Ap5A and ??meATP, showed a complex relationship between conformational change and desensitization. These results highlight that different agonists induce distinct conformational changes, kinetics and recovery from desensitization at P2X1Rs.
Project description:1. Fast-desensitizing P2X(3) receptors of nociceptive dorsol root ganglion (DRG) neurons are thought to mediate pain sensation. Since P2X(3) receptor efficiency is powerfully modulated by desensitization, its underlying properties were studied with patch-clamp recording. 2. On rat cultured DRG neurons, 2 s application of ATP (EC(50)=1.52 microm), ADP (EC(50)=1.1 microm) or alpha,beta-meATP (EC(50)=1.78 microm) produced similar inward currents that fully desensitized, at the same rate, back to baseline. Recovery from desensitization was much slower after ATP and ADP than after alpha,beta-meATP and, in all cases, it had sigmoidal time course. 3. By alternating the application of ATP and alpha,beta-meATP, we observed complete cross-desensitization indicating that these agonists activated the same receptors. This notion was confirmed by the similar antagonism induced by 2', 3'-O-(2,4,6,trinitrophenyl)-adenosine triphosphate (TNP-ATP). 4. Recovery from desensitization elicited by ATP was unexpectedly shaped by transient application of alpha,beta-methylene-adenosine triphosphate (alpha,beta-meATP), and vice versa. Thus, short-lasting, full desensitization produced by alpha,beta-meATP protected receptors from long-lasting desensitization induced by subsequent ATP applications. ATP and ADP had similar properties of recovery from desensitization. 5. Low nm concentrations of alpha,beta-meATP (unable to evoke membrane currents) could speed up recovery from ATP-induced desensitization, while low nm concentrations of ATP enhanced it. Ambient ATP levels were found to be in the pm range (52+/-3 pm). 6. The phenomenon of cross-desensitization and protection was reproduced by rP2X(3) receptors expressed by rat osteoblastic cell 17/2.8 or human embryonic kidney cell 293 cells, indicating P2X(3) receptor specificity. 7. It is suggested that transient application of an agonist that generates rapid recovery from desensitization, is a novel, powerful tool to modulate P2X(3) receptor responsiveness to the natural agonist ATP.
Project description:A calixarene conjugate possessing a tetrapyrenyl moiety at its upper rim (R) is designed as a receptor for sensing trinitrophenol (TNP). To understand the role of the calixarene platform and that of pyrenyl moieties in R, two other control molecules were synthesized. These are as follows: the one possessing a tetraphenyl moiety in place of tetrapyrenyl (R 1 ) and the other one is a p-pyrenyl-hydroxy benzene (R 2 ) that is devoid of the calixarene platform. The R shows high sensitivity toward TNP in tetrahydrofuran (THF) over eleven other nitroaromatic compounds (NACs) studied by exhibiting large fluorescence enhancement and hence is selective to TNP over the other NACs studied. However, the control molecules R 1 and R 2 showed only marginal fluorescence enhancement, supporting the need of a calixarene platform and the presence of a tetrapyrenyl moiety in the receptor system for the selective sensing of TNP. Further, R 1 and R 2 are not suitable for sensing, since these exhibit similar fluorescence response over several NACs studied. The binding of TNP by R has been addressed by fluorescence titration and isothermal titration calorimetry. The nature of the complexation of TNP by R has been revealed by the computational calculations, wherein the data showed the entrapment of TNP by two adjacent pyrene moieties via ?-? stacking interactions. Such host-guest complexation is expected to restrict the mobility of the pyrene moieties present in R. The reduction of the flexibility of the pyrenyl moieties of R upon TNP binding is evidenced by the 1H NMR spectral study, wherein this acts as an additional evidence for the complexation. In the present study, the sensing of TNP by R has been shown in THF solution, on the surface of silica gel and the cellulose paper to result in lowest detection limits (LODs) of 1.5, 3.5, and 6.5 ?M, respectively. Even the solid mixture of R and TNP showed LOD of 2.1 ?mol. Since R is expected to show supramolecular aggregation that is dependent on the guest species, the corresponding details were probed by microscopy techniques, using scanning electron microscopy, atomic force microscopy, and transmission electron microscopy methods, and significant changes in the aggregation of R upon interaction with TNP were found. Such aggregation is responsible for the observed fluorescence enhancement. Thus, the tetrapyrenyl calixarene conjugate (R) acts as a sensitive and robust platform for selective detection of TNP from a mixture of nitroaromatic compounds (NACs) wherein the fluorescence intensities can be imaged and managed by a cellular phone.
Project description:Adenosine 5' triphosphate (ATP) is a ubiquitous extracellular signaling messenger. Here, we describe a method for in-vivo imaging of extracellular ATP with high spatiotemporal resolution. We prepared a comprehensive set of cysteine-substitution mutants of ATP-binding protein, Bacillus FoF1-ATP synthase ? subunit, labeled with small-molecule fluorophores at the introduced cysteine residue. Screening revealed that the Cy3-labeled glutamine-105 mutant (Q105C-Cy3; designated ATPOS) shows a large fluorescence change in the presence of ATP, with submicromolar affinity, pH-independence, and high selectivity for ATP over ATP metabolites and other nucleotides. To enable in-vivo validation, we introduced BoNT/C-Hc for binding to neuronal plasma membrane and Alexa Fluor 488 for ratiometric measurement. The resulting ATPOS complex binds to neurons in cerebral cortex of living mice, and clearly visualized a concentrically propagating wave of extracellular ATP release in response to electrical stimulation. ATPOS should be useful to probe the extracellular ATP dynamics of diverse biological processes in vivo.
Project description:PURPOSE. To identify the type of purinergic receptors activated by adenosine triphosphate (ATP) in rat lacrimal gland and to determine their role in protein secretion. METHODS. Purinergic receptors were identified by RT-PCR, Western blot analysis, and immunofluorescence techniques. Acini from rat lacrimal gland were isolated by collagenase digestion. Acini were incubated with the fluorescence indicator fura-2 tetra-acetoxylmethyl ester, and intracellular [Ca(2+)] ([Ca(2+)](i)) was determined. Protein secretion was measured by fluorescence assay. RESULTS. The authors previously showed that P2X(7)receptors were functional in the lacrimal gland. In this study, they show that P2X(1-4) and P2X(6)receptors were identified in the lacrimal gland by RT-PCR, Western blot, and immunofluorescence analyses. P2X(5) receptors were not detected. ATP increased [Ca(2+)](i) and protein secretion in a concentration-dependent manner. Removal of extracellular Ca(2+) significantly reduced the ATP-stimulated increase in [Ca(2+)](i). Repeated applications of ATP caused desensitization of the [Ca(2+)](i) response. Incubation with the P2X(1) receptor inhibitor NF023 did not alter ATP-stimulated [Ca(2+)](i). Incubation with zinc, which potentiates P2X(2) and P2X(4) receptor responses, or lowering the pH to 6.8, which potentiates P2X(2) receptor responses, did not alter the ATP-stimulated [Ca(2+)](i). P2X(3) receptor inhibitors A-317491 and TNP-ATP significantly decreased ATP-stimulated [Ca(2+)](i) and protein secretion, whereas the P2X(3) receptor agonist ?,? methylene ATP significantly increased them. The P2X(7) receptor inhibitor A438079 had no effect on ATP-stimulated [Ca(2+)](i) at 10(-6) M but did have an effect at 10(-4) M. CONCLUSIONS. Purinergic receptors P2X(1-4) and P2X(6) are present in the lacrimal gland. ATP uses P2X(3) and P2X(7) receptors to stimulate an increase in [Ca(2+)](i) and protein secretion.