Lack of evidence for direct phosphorylation of recombinantly expressed P2X2 and P2X3 receptors by protein kinase C P2X3 and P2X2 +3 receptors are present on sensory neurons , where they contribute not only to transient nociceptive responses , but also to hypersensitivity underlying pathological pain states elicited by nerve injuries . Increased signalling through P2X3 and P2X2 +3 receptors may arise from an increased routing to the plasma membrane and/or gain of function of pre-existing receptors . An obvious effector mechanism for functional modulation is protein kinase C -LRB- PKC -RRB- - mediated phosphorylation , since all P2X family members share a conserved consensus sequence for PKC , TXR/K , within the intracellularly located N-terminal domain . Contradictory reports have been published regarding the exact role of this motif . In the present study , we confirm that site-directed elimination of the potential phosphor-acceptor threonine or the basic residue in the P +2 position of the TXR/K sequence accelerates desensitization of P2X2 receptors and abolishes P2X3 receptor function . Moreover , the PKC activator phorbol 12-myristate 13-acetate increased P2X3 -LRB- but not P2X2 -RRB- receptor-mediated currents . Biochemically , however , we were unable to demonstrate by various experimental approaches a direct phosphorylation of wild-type P2X2 and P2X3 receptors expressed in both Xenopus laevis oocytes and HEK293 cells . In conclusion , our data support the view that the TXR/K motif plays an important role in P2X function and that phorbol 12-myristate 13-acetate is capable of modulating some P2X receptor subtypes . The underlying mechanism , however , is unlikely to involve direct PKC-mediated P2X receptor phosphorylation . Introduction P2X receptors constitute an abundant class of ligand-gated ion channels , which respond to extracellular ATP and related nucleotides with the opening of an intrinsic pore permeable to Na + , K + and Ca2 + -LSB- 1 , 2 -RSB- . A family encompassing seven P2X subunit genes , designated P2X1 -- 7 , has been identified in rodents and mammals . Six of the seven subunit isoforms -LRB- P2X1-P2X5 and P2X7 -RRB- are able to assemble into homotrimeric receptor channels -LSB- 3 -- 8 -RSB- with distinct pharmacological and electrophysiological phenotypes -LSB- 9 -RSB- . According to their sensitivity to the synthetic ATP analogue α,β-methylene adenosine 5 ′ - triphosphate -LRB- αβ-meATP -RRB- and the rate of current desensitization , P2X receptors are generally subgrouped into at least two categories : -LRB- 1 -RRB- rapidly desensitizing and αβ-meATP sensitive -LRB- P2X1 and P2X3 -RRB- and -LRB- 2 -RRB- slowly or non-desensitizing and αβ-meATP insensitive -LRB- P2X2 , P2X4 and P2X7 -RRB- receptors . The term `` non-desensitizing '' means that the currents are maintained for at least a few seconds in the continuous presence of agonist . A peculiar phenotype is inherent to heteromeric P2X2/P2X3 -LRB- P2X2 +3 -RRB- receptors , which feature αβ-meATP sensitivity combined with a non-desensitizing current response . P2X receptors are found on the surface of a large variety of cells , where they are involved in numerous sensory processes including nociception under both physiological and pathological processes -LSB- 10 -- 15 -RSB- . On nociceptive sensory neurons , extracellular ATP acts as a pain-producing neurotransmitter predominantly through homotrimeric P2X3 receptors or heterotrimeric P2X2 +3 receptors . Conditions and mediators that facilitate P2X3 and P2X2 +3 receptor-mediated signalling , apparently by potentiating ionic currents , include -LRB- 1 -RRB- substance P and bradykinin -LSB- 16 -RSB- , -LRB- 2 -RRB- nerve injury -LSB- 17 -RSB- and -LRB- 3 -RRB- calcitonin gene-related peptide -LRB- CGRP -RRB- , a potent vasodilator and proinflammatory agent -LSB- 18 -RSB- . How this increase in receptor function occurs has not been fully solved . One obvious candidate mechanism is protein kinase C -LRB- PKC -RRB- - mediated phosphorylation , since all P2X family members share a conserved consensus sequence for PKC-mediated phosphorylation -LRB- TXR/K -RRB- within the intracellularly located N-terminal domain -LRB- Fig. 1a , b -RRB- . Indeed , results obtained with pharmacological activators and inhibitors are consistent with PKC being involved in P2X3 receptor potentiation by substance P and bradykinin -LSB- 16 -RSB- , as well as the calcitonin gene-related peptide -LSB- 18 -RSB- . Direct PKC-mediated phosphorylation has so far been demonstrated only for the P2X2 receptor , with 18T of the 18TPK20 sequence as the phospho-acceptor site -LSB- 19 -RSB- . Mutational analysis has further shown that the TXR/K sequence of the P2X1 , P2X2 and P2X3 receptors plays an important role in controlling the rate of receptor desensitization -LSB- 16 , 19 , 20 -RSB- . Changes of the rate of desensitization could profoundly influence the efficiency of synaptic transmission and thus contribute to pathological pain states . Overall , there is significant interest in a better understanding of the mechanisms involved in the short-term and long-term regulation of P2X receptors . Fig. 1Effect of site-directed modification of a putative PKC motif on currents mediated by ATP-activated P2X2 and P2X3 receptors . a Schematic model of the transmembrane topology of the rat P2X3 subunit illustrating the N-terminal position of the 12TTK14 sequence . b Alignment of intracellular N-terminal amino acid sequences of the seven P2X subunit isoforms reveals a highly conserved consensus motif , TXR/K . c Typical current traces elicited by applying 10-s pulses of 100 μM ATP to oocytes expressing the indicated wild-type or mutant P2X2 receptors . d Typical current traces elicited by applying 10-s pulses of 100 μM ATP to oocytes expressing the indicated wild-type or mutant P2X3 receptors . Gray areas indicate the duration of ATP application . e All the P2X2 and P2X3 receptors and receptor mutants were expressed efficiently at the cell surface . Intact , healthy oocytes expressing the indicated proteins for 2 days were surface-labelled with the membrane impermeant reactive Cy5 dye and then extracted with dodecylmaltoside . Recombinant proteins were isolated by Ni2 + chelate chromatography and resolved by reducing SDS-PAGE . Shown is a fluorescence scan of an SDS-PAGE gel The aim of this study was to assess biochemically the conditions in which P2X2 and P2X3 receptors become directly phosphorylated by PKC in a heterologous system . While this work was in progress , a similar study was published for the P2X3 receptor that arrived at essentially the same conclusion , namely that the P2X3 receptor is unlikely to be a direct PKC substrate -LSB- 21 -RSB- . Our results complement and extend this finding by demonstrating that the P2X2 receptor also does not serve as a substrate for direct PKC-mediated phosphorylation . Materials and methods Materials The sources of antibodies , enzymes or peptide substrates are specified at the appropriate places in the text . Chemicals not otherwise specified were purchased in the highest available quality from Sigma-Aldrich -LRB- Taufkirchen , Germany -RRB- or Merck -LRB- Darmstadt , Germany -RRB- . Peptide-specific antibodies to P2X1 , P2X2 and P2X3 subunits Peptides ATSSTLGLQENMRTS -LRB- residues 385-399 of the P2X1 subunit -RRB- , QQDSTSTDPKGLAQL -LRB- residues 458 -- 472 of the P2X2 subunit -RRB- and VEKQSTDSGAYSIGH -LRB- residues 383 -- 397 of the P2X3 subunit -RRB- were selected based on their lack of similarity with known proteins and predicted hydrophilicity for raising polyclonal antibodies against the C-terminal end of the rat subunits P2X1 , P2X2 and P2X3 , respectively . Rabbit polyclonal antibodies were ordered through the Custom Antibody Production Services of Eurogentec -LRB- Seraing , Belgium -RRB- . After 3 months and a total of four immunizations , rabbits were sacrificed by complete bleeding . Peptide-specific antibodies were purified from the serum by affinity columns coupled with the peptides used for immunization . Immunoblotting showed that the antibodies recognized only the receptor against which they were raised , thus demonstrating isoform specificity of these antibodies -LRB- results not shown -RRB- . The antibodies were aliquoted at 0.12 -- 0.16 mg/ml in phosphate-buffered saline -LRB- PBS -RRB- containing 0.1 % bovine serum albumin -LRB- BSA -RRB- and 0.01 % thimerosal and stored at − 80 °C . cDNA constructs Plasmids encoding wild-type and N-terminal hexahistidine-tagged -LRB- His-tagged -RRB- versions of rat P2X1 subunits -LRB- rP2X1 , GenBank accession no . X80477 -RRB- , P2X2 subunits -LRB- rP2X2 , GenBank accession no . U14414 -RRB- or rat P2X3 subunits -LRB- rP2X3 , GenBank accession no . X90651 -RRB- in the oocyte expression vector pNKS2 -LSB- 22 -RSB- were available from previous studies -LSB- 3 , 8 -RSB- . Insertion and replacement mutations were introduced by QuikChange site-directed mutagenesis -LRB- Stratagene , La Jolla , CA , USA -RRB- . For protein expression in mammalian cells , the receptor cDNAs were subcloned from the oocyte expression vector pNKS2 into pcDNA3 .1 . The plasmid His-rP2X2-EGFP-pcDNA3.1 with a C-terminal enhanced green fluorescent protein -LRB- EGFP -RRB- fusion was generated by eliminating the stop codon in His-rP2X2 and adding the coding sequence of EGFP -LRB- Clontech , Palo Alto , CA , USA -RRB- in frame . All constructs were verified by restriction analysis and nucleotide sequencing . A pEGFP-C1 plasmid harbouring the N-terminally EGFP-tagged splicing factor SF3B1 was kindly provided by our colleague Dr. Walter Becker -LSB- 23 -RSB- . P2X receptor expression in Xenopus laevis oocytes Capped cRNAs were synthesized from plasmids linearized downstream to the 100 bp long polyA tail provided by the oocyte expression vector pNKS2 -LSB- 22 -RSB- and injected at 0.5 μg/μl in 50-nl aliquots into collagenase-defolliculated X. laevis oocytes using a Nanoliter 2000 injector -LRB- WPI , Sarasota , FL , USA -RRB- , as detailed previously -LSB- 24 -RSB- . Oocytes were maintained at 19 °C in sterile oocyte Ringer 's solution -LSB- ORi : 90 mM NaCl , 1 mM KCl , 1 mM CaCl2 , 1 mM MgCl2 and 10 mM hydroxyethylpiperazine ethanesulfonic acid -LRB- HEPES -RRB- , pH 7.4 -RSB- supplemented with 50 μg/ml of gentamicin . Heterologous expression in HEK293 cells cDNAs encoding P2X subunits in pcDNA3 .1 vector -LRB- Invitrogen , Karlsruhe , Germany -RRB- or the splicing factor SF3B1 in EGFP-C1 vector were transiently transfected into HEK293 cells using Lipofectamine LTX -LRB- Invitrogen , Karlsruhe , Germany -RRB- . Cells were cultured at 37 °C in Dulbecco 's modified Eagle 's medium -LRB- DMEM -RRB- -LRB- Invitrogen -RRB- supplemented with 10 % -LRB- v/v -RRB- fetal calf serum -LRB- FCS -RRB- -LRB- PAA Laboratories , Linz , Austria -RRB- , 100 U/ml of penicillin G and 100 μg/ml of streptomycin . Metabolic -LSB- 35S -RSB- methionine labelling of oocytes For metabolic -LSB- 35S -RSB- methionine labelling of oocyte-expressed recombinant proteins , cRNA-injected oocytes and non-injected controls were incubated overnight with L - -LSB- 35S -RSB- methionine -LRB- > 40 TBq/mmol ; PerkinElmer , Boston , MA , USA -RRB- at about 100 MBq/ml -LRB- 0.4 MBq per oocyte -RRB- in ORi at 19 °C , and then chased for 24 h. Surface fluorescence labelling of oocytes Selective fluorescence labelling of plasma membrane-bound P2X receptors was achieved by using the amino-reactive fluorescent Cy5 dye -LRB- GE Healthcare , Freiburg , Germany -RRB- , which is membrane impermeant due to its two sulfonic acid groups . Two days after cRNA injection , oocytes were washed with oocyte-PBS pH 8.5 -LRB- 20 mM Na phosphate , 110 mM NaCl , 1 mM MgCl2 -RRB- , and then incubated for 30 min at ambient temperature with Cy5 dye , which was diluted 200-fold to a final concentration of 50 μg/ml from a dimethyl sulfoxide -LRB- DMSO -RRB- stock solution . The reaction was terminated by washing the cells with oocyte-PBS , followed by membrane protein extraction with dodecylmaltoside and receptor purification -LRB- see below -RRB- . Isolation of recombinant P2X receptors from X. laevis oocytes or HEK293 cells His-tagged receptors were purified by Ni2 + nitrilotriacetic acid -LRB- NTA -RRB- agarose -LRB- Qiagen , Hilden , Germany -RRB- affinity chromatography from non-ionic detergent extracts of oocytes or HEK293 cells in the presence of iodoacetamide essentially as described -LSB- 3 , 8 , 25 -RSB- . Slight modifications were that buffers were supplemented with ethylenediaminetetraacetate -LRB- EDTA -RRB- - free Halt ™ protease inhibitor cocktail -LRB- Pierce , Rockford , IL , USA -RRB- , 50 mM NaF and 50 mM Na4P2O7 for phosphatase inhibition , and that digitonin was replaced by 0.2 % and 0.05 % dodecylmaltoside for membrane protein extraction and repetitive washing of resin-bound proteins , respectively . Proteins were released from the washed Ni2 + NTA agarose beads with non-denaturing elution buffer consisting of 250 mM imidazole/HCl -LRB- pH 7.4 -RRB- and 0.02 % dodecylmaltoside , and then kept at 0 °C until analysed on the day of purification . In addition , immunoprecipitation assays were performed using either cRNA-injected Xenopus oocytes or transiently transfected HEK293 cells . Cells were lysed in ice-cold immunoprecipitation lysis buffer containing 150 mM NaCl , 25 mM Tris/HCl pH 7.4 , 20 mM NaF , 20 mM Na4P2O7 , 10 μl/ml Halt ™ protease inhibitor cocktail without EDTA -LRB- Pierce , Rockford , IL , USA -RRB- , 1 % sodium dodecyl sulfate -LRB- SDS -RRB- -LRB- w/v -RRB- and 50 mM iodoacetamide . For efficient solubilization , lysates were incubated for 30 min on ice with occasional vortexing , and then cleared by centrifugation for 15 min at 16,100 g and 4 °C . The cleared SDS extract was diluted 1:1 with immunoprecipitation binding buffer -LSB- 150 mM NaCl , 20 mM Tris/HCl pH 7.4 , 0.4 mM phenylmethylsulfonyl fluoride -LRB- PMSF -RRB- , 0.5 % Triton X-100 v/v and 25 mM iodoacetamide -RSB- , supplemented 1:1,000 with the desired peptide-specific P2X2 or P2X3 antibodies or a polyclonal goat anti-EGFP antibody -LRB- Rockland , Gilbertsville , PA , USA -RRB- and incubated for 2 h on ice . Immunocomplexes were bound to the protein A Sepharose by overnight head-over-head rotation at 8 °C , then washed 5 times with immunoprecipitation binding buffer containing 0.1 % Triton X-100 and finally eluted by incubating the beads for 15 min at 56 °C in reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis -LRB- SDS-PAGE -RRB- sample buffer . In vitro PKC phosphorylation assay In vitro phosphorylation assays were performed by using P2X receptors as substrates , purified as detailed above . In addition , a selective peptide PKC substrate -LRB- RRGRTGRGRRGIYR , Calbiochem-Merck , Darmstadt , Germany , final concentration 150 μM -RRB- corresponding to the amino acid sequence 1487-1500 of the hepatitis C virus polyprotein -LSB- 26 -RSB- was used as a positive control . The proteins or the peptide were incubated for 30 min at 30 °C with 0.02 unit -LRB- 0.02 μg -RRB- of purified catalytic PKC subunit from rat brain and 50 μM -LSB- γ-32P -RSB- ATP -LRB- 10 mCi/ml , Amersham Biosciences GE Healthcare , Freiburg , Germany -RRB- in phosphorylation buffer containing 6 mM CaCl2 , 10 mM MgCl2 , 20 mM Tris/HCl pH 7.5 , 1 mM EDTA , 1 mM mercaptoethanol , 0.05 % Triton X-100 -LRB- v/v -RRB- and 0.1 mg/ml BSA . The reaction was terminated by boiling in reducing SDS-PAGE sample buffer for 30 s. Proteins were resolved by reducing SDS-PAGE and visualized by immunoblotting or phosphorimaging as detailed below . In vivo phosphorylation assay Transiently transfected HEK293 cells were metabolically labelled by a 3-h incubation with 500 μCi/ml of -LSB- 32P -RSB- orthophosphate -LRB- Amersham Biosciences GE Healthcare , Freiburg , Germany -RRB- in phosphate-free oocyte Ringer solution or in phosphate-free DMEM -LRB- Invitrogen , Karlsruhe , Germany -RRB- as appropriate . Cells were treated with or without 100 nM phorbol 12-myristate 13-acetate -LRB- PMA ; Sigma-Aldrich , Taufkirchen , Germany -RRB- or 100 nM calyculin A -LRB- Tocris Bioscience , Ellisville , MO , USA -RRB- for 10 min at ambient temperature . Non-incorporated radioactivity was removed by rinsing the cells twice with tracer-free medium . Affinity purification or immunoprecipitation was performed as described above followed by SDS-PAGE and immunoblotting or phosphorimaging . SDS-PAGE Samples for SDS-PAGE were denatured by incubating in reducing SDS-PAGE sample buffer for 15 min at 56 °C and then electrophoresed on a Laemmli SDS-PAGE gel -LRB- 10 % acrylamide -RRB- in parallel with -LSB- 14C -RSB- - labelled molecular mass markers -LRB- Rainbow ™ , Amersham Biosciences GE Healthcare , Freiburg , Germany -RRB- . Cy5-labelled proteins were visualized by scanning the wet SDS-PAGE gel with a fluorescence scanner -LRB- Typhoon , GE Healthcare , Freiburg , Germany -RRB- . For visualization of radiolabelled or non-labelled proteins , SDS-PAGE gels were either fixed , dried and exposed to a PhosphorImager screen , and scanned using a Storm 820 PhosphorImager -LRB- Amersham Biosciences GE Healthcare , Freiburg , Germany -RRB- or/and blotted to polyvinylidene fluoride -LRB- PVDF -RRB- membranes . Immunoblotting Proteins separated by SDS-PAGE were electroblotted onto PVDF membranes . Membranes were blocked for 1 h with 5 % non-fat milk in PBS -LRB- or , alternatively , with 5 % BSA if the phosphothreonine monoclonal antibody was used for immunoblotting -RRB- , washed 3 × 5 min with PBST -LRB- 0.1 % Tween 20 in PBS -RRB- and incubated for 2 h at 4 °C in PBS containing the appropriate primary antibody . Phosphorylated proteins were detected by immunoblotting with a phosphothreonine monoclonal antibody -LRB- Cell Signaling Technology , Beverly , MA , USA -RRB- . Alternatively , the peptide-specific antibodies to P2X2 and P2X3 subunits were used to verify receptor expression levels . Following washing in PBST , an appropriate horseradish peroxidase-conjugated secondary antibody -LRB- goat anti-mouse or goat anti-rabbit , Pierce , Rockford , IL , USA -RRB- was applied for 1 h in PBS supplemented with 1 % non-fat milk . After another 3 × 5 min washes with PBST , immunoreactive bands were visualized by chemiluminescence imaging using a Fujifilm LAS-3000 system -LRB- Tokyo , Japan -RRB- and the AIDA Image Analyser Software Version 4.08 -LRB- Raytest , Straubenhardt , Germany -RRB- . Two-electrode voltage clamp current recordings and electrical capacitance measurements Two-microelectrode voltage clamp measurements of X. laevis oocytes were performed 2 days after cRNA injection using a Turbo TEC-05 amplifier -LRB- npi electronics , Tamm , Germany -RRB- interfaced by an INT-20X AD/DA converter -LRB- npi electronics , Tamm , Germany -RRB- to a personal computer running the CellWorks Lite 5.1 software -LRB- npi electronics , Tamm , Germany -RRB- , as detailed previously -LSB- 27 -- 29 -RSB- . Current traces were elicited by 100 μM ATP at 1 - to 5-min intervals -LRB- Sigma -- Aldrich , Taufkirchen , Germany -RRB- at a holding potential of − 60 mV in nominally Ca2 + - free bath medium -LRB- designated Mg-ORi -RRB- to avoid activation of endogenous Ca2 + - activated Cl − channels . ATP was prepared in Mg − ORi at its final concentration and applied by gravity-fed perfusion at a flow rate of 10 ml/min to an oocyte held by the two microelectrodes in the bath chamber -LRB- volume ∼ 10 μl -RRB- . Switching between different perfusion solutions was achieved with electromagnetic valves controlled by the CellWorks Lite 5.1 software . Electrophysiological recordings were analysed using the Origin 6.0 software -LRB- Microcal Software Inc. , Northampton , MA , USA -RRB- . Single exponentials were fitted to the decay phase of the macroscopic currents with a nonlinear least squares method . The time constant τ was taken as an estimate of the rate of receptor desensitization . To monitor PMA-induced changes in the oocyte surface area , depolarizing voltage steps of ΔV = 10 mV were applied from the holding potential to elicit a capacitive transient Icap , the integral of which yields the charge movement Qcap and thus the membrane capacitance Cm = Qcap/ΔV -LSB- 30 -- 32 -RSB- . PMA was dissolved in DMSO to a stock solution of 10 mM and stored at − 20 °C . On the day of the experiment , the stock solution was pre-diluted to 100 μM in DMSO and then further diluted with Mg-ORi to the final concentration of 100 nM . Vehicle control experiments with a final concentration of 0.1 % DMSO did not reveal any effects on P2X2 receptor-mediated currents measured with X. laevis oocytes -LRB- results not shown -RRB- . All measurements were made at room temperature -LRB- 21 -- 23 °C -RRB- . Data analysis Data are shown as means ± SEM . The paired Student 's t-test -LRB- two-tailed -RRB- was used to compare current amplitudes with statistical significance set to p < 0.05 . Results Site-directed modification of a putative N-terminal PKC site affects the electrophysiological phenotype of P2X2 and P2X3 receptor channels First , we tested each of the non-mutated and mutant receptor constructs for expression of an ATP-gated inward current in cRNA-injected X. laevis oocytes . Application of 100 μM ATP induced typical non-desensitizing currents through oocyte-expressed P2X2 receptors -LRB- Fig. 1c , left current trace -RRB- . The P2X2 subunit shares with other P2X subunit isoforms a conserved consensus site for PKC phosphorylation -LRB- TXR/K , where T is the phosphorylation site and X denotes any amino acid ; see Fig. 1a , b -RRB- . Disruption of the conserved N-terminal PKC site by mutating the phosphor-acceptor 18T to A resulted in a rapidly desensitizing channel -LRB- Fig. 1c , middle current trace -RRB- . Also , replacement of 20K by T resulted in a desensitizing phenotype -LRB- Fig. 1c , right current trace -RRB- . Since PKC preferentially phosphorylates threonine -LRB- or serine -RRB- residues that are close to a C-terminal basic residue , replacement of 20K in the P +2 position by a non-basic residue such as T will destroy the putative PKC site despite preserving the phosphor-acceptor 18T . Virtually identical findings had previously been reported by Boue-Grabot et al. -LSB- 19 -RSB- . The average half-times of desensitization -LRB- τ -RRB- of the 18A and 20T P2X2 mutants were quite similar , with mean τ values -LRB- ± SEM -RRB- of 4.5 ± 0.4 s and 7.3 ± 0.5 s , respectively , of eight oocytes from two females per value . ATP applied to oocytes expressing the P2X3 receptor elicited a rapidly desensitizing inward current -LRB- Fig. 1d , left current trace -RRB- as is typical for this P2X receptor subtype . Incorporation of similar mutations , as in the P2X2 subunit in the analogous positions of the P2X3 receptor T12A or K14T , completely abolished P2X3 receptor function -LRB- Fig. 1d , middle and right current traces -RRB- . Visualization of plasma membrane-bound fluorescence-labelled P2X receptors by affinity purification combined with SDS-PAGE and fluorescence scanning verified that all the receptors were efficiently exported to the oocyte surface -LRB- Fig. 1e -RRB- . The PKC activator PMA augments P2X3 receptor-mediated currents , yet leaves P2X2-mediated currents unchanged Since the mutagenesis data for the P2X2 and P2X3 receptor were consistent with the possibility of a regulatory role for PKC in P2X receptor function , we examined whether the PKC activator PMA was capable of affecting the electrophysiological phenotype of P2X2 or P2X3 receptors . In planning the experiments , we considered that PMA treatment of X. laevis oocytes is known to induce a pronounced endocytotic reduction of the cell surface area , as evidenced microscopically and electrophysiologically by the disappearance of virtually all microvilli and a concomitant decrease of the electrical capacitance of the plasma membrane , respectively -LSB- 31 , 32 -RSB- . Associated with oocyte surface reduction is a proportional internalization of membrane proteins , such as endogenous Na + - K + pumps , suggesting that PMA treatment would stimulate internalization of recombinant P2X receptors as well . Re-examination of the time course of the PMA-induced reduction of the oocyte surface area showed that the electrical capacitance did not start to decline significantly before 10 min after PMA application -LRB- Fig. 2a -RRB- . Accordingly , all current recordings were done within a time window of 10 min after PMA application to prevent any contribution of receptor internalization to the PMA effect on P2X receptor-dependent currents . Fig. 2Effect of PMA on ATP-induced currents mediated by expressed P2X2 and P2X3 receptors in X. laevis oocytes . a The electrical membrane capacitance was monitored as a measure of the oocyte surface area during sustained stimulation with PMA . Each bar represents the mean Cm ± SEM calculated from the areas under the current transients elicited in 10-s intervals by five consecutive 10-mV depolarizing steps . b , c Representative current traces elicited by 10-s lasting pulses of 100 μM ATP applied in 10-min intervals to oocytes expressing the indicated P2X receptors or P2X receptor mutants . Where denoted by a black bar , oocytes were pre-incubated with 100 nM PMA before ATP was co-applied . PMA was without effect on the P2X2 receptor-mediated current -LRB- b -RRB- , but induced a marked increase of the current amplitude mediated by P2X3 receptors -LRB- c -RRB- . Gray areas indicate the duration of ATP application Figure 2b and c show typical current traces elicited by ATP from P2X2 or P2X3 receptor-expressing oocytes before and after a 10-min exposure to PMA . PMA neither affected the shape nor the amplitude of P2X2 receptor-mediated current -LRB- Fig. 2b -RRB- . However , PMA produced a marked increase of the current amplitude mediated by the P2X3 receptor -LRB- Fig. 2c -RRB- . In one experiment with four oocytes , a statistically significant increase of the P2X3-mediated current amplitude by PMA of 155 ± 42 % -LRB- SEM -RRB- was determined . P2X2 and P2X3 receptors exist in a non-phosphorylated state in both X. laevis oocytes and HEK293 cells To explain the accelerated desensitization upon site-directed perturbation of the 18TXK20 motif , it has been suggested that the wild-type P2X2 receptor is constitutively phosphorylated at 18T and that this phosphorylation is responsible for the slow desensitization rate -LSB- 19 -RSB- . To address this issue , we next examined biochemically whether P2X3 receptors also exist in a constitutively phosphorylated state ; the P2X2 receptor was also analysed to allow for a direct comparison of the results . Both parent P2X receptors and mutants were purified by Ni2 + chelate affinity chromatography from dodecylmaltoside extracts of -LSB- 35S -RSB- methionine-labelled oocytes , resolved by SDS-PAGE , and blotted onto a PVDF membrane . However , probing with a phosphothreonine-specific monoclonal antibody revealed no discernible signal -LRB- Fig. 3a -RRB- despite the presence of significant amounts of -LSB- 35S -RSB- methionine-labelled P2X2 and P2X3 protein in the SDS-PAGE gel -LRB- Fig. 3b -RRB- . Fig. 3Immunoblots show no constitutive phosphorylation of oocyte-expressed P2X2 or P2X3 receptors . The indicated wild-type or mutant P2X subunits were purified by Ni2 + chelate chromatography from -LSB- 35S -RSB- methionine-labelled X. laevis oocytes and resolved by denaturing SDS-PAGE . a Shown is a representative immunoblot probed with a phosphothreonine-specific monoclonal antibody and a peroxidase-conjugated secondary antibody . No phosphorylation signal could be detected at the SDS-PAGE migration position of P2X2 and P2X3 subunits , which are indicated by the adjacent PhosphorImager scan in panel b. b Direct PhosphorImager visualization of metabolically -LSB- 35S -RSB- methionine-labelled parent and mutant P2X subunits resolved by SDS-PAGE of the same samples as in panel a. cLeft panel : representative immunoblot of P2X2 subunits tagged with either an N-terminal or C-terminal hexahistidine sequence -LRB- designated His-P2X2 or P2X2-His , respectively -RRB- and probed with a phosphothreonine-specific monoclonal antibody and a peroxidase-conjugated secondary antibody . No phosphorylation signal could be detected at the SDS-PAGE migration position of P2X2 subunits , which were visualized by immunoblotting with the P2X2 subunit polyclonal antibody -LRB- middle panel -RRB- or PhosphorImager scanning of incorporated -LSB- 35S -RSB- methionine -LRB- right panel -RRB- To exclude the possibility that the apparent lack of receptor phosphorylation resulted from an interference between the N-terminal His tag and the PKC site of P2X3 or P2X2 receptor subunits , which are separated by only 12 or 20 residues , respectively , we performed analogous experiments with a C-terminally His-tagged P2X2 receptor , P2X2-His . As apparent from Fig. 3c , neither N-terminally nor C-terminally His-tagged P2X2 receptor became phosphorylated , as evidenced by immunoblotting with a phosphor-specific anti-threonine antibody -LRB- Fig. 3c -RRB- . The presence of P2X2 receptor protein was verified by immunoblotting and metabolic labelling with -LSB- 35S -RSB- methionine -LRB- Fig. 3c , middle and right panel -RRB- . Boue-Grabot et al. -LSB- 19 -RSB- have demonstrated phosphorylation of P2X2 receptors expressed in HEK293 cells . To address the possibility that P2X receptor phosphorylation is host cell specific , we analysed HEK293 cells transiently transfected with P2X2 or P2X3 expression plasmids . P2X receptor proteins were isolated by immunoprecipitation and resolved by reducing SDS-PAGE . The efficient expression of the P2X3 receptor or the C-terminally EGFP-tagged P2X2 receptor in HEK293 cells was verified by immunoblotting with subunit bands migrating at 54 kDa or 83 kDa -LRB- 27 kDa contributed to the P2X2 subunit by the EGFP tag -RRB- , respectively -LRB- Fig. 4b -RRB- . However , phosphorylated P2X2 or P2X3 subunits could not be detected by immunoblotting with a phosphothreonine-specific antibody -LRB- Fig. 4a -RRB- . In contrast , the PKC-phosphorylated splicing factor SF3B1 , which was expressed in HEK293 cells as a positive control substrate and immunoprecipitated with an EGFP antibody , was efficiently phosphorylated under identical conditions , assuring that the assay was able to detect phosphorylated proteins -LRB- Fig. 4a , lane 1 -RRB- . Taken together , these results suggest that both P2X2 and P2X3 receptors exist constitutively in a non-phosphorylated state in both X. laevis oocytes and HEK293 cells . Fig. 4Immunoblotting reveals no constitutive phosphorylation of HEK293 cell-expressed P2X2 and P2X3 receptors . The indicated proteins were isolated by immunoprecipitation using the peptide-specific P2X3 subunit polyclonal antibody or an EGFP-specific polyclonal antibody as appropriate and resolved by reducing SDS-PAGE . a Shown is an immunoblot probed by phosphothreonine-specific monoclonal antibody and a peroxidase-conjugated secondary antibody . Note that phosphorylation of the P2X3 receptor or the EGFP-tagged P2X2 receptor is not detectable , whereas a positive control -- the EGFP-tagged splicing factor SF3B1 -LRB- migration mass 97 kDa -LSB- 23 -RSB- -RRB- -- was detected by the antibody . b The same samples as in panel a were immunoblotted using peptide-specific P2X2 and P2X3 subunit polyclonal antibodies to verify the expression of the transfected genes PMA-activated PKC does not drive the phosphorylation of P2X2 or P2X3 receptors A majority of protein kinases are quiescent unless their activity is stimulated by specific stimuli and effectors . To drive phosphorylation by active endogenous PKC , we incubated non-denatured recombinant His-tagged P2X1 , P2X2 and P2X3 receptors isolated by Ni2 + NTA agarose affinity chromatography from dodecylmaltoside extracts of X. laevis oocytes with PMA-stimulated lysates of X. laevis oocytes -LRB- Fig. 5a -RRB- or HEK293 cells -LRB- Fig. 5b -RRB- in the presence of -LSB- γ-32P -RSB- ATP . As a positive control , a specific peptide PKC substrate RRGRTGRGRRGIYR was incubated with the lysates . This peptide was equally well phosphorylated by addition of either cell lysate or a commercial preparation of the rat brain PKC subunit -LRB- Fig. 5a , b -RRB- . In contrast , however , no 32P incorporation could be detected in the SDS-PAGE migration position of the P2X1 , P2X3 or P2X2 receptor subunit at 56 kDa , 54 kDa or 66 kDa , respectively . The presence of purified P2X1 , P2X2 and P2X3 receptor subunit protein in this assay was verified by immunoblotting using P2X1 - , P2X2 - or P2X3-specific antibodies -LRB- Fig. 5c -RRB- . The phosphorylation of the synthetic peptide clearly demonstrates that both X. laevis oocytes and HEK293 cells contain sufficient endogenous PKC to drive at least the phosphorylation of a synthetic peptide substrate . Fig. 5Lysates of X. laevis oocytes or HEK293 cells contain endogenous PKC , but do not support PMA-driven phosphorylation of P2X2 or P2X3 receptors . The indicated wild-type and mutant P2X receptors were purified by non-denaturing Ni2 + chelate chromatography from X. laevis oocytes , supplemented with -LSB- γ-32P -RSB- ATP , 100 nM PMA and oocyte lysate -LRB- a -RRB- or HEK293 cell lysate -LRB- b -RRB- , and incubated for 30 min at ambient temperature . In parallel , a peptide PKC substrate -LRB- final concentration 0.5 μg/μl -RRB- was incubated with -LSB- γ-32P -RSB- ATP , PMA and either of the two cell lysates or the purified rat brain PKC catalytic subunit as indicated . The proteins were resolved by reducing SDS-PAGE and visualized by phosphorimaging . Note that the band pattern was similar irrespective of whether protein of mock-injected oocytes or P2X receptor-expressing oocytes was used as a substrate , arguing against specific labelling of either P2X receptor . Arrows indicate migration positions of P2X1 , P2X2 and P2X3 subunits . Arrowhead in a , dominant background band that is present in all oocyte samples . The peptide PKC substrate was similarly labelled by lysates or purified rat brain PKC , confirming the presence of endogenous PKC in X. laevis oocytes and HEK293 cells . c The same samples as in panels a and b were resolved on a separate SDS-PAGE gel . The indicated P2X subunits were detected by immunoblotting using the appropriate P2X subunit-specific polyclonal antibodies . A sample of mock-transfected cells was simultaneously probed with the P2X2 and P2X3 subunit-specific polyclonal antibodies Discernible phosphorylation of P2X2 and P2X3 receptors might be obscured by a low level of endogenous PKC . Therefore , an analogous experiment was performed , in which the purified P2X2 and P2X3 receptors were incubated with purified rat brain PKC catalytic subunit rather than cell lysate in the presence of -LSB- γ-32P -RSB- ATP . Again , the specific peptide PKC substrate was efficiently phosphorylated as demonstrated both by direct phosphorimaging -LRB- Fig. 6a -RRB- and immunoblotting with the phosphothreonine monoclonal antibody -LRB- Fig. 6b -RRB- . Phosphorylated P2X2 or P2X3 subunits could not be detected by either method -LRB- Fig. 6a , b -RRB- despite the presence of abundant amounts of the respective P2X subunits in the assays -LRB- Fig. 6c -RRB- . Fig. 6PKC phosphorylates a specific PKC substrate in vitro , but does not phosphorylate P2X2 or P2X3 receptors . The immunoprecipitated recombinant P2X2 or P2X3 receptor from X. laevis oocytes or a synthetic peptide -LRB- 1 or 10 μg -RRB- derived from the non-structural protein 3 of hepatitis C virus , which serves as a specific substrate for PKC , were incubated for 30 min with purified rat brain PKC catalytic subunit -LRB- 0.02 μg corresponding to 0.02 U/μl -RRB- in the presence of 50 μM -LSB- γ-32P -RSB- ATP -LRB- 10 mCi/ml -RRB- , and then subjected to SDS-PAGE and phosphorimaging . The rat brain PKC catalytic subunit does not require Ca2 + and phosphatidylserine for activity -LSB- 42 -RSB- . 32P was incorporated into the PKC substrate -LRB- left panel -RRB- and was also detected by immunoblotting with an anti-phosphothreonine antibody -LRB- middle panel -RRB- . However , there was no detectable 32P incorporation at a migration position of ∼ 66 kDa or ∼ 54 kDa of the P2X2 or P2X3 subunit , respectively , the presence of which could be verified by immunoblotting -LRB- right panel -RRB- PMA or calyculin A do not enhance phosphorylation of P2X3 receptors in intact HEK cells In a final set of experiments , we attempted to directly demonstrate phosphorylation in vivo by using metabolic -LSB- 32P -RSB- orthophosphate labelling combined with immunoprecipitation analysis as a read out . Mock-transfected and P2X3 receptor-transfected HEK293 cells were incubated with -LSB- 32P -RSB- orthophosphate in phosphate-free DMEM , followed by incubation with the PKC activator PMA and/or the phosphoserine/phosphothreonine phosphatase inhibitor calyculin A . The PhosphorImager scan revealed an identical pattern of phosphorylated bands almost irrespective of the experimental conditions , with no evidence for a significant 32P-labelling of the P2X3 subunit -LRB- Fig. 7 , top panel -RRB- . P2X3 receptor expression was verified by immunoblotting -LRB- Fig. 7 , bottom panel -RRB- . Fig. 7PMA and calyculin A do not induce phosphorylation of P2X3 receptors in intact HEK cells . Mock - and P2X3 receptor-transfected HEK293 cells were incubated with -LSB- 32P -RSB- orthophosphate -LRB- 500 μCi/ml -RRB- for 3 h in phosphate-free DMEM , followed by a 10-min incubation with 100 nM of each , the PKC activator PMA and/or the phosphatase inhibitor calyculin A. Triton X-100 extracts were then prepared from the cells and subjected to immunoprecipitation using the peptide-specific P2X3 subunit polyclonal antibody , followed by reducing SDS-PAGE and phosphorimaging . The migration position of the P2X3 subunit is marked by an arrow , indicating the absence of 32P incorporation into the P2X3 subunit . P2X3 receptor expression was verified by immunoblotting with the P2X3 subunit polyclonal antibody -LRB- bottom panel -RRB- Discussion Both P2X3 and P2X2 +3 receptors are present on sensory neurons , where they play an important role in nociception . In addition to mediating transient nociceptive responses , these receptors contribute critically to the hyperexcitability that underlies abnormal pain states elicited by nerve injuries . One mechanism for the increased responsiveness to ATP is sensitization of P2X3 and P2X2 +3 receptors arising from a significant enhancement in the trafficking of P2X3 subunit-containing receptors and/or an increase in function of pre-existing receptors -LSB- 17 -RSB- . An obvious candidate mechanism for functional modulation is phosphorylation , which is believed to occur universally in all ligand-gated ion channels as reviewed by Levitan -LSB- 33 -RSB- . Evidence for a control of P2X receptor channel function by PKC-mediated phosphorylation Site-directed elimination of the putative N-terminal PKC site has already previously been shown to severely affect the electrophysiological phenotype of several P2X receptor subtypes , including P2X1 , P2X2 and P2X3 receptors from rat and/or human -LSB- 16 , 19 , 20 , 34 -RSB- and P2X5 receptors from frog -LSB- 35 -RSB- . Replacing the putative phospho-acceptor residue 18T or the basic residue in the P +2 position -LRB- 20K -RRB- by A or a non-basic residue , respectively , imposed rapidly desensitizing properties onto the non-desensitizing wt P2X2 receptor without grossly affecting the current amplitude . In contrast , when analogous mutations were introduced into the rapidly desensitizing receptor subtypes P2X1 or P2X3 , peak currents declined to low or undetectable levels , respectively -LSB- 16 ,20 , 34 -RSB- ; the same observation was also made with the slower desensitizing frog P2X5 receptor -LSB- 35 -RSB- . Overall , the results obtained by site-directed elimination of the N-terminal TXR/K motif can be plausibly reconciled with the hypothesis that the rate of P2X receptor channel desensitization is controlled by a phosphorylation-dephosphorylation mechanism , with phosphorylation at this site being associated with a decreased rate of desensitization . Additional evidence for a possible involvement of PKC-dependent phosphorylation comes from experiments with the PKC activator PMA , which was consistently found to elicit a severalfold increase in currents mediated by the P2X1 receptor -LSB- 36 -RSB- or the P2X3 receptor -LSB- 16 , 18 , 21 -RSB- without changing the rate of desensitization . A PMA-induced change of the current desensitization was noted for C-terminally truncated P2X2 receptors , which are known to exhibit a desensitizing phenotype in contrast to the parent full-length P2X2 receptors -LSB- 19 , 37 -RSB- . PMA treatment converted the rapidly desensitizing phenotype of truncated P2X2 receptors to slowly desensitizing , but did not affect the kinetic properties or current amplitudes of the full-length P2X2 receptors . The same lack of effect of PMA has been observed with wild-type P2X2 receptors stably expressed in HEK293 -LSB- 38 -RSB- . It has been inferred from these data that P2X2 receptors are constitutively phosphorylated and that this phosphorylation is responsible for their non-desensitizing phenotype . Consistent with this view , immunoblot analysis with a phosphothreonine-proline-specific monoclonal antibody efficiently detected wild-type P2X2 receptors with an intact PKC site , but not P2X2 receptor mutants with a disrupted PKC site -LSB- 19 -RSB- . Constitutive phosphorylation has been also demonstrated for the P2X1 receptor expressed in HEK293 cells -LSB- 34 -RSB- . Further studies demonstrated that exogenously added uridine triphosphate -LRB- UTP -RRB- potentiated the current mediated by endogenous and expressed P2X3 receptors in response to αβ-meATP as agonist . Substitution of phospho-acceptor residues of PKC consensus sites located in the ectodomain of the P2X3 receptor abolished this UTP-induced current potentiation , suggesting a role of ecto-protein kinase C in P2X3 receptor regulation -LSB- 39 , 40 -RSB- . Evidence against a control of P2X receptor channel function by PKC-mediated phosphorylation In direct contrast to the results of Boue-Grabot et al. -LSB- 19 -RSB- , we were unable to demonstrate a direct phosphorylation of wild-type P2X2 receptors expressed in X. laevis oocytes and HEK293 cells . The various experimental approaches for phosphorylation detection that we applied included immunoblot analysis of purified P2X2 receptors . In the experiments shown , we used a phosphothreonine-specific monoclonal antibody instead of a phosphothreonine-proline-specific antibody , because the TXK motif of the P2X3 receptor contains a threonine in the - X-position instead of the proline found in the amino acid sequence of the P2X1 and P2X2 subunit -LRB- cf. Fig. 1b -RRB- . However , even when we used the same phosphothreonine-proline-specific antibody as Boue-Grabot et al. -LSB- 19 -RSB- , we could not demonstrate phosphorylation of P2X2 receptors in our experiments -LRB- data not shown -RRB- . Recently , it has been shown that a proline in the P +1 position strongly disfavours substrate recognition by PKC -LSB- 41 -RSB- , suggesting that our negative results with P2X2 receptors may not result from technical limitations , but reflect the inherent inability of P2X2 receptors to serve as substrates for PKC . Notably , P2X3 receptors , despite harbouring a T in the P +1 position , could also not be demonstrated to become phosphorylated by PKC in our experiments , as well as in a recently published paper -LSB- 21 -RSB- . Further evidence against a direct PKC-mediated P2X receptor phosphorylation comes from co-expression studies of P2X1 and G protein-coupled receptors . Co-expression of the metabotropic glutamate receptor mGluR1α with P2X1 receptors produced a 2.5-fold increase of the P2X1 receptor-mediated current that was blocked by the PKC inhibitor staurosporine and could be mimicked by PMA treatment of singly expressed P2X1 receptors . Although the pharmacological data are basically consistent with PKC-mediated receptor phosphorylation being involved , mutant P2X1 receptors with a disrupted N-terminal PKC site -LRB- T18V -RRB- still showed PMA-mediated and mGluR1α-mediated potentiation . Also , PMA treatment did not lead to an increase in the -LRB- rather weak -RRB- basal phosphorylation of the P2X1 receptor -LSB- 36 -RSB- . Functional data supporting a role of PKC in P2X receptor regulation have been detailed above . There are , however , also functional data that are inconsistent with the view that the TXR/K motif represents a true PKC site : -LRB- 1 -RRB- replacement of the phospho-acceptor threonine by an acidic residue to mimic incorporation of a negatively charged phosphate group by constitutive phosphorylation was unable to restore the function of P2X1 receptors -LSB- 34 -RSB- and P2X3 receptors -LSB- 16 -RSB- ; -LRB- 2 -RRB- the PMA-induced potentiation of hP2X1 receptor currents could be blocked by the PKC inhibitor staurosporine , but surprisingly , not by site-directed elimination of the PKC site -LSB- 36 -RSB- ; thus it is much more likely that PMA exerts its effect on the P2X1 receptor -LRB- and the P2X3 receptor -RRB- through a yet to be identified accessory protein . Taken together , there is no doubt that the TXR/K motif plays an important role in the functioning of P2X receptors , and that PMA is capable of modulating P2X1 - and P2X3-mediated receptor currents . The underlying mechanism , however , is unlikely to involve direct PKC-mediated phosphorylation of P2X receptors . The existence of a proline in the P +1 position in several P2X subunit isoforms -LRB- P2X1 , P2X2 , P2X4 -RRB- might be one causative factor preventing direct phosphorylation by PKC . Additional steric constraints may explain why P2X3 receptors also do not serve as PKC substrates , despite lacking the proline in P +1 . The exact role of the TXR/K motif awaits further clarification .

O O O O O B O O O B O B O O B I I B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O B O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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