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• W2113370189 abstract "Type II cGMP-dependent protein kinase (cGKII) isolated from pig intestinal brush borders and type Iα cGK (cGKI) purified from bovine lung were compared for their ability to activate the cystic fibrosis transmembrane conductance regulator (CFTR)-Cl− channel in excised, inside-out membrane patches from NIH-3T3 fibroblasts and from a rat intestinal cell line (IEC-CF7) stably expressing recombinant CFTR. In both cell models, in the presence of cGMP and ATP, cGKII was found to mimic the effect of the catalytic subunit of cAMP-dependent protein kinase (cAK) on opening CFTR-Cl− channels, albeit with different kinetics (2-3-min lag time, reduced rate of activation). By contrast, cGKI or a monomeric cGKI catalytic fragment was incapable of opening CFTR-Cl− channels and also failed to potentiate cGKII activation of the channels. The cAK activation but not the cGKII activation was blocked by a cAK inhibitor peptide. The slow activation by cGKII could not be ascribed to counteracting protein phosphatases, since neither calyculin A, a potent inhibitor of phosphatase 1 and 2A, nor ATPγS (adenosine 5′-O-(thiotriphosphate)), producing stable thiophosphorylation, was able to enhance the activation kinetics. Channels preactivated by cGKII closed instantaneously upon removal of ATP and kinase but reopened in the presence of ATP alone. Paradoxically, immunoprecipitated CFTR or CF-2, a cloned R domain fragment of CFTR (amino acids 645-835) could be phosphorylated to a similar extent with only minor kinetic differences by both isotypes of cGK. Phosphopeptide maps of CF-2 and CFTR, however, revealed very subtle differences in site-specificity between the cGK isoforms. These results indicate that cGKII, in contrast to cGKIα, is a potential activator of chloride transport in CFTR-expressing cell types. Type II cGMP-dependent protein kinase (cGKII) isolated from pig intestinal brush borders and type Iα cGK (cGKI) purified from bovine lung were compared for their ability to activate the cystic fibrosis transmembrane conductance regulator (CFTR)-Cl− channel in excised, inside-out membrane patches from NIH-3T3 fibroblasts and from a rat intestinal cell line (IEC-CF7) stably expressing recombinant CFTR. In both cell models, in the presence of cGMP and ATP, cGKII was found to mimic the effect of the catalytic subunit of cAMP-dependent protein kinase (cAK) on opening CFTR-Cl− channels, albeit with different kinetics (2-3-min lag time, reduced rate of activation). By contrast, cGKI or a monomeric cGKI catalytic fragment was incapable of opening CFTR-Cl− channels and also failed to potentiate cGKII activation of the channels. The cAK activation but not the cGKII activation was blocked by a cAK inhibitor peptide. The slow activation by cGKII could not be ascribed to counteracting protein phosphatases, since neither calyculin A, a potent inhibitor of phosphatase 1 and 2A, nor ATPγS (adenosine 5′-O-(thiotriphosphate)), producing stable thiophosphorylation, was able to enhance the activation kinetics. Channels preactivated by cGKII closed instantaneously upon removal of ATP and kinase but reopened in the presence of ATP alone. Paradoxically, immunoprecipitated CFTR or CF-2, a cloned R domain fragment of CFTR (amino acids 645-835) could be phosphorylated to a similar extent with only minor kinetic differences by both isotypes of cGK. Phosphopeptide maps of CF-2 and CFTR, however, revealed very subtle differences in site-specificity between the cGK isoforms. These results indicate that cGKII, in contrast to cGKIα, is a potential activator of chloride transport in CFTR-expressing cell types. INTRODUCTIONGuanosine 3′,5′-cyclic monophosphate (cGMP) has been identified as an important intracellular mediator of salt and water secretion in intestinal epithelium(1Field M. Graf L.H. Laird W.J. Smith P.L. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 2800-2804Crossref PubMed Scopus (432) Google Scholar, 2Hughes J.M. Murad F. Chang B. Guerrant R.L. Nature. 1978; 271: 755-756Crossref PubMed Scopus (252) Google Scholar, 3Field M. Rao M.C. Chang E.B. N. Engl. J. Med. 1989; 321 (879-883): 800-806Crossref PubMed Scopus (232) Google Scholar). Secretagogues acting through the cGMP-signaling pathway include the family of heat-stable enterotoxins (STs), 1The abbreviations used are: STheat-stable enterotoxinCFTRcystic fibrosis transmembrane conductance regulatorCFcystic fibrosiscGKcGMP-dependent protein kinasecAKcAMP-dependent protein kinasePKIWalsh inhibitor peptide (PKI(5-24)-amide)ATPγSadenosine 5′-O-(thiotriphosphate)PAGEpolyacrylamide gel electrophoresis. low molecular weight peptides secreted by enteropathogenic bacteria, and guanylin, a recently discovered endogenous ST-like peptide hormone(3Field M. Rao M.C. Chang E.B. N. Engl. J. Med. 1989; 321 (879-883): 800-806Crossref PubMed Scopus (232) Google Scholar, 4Currie M.G. Fok K.F. Kato J. Moore R.J. Hamra F.K. Duffin K.L. Smith C.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 947-951Crossref PubMed Scopus (502) Google Scholar). Binding of ST or guanylin to the receptor domain of an intestine-specific isoform of guanylyl cyclase (GC-C) triggers cyclase activation, cGMP accumulation, and stimulation of net fluid secretion through the activation of apical Cl− channels in parallel with inhibition of coupled NaCl transporters(3Field M. Rao M.C. Chang E.B. N. Engl. J. Med. 1989; 321 (879-883): 800-806Crossref PubMed Scopus (232) Google Scholar, 5Schultz S. Green C.K. Yuen P.S.T. Garbers D.L. Cell. 1990; 63: 941-948Abstract Full Text PDF PubMed Scopus (516) Google Scholar, 6Vaandrager A.B. Schulz S. De Jonge H.R. Garbers D.L. J. Biol. Chem. 1993; 268: 2171-2179Google Scholar). The cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial Cl− channel mutated in CF patients(7Collins F.S. Science. 1992; 256: 774-779Crossref PubMed Scopus (712) Google Scholar, 8Welsh M.J. Smith A.E. Cell. 1993; 73: 1251-1254Abstract Full Text PDF PubMed Scopus (1216) Google Scholar), appears to be involved in the Cl− secretory response to ST and cGMP analogues, as evidenced by the absence of this response in CF intestine(9De Jonge H.R. Bijman J. Sinaasappel M. Pediatr. Pulmonol. Suppl. 1987; 1: 54-57Google Scholar, 10Baxter P.S. Goldhill J. Hardcastle P.T. Taylor C.J. Nature. 1988; 335: 211Crossref PubMed Scopus (39) Google Scholar).Several mechanisms have been proposed to link cGMP to the CFTR-Cl− channels, including (i) cGMP cross-activation of cAMP-dependent protein kinase (11Forte L.R. Thorne P.K. Eber S.L. Krause W.J. Freeman R.H. Francis S. Corbin J.D. Am. J. Physiol. 1992; 263: C607-C615Crossref PubMed Google Scholar, 12Tien X.-Y. Brasitus T.A. Kaetzel M.A. Dedman J.R. Nelson D.J. J. Biol. Chem. 1994; 269: 51-54Abstract Full Text PDF PubMed Google Scholar, 13Chao A.C. de Sauvage F.J. Dong Y.-J. Wagner J.A. Goeddel D.V. Gardner P. EMBO J. 1994; 13: 1065-1072Crossref PubMed Scopus (231) Google Scholar) followed by multisite-phosphorylation of CFTR(14Cheng S.H. Rich D.P. Marshall J. Gregory R.J. Welsh M.J. Smith A.E. Cell. 1991; 66: 1027-1036Abstract Full Text PDF PubMed Scopus (527) Google Scholar), (ii) direct interaction of cGMP with the CFTR protein(15Sullivan S.K. Agellon L.B. Schick R. Gregory R.J. Paul S. Pediatr. Pulmonol. Suppl. 1994; 10: 187-188Google Scholar), and (iii) cGMP activation of an intestine-specific isoform of cGMP-dependent protein kinase (type II cGK; (16De Jonge H.R. Nature. 1976; 262: 590-593Crossref Scopus (60) Google Scholar, 17De Jonge H.R. Adv. Cyclic Nucleotide Res. 1981; 14: 315-333PubMed Google Scholar, 18De Jonge H.R. Rao M.C. Lebenthal E. Duffey M. Textbook of Secretory Diarrhea. Raven Press, New York1990: 191-207Google Scholar, 19Vaandrager A.B. De Jonge H.R. Adv. Pharmacol. 1994; 26: 253-283Crossref PubMed Scopus (43) Google Scholar)). cGKII was discovered as a cGMP-sensitive 86-kDa phosphoprotein localized in intestinal brush border membranes (16De Jonge H.R. Nature. 1976; 262: 590-593Crossref Scopus (60) Google Scholar), which comigrated with a cGMP receptor protein on one- and two-dimensional gels(17De Jonge H.R. Adv. Cyclic Nucleotide Res. 1981; 14: 315-333PubMed Google Scholar, 18De Jonge H.R. Rao M.C. Lebenthal E. Duffey M. Textbook of Secretory Diarrhea. Raven Press, New York1990: 191-207Google Scholar). The intestinal isoform is clearly distinct from the homodimeric type Iα and Iβ cGK (153-156 kDa) identified in other mammalian tissues, as illustrated by differences in subcellular localization, subunit composition, isoelectric point, phosphopeptide maps, immunoreactivity, and affinity for cyclic nucleotide analogues(17De Jonge H.R. Adv. Cyclic Nucleotide Res. 1981; 14: 315-333PubMed Google Scholar, 18De Jonge H.R. Rao M.C. Lebenthal E. Duffey M. Textbook of Secretory Diarrhea. Raven Press, New York1990: 191-207Google Scholar, 19Vaandrager A.B. De Jonge H.R. Adv. Pharmacol. 1994; 26: 253-283Crossref PubMed Scopus (43) Google Scholar). Recently, molecular cloning of cGKII from mouse brain (20Uhler M.D. J. Biol. Chem. 1993; 268: 13586-13591Abstract Full Text PDF PubMed Google Scholar) and rat intestine (21Jarchau T. Häusler C. Markert T. Pöhler D. Vandekerckhove J. De Jonge H.R. Lohmann S.M. Walter U. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9426-9430Crossref PubMed Scopus (137) Google Scholar) demonstrated that cGKII is a different gene product than cGKIα and Iβ(22Wernet W. Flockerzi V. Hofmann F. FEBS Lett. 1989; 251: 191-196Crossref PubMed Scopus (161) Google Scholar, 23Sandberg M. Natarajan V. Ronander I. Kalderon D. Walter U. Lohmann S.M. Jahnsen T. FEBS Lett. 1989; 255: 321-329Crossref PubMed Scopus (115) Google Scholar).In the present study, evidence for a functional difference between cGK isoenzymes was obtained from studies of the activation of CFTR-Cl− channels in excised membrane patches of an intestinal cell line (IEC-CF7; (24Bijman J. Dalemans W. Kansen M. Keulemans J. Verbeek E. Hoogeveen A. De Jonge H.R. Wilke M. Dreyer D. Lecocq J.-P. Favirani A. Scholte B. Am. J. Physiol. 1993; 264: L229-L235Crossref PubMed Google Scholar)) or NIH-3T3 fibroblasts stably expressing recombinant CFTR(25Anderson M.P. Berger H.A. Rick D.P. Gregory R.J. Smith A.E. Welsh M.J. Cell. 1993; 67: 775-784Abstract Full Text PDF Scopus (408) Google Scholar). In both models, exposure of patches to a combination of cGMP and ATP failed to elicit Cl− channel activity. However, the further addition of purified cGKII, but not cGKIα, resulted in almost full activation of CFTR-Cl− currents.Differential activation of the CFTR-Cl− channel by cGKII is the first example of isotype specificity in cGK regulation of cellular functions and provides a plausible explanation for the prominent role of cGMP as a regulator of Cl− transport in intestinal epithelium in comparison to other CFTR-expressing cell types in which cGKII expression is marginal or absent(19Vaandrager A.B. De Jonge H.R. Adv. Pharmacol. 1994; 26: 253-283Crossref PubMed Scopus (43) Google Scholar, 20Uhler M.D. J. Biol. Chem. 1993; 268: 13586-13591Abstract Full Text PDF PubMed Google Scholar, 21Jarchau T. Häusler C. Markert T. Pöhler D. Vandekerckhove J. De Jonge H.R. Lohmann S.M. Walter U. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9426-9430Crossref PubMed Scopus (137) Google Scholar).EXPERIMENTAL PROCEDURESMaterialsCalyculin A was obtained from Calbiochem, San Diego, CA. PKI, the Walsh inhibitor peptide (PKI(5-24)-amide) was obtained from Dr. U. Walter, Würzburg, Germany. CF-2, a cloned R domain peptide of CFTR (AA645-835), was produced in bacteria and purified as described(26Picciotto M.R. Cohn J.A. Bertuzzi G. Greengard P. Nairn A.C. J. Biol. Chem. 1992; 267: 12742-12752Abstract Full Text PDF PubMed Google Scholar). Adenosine 5′-triphosphate, sodium salt (ATP), and cGMP were obtained from Boehringer Mannheim. [γ-32P]ATP was obtained from Amersham, UK. All other chemicals were from Sigma.Cells and CFTR Expression SystemsTwo cell types that stably express CFTR were prepared and maintained as described previously(24Bijman J. Dalemans W. Kansen M. Keulemans J. Verbeek E. Hoogeveen A. De Jonge H.R. Wilke M. Dreyer D. Lecocq J.-P. Favirani A. Scholte B. Am. J. Physiol. 1993; 264: L229-L235Crossref PubMed Google Scholar, 27Anderson M.P. Gregory R.J. Thompson S. Souza D.W. Paul S. Mulligan R.C. Smith A.E. Welsh M.J. Science. 1991; 253: 202-205Crossref PubMed Scopus (875) Google Scholar). IEC-CF7 cells were obtained by stable transfection of the rat fetal intestine-derived IEC-6 cell line with a plasmid encoding CFTR(24Bijman J. Dalemans W. Kansen M. Keulemans J. Verbeek E. Hoogeveen A. De Jonge H.R. Wilke M. Dreyer D. Lecocq J.-P. Favirani A. Scholte B. Am. J. Physiol. 1993; 264: L229-L235Crossref PubMed Google Scholar); NIH-3T3 cells expressed CFTR after infection with a retroviral vector encoding CFTR(27Anderson M.P. Gregory R.J. Thompson S. Souza D.W. Paul S. Mulligan R.C. Smith A.E. Welsh M.J. Science. 1991; 253: 202-205Crossref PubMed Scopus (875) Google Scholar).Isolation of Protein KinasescGKII was purified from the small intestine of adult pigs (donated by the Department of Experimental Cardiology, Erasmus University). The small intestine was dissected from anesthetized pigs, rinsed with ice-cold 0.9% NaCl, and frozen in liquid nitrogen. Brush border membrane vesicles were prepared from the intestinal pieces by a freeze-thaw procedure and subsequent differential Mg2+ precipitation and centrifugation as described previously(28Van Dommelen F.S. Hamer C.M. De Jonge H.R. Biochem. J. 1986; 236: 771-778Crossref PubMed Scopus (21) Google Scholar). cGKII extraction from the vesicles and purification by affinity chromatography on 8-(2-aminoethyl)-amino-cAMP-Sepharose was performed essentially as described (17De Jonge H.R. Adv. Cyclic Nucleotide Res. 1981; 14: 315-333PubMed Google Scholar, 18De Jonge H.R. Rao M.C. Lebenthal E. Duffey M. Textbook of Secretory Diarrhea. Raven Press, New York1990: 191-207Google Scholar) with a slight modification. To obtain detergent-free enzyme for use in the patch clamp experiments, elution of cGKII from the affinity gel with 1 mM cGMP was performed in the presence of 4 mM octyl glucoside rather than Triton X-100. Subsequently, cGMP and octyl glucoside were removed by dialysis in detergent-free patch clamp medium (see below).The catalytic subunit of type II cAK and cGKI (characterized by antibody analysis to be primarily the Iα isoform; (29Keilbach A. Ruth P. Hofmann F. Eur. J. Biochem. 1992; 208: 467-473Crossref PubMed Scopus (98) Google Scholar)) were purified from bovine heart and bovine lung, respectively, as described (30Kaczmarek L.K. Jenning K.R. Strumwasser F. Nairn A.C. Walter U. Wilson F.D. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 7487-7491Crossref PubMed Scopus (172) Google Scholar, 31Walter U. Miller P. Wilson F. Menkes D. Greengard P. J. Biol. Chem. 1980; 255: 3757-3763Abstract Full Text PDF PubMed Google Scholar). The specific activities (units/mg protein) of the purified protein kinases as determined by the Kemptide phosphorylation assay (32Kemp B.E. Graves D.J. Benjamini E. Krebs E.G. J. Biol. Chem. 1977; 252: 4888-4894Abstract Full Text PDF PubMed Google Scholar) were 4.2 (cAK), 2.0 (cGKI), and 1.6 (cGKII), respectively. A monomeric constitutively active cGKI fragment was obtained by limited trypsinization as described(33Monken C.E. Gill G.N. J. Biol. Chem. 1980; 255: 7067-7070Abstract Full Text PDF PubMed Google Scholar).Patch Clamp TechniquePatch clamp experiments were performed as described by Hamill et al.(34Hamill O.P. Marty A. Neher E. Sackmann B. Sigworth F.J. Pfluegers Arch. 1981; 395: 85-100Crossref Scopus (15094) Google Scholar). Glass (borosilicate) pipettes were pulled to a resistance of 308 megohms and heat polished. Pipette potential refers to the voltage applied to the pipette interior with reference to the bath potential. Upward deflections denote negative charge flowing out of the pipette. A List EPC-7 amplifier was used for current amplification and voltage clamping. Membrane voltage was continuously clamped at −40 mV, except when performing current-voltage relationships. Data were monitored on an oscilloscope and stored on a VCR. The recorded data were filtered at 50 or 100 Hz, digitized at 200 Hz, and analyzed on a personal computer. Data analysis was performed as described by Kansen et al.(35Kansen M. Bajnath R. Groot J. De Jonge H.R. Scholte B.J. Hoogeveen A.T. Bijman J. Pfluegers Arch. 1993; 422: 539-545Crossref PubMed Scopus (7) Google Scholar). In view of the fact that the high density of CFTR-Cl− channels in membrane patches of the 3T3-CFTR fibroblasts hinders the accurate determination of open state probability or number of channels, channel activity in these patches was expressed in pA, rather than as the number of open channels. The composition of the bath and pipette solutions was (in mM): 140 N-methyl-D-glucamine, 1 EGTA, 3 MgCl2, and 10 Hepes-HCl (pH 7.3, final Cl− concentration 147 mM). In some experiments a low Cl− pipette buffer was used containing (in mM): 140 N-methyl-D-glucamine, 100 L-aspartic acid, 2 MgCl2, 5 CaCl2, and 10 Hepes-HCl (pH 7.3; final Cl− concentration 49 mM). Excised patches were studied in a solution exchange compartment (volume 1 ml), as described previously by Kansen et al.(36Kansen M. Keulemans J. Hoogeveen A.T. Scholte B.J. Vaandrager A.B. Van der Kamp A.W.M. Sinaasappel M. Bot A.G.M. De Jonge H.R. Bijman J. Biochim. Biophys. Acta. 1992; 1139: 49-56Crossref PubMed Scopus (9) Google Scholar). Experiments were performed at room temperature.In Vitro Phosphorylation of CFTR and CF-2 and Phosphopeptide MappingCFTR was immunoprecipitated from T84 cells using specific CFTR antibodies and protein A-Sepharose beads as described(26Picciotto M.R. Cohn J.A. Bertuzzi G. Greengard P. Nairn A.C. J. Biol. Chem. 1992; 267: 12742-12752Abstract Full Text PDF PubMed Google Scholar). CFTR attached to protein A-Sepharose beads (10 μl of suspension) or CFTR antibody plus beads alone was incubated at 30°C for 40 min in 100 μl of buffer containing 10 mM MgCl2, 1 mM EGTA, 10 mM Hepes, pH 7.3, 50 μM MgATP, 20 μM cGMP, 10 Ci/mmol [γ-32P]ATP, and purified protein kinases (catalytic subunit of cAK, 2 milliunits/ml; cGKI, 7.5 milliunits/ml; cGKII, 9.4 milliunits/ml). The phosphorylated samples were washed, resuspended in SDS-stop solution, and analyzed on 6% SDS-PAGE as described previously(26Picciotto M.R. Cohn J.A. Bertuzzi G. Greengard P. Nairn A.C. J. Biol. Chem. 1992; 267: 12742-12752Abstract Full Text PDF PubMed Google Scholar). Phosphorylation conditions for CF-2 (5 μM) were similar to those for CFTR. For kinetic experiments, linear incorporation of 32P with time was ensured by (i) restricting the incubation time to 5 min, (ii) varying the CF-2 concentrations between 0.1 and 0.5 μM, (iii) increasing the [γ-32P]ATP concentration to 500 μM (1 Ci/mmol), and (iv) using equal concentrations (25 nM) of each protein kinase. Reactions were terminated by the addition of 20 μl of 70% trichloroacetic acid, and protein pellets were washed three times with 0.2 ml of ice-cold H2O, suspended in 50 μl of SDS-stop buffer, boiled for 2 min, and subjected to 12% SDS-PAGE as described(26Picciotto M.R. Cohn J.A. Bertuzzi G. Greengard P. Nairn A.C. J. Biol. Chem. 1992; 267: 12742-12752Abstract Full Text PDF PubMed Google Scholar). 32P-Labeled CF-2 or CFTR were excised from the dried gels, washed with two changes of 10% acetic acid/30% methanol and three changes of 50% methanol, and lyophilized. In the kinetic experiments, the incorporation of 32P into CF-2 was quantified by liquid scintillation spectrometry. For two-dimensional phosphopeptide mapping of CF-2 and CFTR, 1 ml of 50 mM NH4CO3, pH 8.0, containing L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (50 μg/ml) was added to the dried gel pieces and the mixture was incubated at 37°C for 20 h(26Picciotto M.R. Cohn J.A. Bertuzzi G. Greengard P. Nairn A.C. J. Biol. Chem. 1992; 267: 12742-12752Abstract Full Text PDF PubMed Google Scholar). The gel pieces were washed with 0.5 ml of 50 mM NH4HCO3 at 37°C for 4 h, and the collected supernatants were lyophilized. Phosphopeptides were separated on thin layer cellulose sheets (20 × 20 cm, Eastman Kodak Co.) by electrophoresis in the first dimension, followed by chromatography in the second dimension. Dried sheets were subjected to autoradiography.RESULTSActivation of CFTR-Cl−Channels by Protein KinasesIn agreement with earlier studies of Cl− channel activation in excised, inside-out membrane patches from 3T3-CFTR fibroblasts(25Anderson M.P. Berger H.A. Rick D.P. Gregory R.J. Smith A.E. Welsh M.J. Cell. 1993; 67: 775-784Abstract Full Text PDF Scopus (408) Google Scholar, 37Gregory R.J. Cheng S.H. Rick D.P. Marshall J. Hekir K. Ostedgaard L. Klinger K.W. Welsh M.J. Smith A.E. Nature. 1990; 347: 382-386Crossref PubMed Scopus (269) Google Scholar, 38Berger H.A. Travis S.M. Welsh M.J. J. Biol. Chem. 1993; 268: 2037-2047Abstract Full Text PDF PubMed Google Scholar), the addition of catalytic subunit of cAK (2 milliunits/ml) to the bath, in the presence of 2 mM MgATP, resulted in rapid activation (lag time < 1 min) of multiple anion-selective channels (average current increase per patch 40 ± 38 pA at −40 mV holding potential; n = 15) showing characteristic properties of the CFTR-Cl− channel (i.e. Cl− selectivity, linear current-voltage relationship in symmetrical Cl− concentrations, 8 pS single channel conductance; results not shown). A similar low conductance channel, occurring at a much lower density (2-6 channels/patch) was activated by cAK in excised patches from the rat intestinal IEC-CF7 cell line stably expressing CFTR(24Bijman J. Dalemans W. Kansen M. Keulemans J. Verbeek E. Hoogeveen A. De Jonge H.R. Wilke M. Dreyer D. Lecocq J.-P. Favirani A. Scholte B. Am. J. Physiol. 1993; 264: L229-L235Crossref PubMed Google Scholar). In addition to cAK, the effect of cGK on CFTR-Cl− channel opening was examined in excised, cell-free patches from both cell lines. Addition of MgATP (2 mM) and cGMP (50 μM) together did not activate current in 3T3-CFTR patches during 5-15 min observation (Fig. 1A). Inclusion of the cGKI isoform purified from bovine lung (10 milliunits/ml) likewise failed to open the CFTR-Cl− channel (Fig. 1A), confirming earlier observations by Berger et al.(38Berger H.A. Travis S.M. Welsh M.J. J. Biol. Chem. 1993; 268: 2037-2047Abstract Full Text PDF PubMed Google Scholar). However, the subsequent addition of saturating concentrations of the cGKII (10 milliunits/ml) elicited a large Cl− current, reaching a value after 15 min that was 78 ± 24% (n = 5) of the maximal current attained upon addition of saturating amounts of cAK (2 milliunits/ml) to the same patch (Fig. 1A). Half-maximal cGKII-activation (48 ± 9% of the maximal cGKII response at 15 min; n = 5) was observed in the presence of 2 milliunits/ml cGKII, whereas the threshold for current activation (10 ± 6%; n = 5) was found at 0.5 milliunits/ml cGKII (data not shown). In comparison to cAK, CFTR-Cl− current activation by cGKII was a relatively slow process (time required to reach half-maximal activation following addition of 10 milliunits/ml cGKII: 8 ± 1 min; for 2 milliunits/ml cAK: 0.7 ± 0.2 min, n = 6; Fig. 1, A and B). This slow activation was unlikely to result from the activity of counteracting protein phosphatases, since neither calyculin A (10−7M), a potent inhibitor of phosphatase 1 and 2A (recently implied in CFTR regulation; (38Berger H.A. Travis S.M. Welsh M.J. J. Biol. Chem. 1993; 268: 2037-2047Abstract Full Text PDF PubMed Google Scholar) and (39Gadsby D.C. Nairn A.C. Trends Biochem. Sci. 1994; 19: 513-518Abstract Full Text PDF PubMed Scopus (82) Google Scholar)), nor the additional presence of ATPγS (1 mM) to produce stable thiophosphorylation, was able to enhance the CFTR activation kinetics (results not shown).The cGKII isoform, but not cGKI, was capable of activating CFTR-Cl− channels also in excised patches from IEC-CF7 cells (Fig. 2). In this low expression model, single-channel events could be monitored (Fig. 2A), which had a linear I-V relation in symmetrical Cl− concentrations, a channel conductance of 8 pS, and a rightward shift in current reversal potential upon lowering of the Cl− concentration in the pipette (Fig. 2B). The mean open probability (Po) of CFTR-Cl− channels measured at the plateau phase of activation by saturating concentrations of cGKII (0.22 ± 0.09; n = 9) did not differ significantly from the Po of cAK-activated channels (0.23 ± 0.12; n = 8).Figure 2Biophysical characteristics of the cGKII-activated channel in excised, inside-out membrane patches from IEC-CF7 cells. Panel A, current tracings of cGKII-activated channels. Left, symmetrical 147/147 mM chloride solutions. Right, pipette buffer was replaced by a low (49 mM) chloride buffer. Tracings were obtained at the indicated voltages. C, all channels closed; dotted line, single-channel current levels. Panel B, I-V characteristics of the channel. The channel conductance was 8.0 ± 0.6 pS (n = 5). •, symmetrical Cl− solution (147/147 mM); ▲, reduction of Cl− in the pipette to 49 mM by replacement with aspartic acid.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Additional proof of the identity of the cGKII-activated channel as CFTR came from the observation that cGKII could not further enhance channel activity in excised patches from 3T3-CFTR cells following their pre-phosphorylation by cAK (2 milliunits/ml) and ATP (2 mM) (results not shown). Another similarity between CFTR-Cl− channel regulation by cAK and cGKII was the observation that the currents rapidly returned to near base-line values upon the removal of either kinase and ATP from the bath, but could be restored almost instantaneously by the readdition of 2 mM ATP alone, confirming the crucial role of ATP in CFTR-Cl− channel functioning (Fig. 1B; cf. Refs. 25, 38, and 40).To eliminate the possibility that cGKII activation of CFTR resulted from a contamination of the cGKII preparation with cAK, a specific peptide inhibitor of cAK, PKI (0.1 μM) was added to the bath. Under this condition CFTR-Cl− channel activation in 3T3 membrane patches by cAK (2 milliunits/ml) was completely abolished, whereas channel activation by cGKII (2 milliunits/ml) was not significantly affected (42 ± 3% of the maximal channel activity evoked by cAK in the same patch following removal of PKI from the bath, as compared to 37 ± 5% in the absence of PKI; n = 5). Moreover, PKI was unable to inhibit phosphorylation of Kemptide or CF-2 by cGKII (not shown).Finally, the possibility was considered that cGKI, in spite of its failure to open CFTR-Cl− channels by itself, could interfere with the activation of CFTR-Cl− channels by the other cGK isoform. However, neither preincubation of 3T3 patches with cGKI (10 milliunits/ml, 15 min; results not shown) nor the simultaneous addition of cGKI (10 milliunits/ml) and cGKII (10 milliunits/ml) had any effect on the rate or extent of CFTR-Cl− channel activation as compared to cGKII alone (Fig. 1C).Phosphorylation of CFTR and CF-2 by cGK IsotypesPhosphorylation studies carried out with CFTR immunoprecipitates (Fig. 3A) and CF-2, the recombinant R domain of CFTR (Fig. 3B) confirmed previous reports that both proteins are excellent in vitro substrates for cAK and cGKI(26Picciotto M.R. Cohn J.A. Bertuzzi G. Greengard P. Nairn A.C. J. Biol. Chem. 1992; 267: 12742-12752Abstract Full Text PDF PubMed Google Scholar, 38Berger H.A. Travis S.M. Welsh M.J. J. Biol. Chem. 1993; 268: 2037-2047Abstract Full Text PDF PubMed Google Scholar). This discrepancy between phosphorylation and functional studies was even more apparent from the kinetics of in vitro phosphorylation of CF-2 by cGKI and cGKII isoenzymes (Fig. 3B). CF-2 was a better substrate for cGKI than for cGKII (similar Km; 3-fold higher Vmax), and the plateau level of phosphate incorporation into CFTR (Fig. 3A) was also higher for cGKI (140 ± 11% of the cGKII level; n = 8).Figure 3Phosphorylation of CFTR (panel A) and CF-2 (panel B) by purified protein kinases. Panel A, CFTR was immunoprecipitated from T84 cells and phosphorylated as described under “Experimental Procedures.” The reactions also contained: catalytic subunit of cAK (2 milliunits/ml; lanes 1 and 2), cGKI (7.5 milliunits/ml; lanes 3 and 4), and cGKII (9.4 milliunits/ml; lanes 5 and 6). Lanes 2, 4, and 6, control experiments in which CFTR was omitted. The 32P-labeled proteins were separated by 6% SDS-PAGE. The gel was dried and exposed to x-ray film. CFTR migrates as a broad band of 180 kDa (“band C”; see (14Cheng S.H. Rich D.P. Marshall J. Gregory R.J. Welsh M.J. Smith A.E. Cell. 1991; 66: 1027-1036Abstract Full Text PDF PubMed Scopus (527) Google Scholar)). The 86- and 74-kDa bands represent residual amounts of autophosphorylated cGKII (lanes 5 and 6, intact 86-kDa form + 74-kDa proteolytic fragment; cf. (17De Jonge H.R. Adv. Cyclic Nucleotide Res. 1981; 14: 315-333PubMed Google Scholar)) and cGKI (lanes 3 and 4, intact 74-kDa form), respectively, remaining following the washing steps. Panel B, Lineweaver-Burk plots of CF-2 phosphorylation by equal concentrations (25 nM) of cAK, cGKI, and cGKII. The experimental conditions needed to ensure linear rates of 32P incorporation are specified under “Experimental Procedures.” The inset shows the kinetic constants (Km, Vmax) calculated from the Lineweaver-Burk plots. A.U., arbitrary units. Data represent the mean of three experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Two-dimensional Phosphopeptide Map" @default.
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• W2113370189 title "Isotype-specific Activation of Cystic Fibrosis Transmembrane Conductance Regulator-Chloride Channels by cGMP-dependent Protein Kinase II" @default.
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