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Cyclic GMP-dependent Protein Kinase Activates Cloned BKCa Channels Expressed in Mammalian Cells by Direct Phosphorylation at Serine 1072

1999; Elsevier BV; Volume: 274; Issue: 16 Linguagem: Inglês

10.1074/jbc.274.16.10927

ISSN

1083-351X

Autores

Mitsuhiro Fukao, Helen S. Mason, Fiona C. Britton, James L. Kenyon, Burton Horowitz, Kathleen D. Keef,

Tópico(s)

Nitric Oxide and Endothelin Effects

Resumo

NO-induced activation of cGMP-dependent protein kinase (PKG) increases the open probability of large conductance Ca2+-activated K+ channels and results in smooth muscle relaxation. However, the molecular mechanism of channel regulation by the NO-PKG pathway has not been determined on cloned channels. The present study was designed to clarify PKG-mediated modulation of channels at the molecular level. The cDNA encoding the α-subunit of the large conductance Ca2+-activated K+ channel,cslo-α, was expressed in HEK293 cells. Whole cell and single channel characteristics of cslo-α exhibited functional features of native large conductance Ca2+-activated K+ channels in smooth muscle cells. The NO-donor sodium nitroprusside increased outward current 2.3-fold in whole cell recordings. In cell-attached patches, sodium nitroprusside increased the channel open probability (NPo) ofcslo-α channels 3.3-fold without affecting unitary conductance. The stimulatory effect of sodium nitroprusside was inhibited by the PKG-inhibitor KT5823. Direct application of PKG-Iα to the cytosolic surface of inside-out patches increased NPo 3.2-fold only in the presence of ATP and cGMP without affecting unitary conductance. A point mutation of cslo-α in which Ser-1072 (the only optimal consensus sequence for PKG phosphorylation) was replaced by Ala abolished the PKG effect on NPo in inside-out patches and the effect of SNP in cell attached patches. These results indicate that PKG activates cslo-α by direct phosphorylation at serine 1072. NO-induced activation of cGMP-dependent protein kinase (PKG) increases the open probability of large conductance Ca2+-activated K+ channels and results in smooth muscle relaxation. However, the molecular mechanism of channel regulation by the NO-PKG pathway has not been determined on cloned channels. The present study was designed to clarify PKG-mediated modulation of channels at the molecular level. The cDNA encoding the α-subunit of the large conductance Ca2+-activated K+ channel,cslo-α, was expressed in HEK293 cells. Whole cell and single channel characteristics of cslo-α exhibited functional features of native large conductance Ca2+-activated K+ channels in smooth muscle cells. The NO-donor sodium nitroprusside increased outward current 2.3-fold in whole cell recordings. In cell-attached patches, sodium nitroprusside increased the channel open probability (NPo) ofcslo-α channels 3.3-fold without affecting unitary conductance. The stimulatory effect of sodium nitroprusside was inhibited by the PKG-inhibitor KT5823. Direct application of PKG-Iα to the cytosolic surface of inside-out patches increased NPo 3.2-fold only in the presence of ATP and cGMP without affecting unitary conductance. A point mutation of cslo-α in which Ser-1072 (the only optimal consensus sequence for PKG phosphorylation) was replaced by Ala abolished the PKG effect on NPo in inside-out patches and the effect of SNP in cell attached patches. These results indicate that PKG activates cslo-α by direct phosphorylation at serine 1072. Large conductance Ca2+-activated K+(BKCa) 1The abbreviations used are: BKCachannel, large conductance Ca2+-activated K+channel; PKG, cGMP-dependent protein kinase; KVchannel, voltage-gated K+ channel; NPo, channel open probability; IBTX, iberiotoxin; SNP, sodium nitroprusside; DETA, diethylenetriamine; PCR, polymerase chain reaction; pS, picosiemens. channels are ubiquitously distributed among tissues and are particularly abundant in smooth muscle (1Nelson M.T. Quayle J.M. Am. J. Physiol. 1995; 268: C799-C822Crossref PubMed Google Scholar, 2Tseng-Crank J. Foster C.D. Krause J.D. Mertz R. Godinot N. DiChiara T.J. Reinhart P.H. Neuron. 1994; 13: 1315-1330Abstract Full Text PDF PubMed Scopus (384) Google Scholar). The activity of BKCachannels is regulated by membrane potential, intracellular Ca2+, and phosphorylation (3Toro L. Wallner M. Merra P. Tanaka Y. News Physiol. Sci. 1998; 13: 112-117PubMed Google Scholar, 4Kaczorowski G.J. Knaus H.G. Leonard R.J. McManus O.B. Garcia M.L J. Bioenerg. Biomembr. 1996; 28: 255-267Crossref PubMed Scopus (266) Google Scholar). Although BKCachannels are usually not involved in setting resting potential, they play a key role as a negative feedback mechanism to limit depolarization and contraction (5Latorre R. Oberhauser A. Labarca P. Alvarez O. Annu. Rev. Physiol. 1989; 51: 385-399Crossref PubMed Scopus (636) Google Scholar, 6Nelson M.T. Cheng H. Rubart M. Santana L.F. Bonev A.D. Knot H.J. Lederer W.J. Science. 1995; 270: 633-637Crossref PubMed Scopus (1208) Google Scholar, 7Taniguchi J. Furukawa K.I. Shigekawa M. Pflügers Arch. 1993; 423: 167-172Crossref PubMed Scopus (169) Google Scholar). Activation of BKCachannels is increased by nitric oxide (NO) and atrial natriuretic peptide, which hyperpolarize the membrane and increase the sensitivity of BKCa channels to Ca2+ (8Sansom S.C. Stockand J.D. Clin. Exp. Pharmacol. Physiol. 1996; 23: 76-82Crossref PubMed Scopus (34) Google Scholar, 9Tanaka Y. Aida M. Tanaka H. Shigenobu K. Toro L. Naunyn-Schmiedeberg's Arch. Pharmacol. 1998; 357: 705-708Crossref PubMed Scopus (33) Google Scholar, 10Robertson B.E. Schubert R. Hescheler J. Nelson M.T. Am. J. Physiol. 1993; 265: C299-C303Crossref PubMed Google Scholar, 11Williams Jr., D.L. Katz G.M. Roy-Contancin L. Reuben J.P. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 9360-9364Crossref PubMed Scopus (114) Google Scholar). Membrane hyperpolarization closes voltage-dependent Ca2+channels, reduces Ca2+ influx, and leads to a reduction in intracellular Ca2+ concentration and relaxation (1Nelson M.T. Quayle J.M. Am. J. Physiol. 1995; 268: C799-C822Crossref PubMed Google Scholar). NO has been reported to stimulate BKCa channels directly as well as through stimulation of guanylate cyclase and the subsequent increase in cGMP (12Bolotina V.M. Najibi S. Palacino J.J. Pagano P.J. Cohen R.A. Nature. 1994; 368: 850-853Crossref PubMed Scopus (1514) Google Scholar, 13Mistry D.K. Garland C.J. Br. J. Pharmacol. 1998; 124: 1131-1140Crossref PubMed Scopus (178) Google Scholar, 14Fujino K. Nakaya S. Wakatsuki T. Miyoshi Y. Nakaya Y. Mori H. Inoue I. J. Pharmacol. Exp. Ther. 1991; 256: 371-377PubMed Google Scholar, 15Archer S.L. Huang J.M. Hampl V. Nelson D.P. Shultz P.J. Weir E.K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7583-7587Crossref PubMed Scopus (746) Google Scholar). In addition, activation of BKCa channels plays an important role in NO-induced relaxation of smooth muscle (16Khan S.A. Mathews W.R. Meisheri K.D. J. Pharmacol. Exp. Ther. 1993; 267: 1327-1335PubMed Google Scholar, 17Bychkov R. Gollasch M. Steinke T. Ried C. Luft F.C. Haller H. J. Pharmacol. Exp. Ther. 1998; 285: 293-298PubMed Google Scholar, 18Jiang F. Li C.G. Rand M.J. Br. J. Pharmacol. 1998; 123: 106-112Crossref PubMed Scopus (12) Google Scholar, 19Bialecki R.A. Stinson-Fisher C. Am. J. Physiol. 1995; 268: L152-L159PubMed Google Scholar, 20Vaali K. Li L. Paakkari I. Vapaatalo H. J. Pharmacol. Exp. Ther. 1998; 286: 110-114PubMed Google Scholar). cGMP activates cGMP-dependent protein kinase (PKG), which phosphorylates various cytosolic and membrane proteins that regulate smooth muscle tone either directly or indirectly (21Butt E. Geiger J. Jarchau T. Lohmann S.M. Walter U. Neurochem. Res. 1993; 18: 27-42Crossref PubMed Scopus (124) Google Scholar,22Lincoln T.M. Cornwell T.L. Blood Vessels. 1991; 28: 129-137PubMed Google Scholar). Recent studies in native cells suggest that PKG activates BKCa channels through phosphorylation of the channel (23Alioua A. Huggins J.P. Rousseau E. Am. J. Physiol. 1995; 268: L1057-L1063PubMed Google Scholar). These results are supported by biochemical studies of cloned BKCa channels, which demonstrate PKG-induced phosphorylation of the channel (24Alioua A. Tanaka Y. Wallner M. Hofmann F. Ruth P. Meera P.F. Toro L. J. Biol. Chem. 1998; 273: 32950-32956Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). The primary sequence of BKCa has been determined using molecular cloning techniques in Drosophila (25Atkinson N.S. Robertson G.A. Ganetzky B. Science. 1991; 253: 551-555Crossref PubMed Scopus (541) Google Scholar) and mammals (26Butler A. Tsunoda S. McCobb D.P. Wei A. Salkoff L. Science. 1993; 261: 221-224Crossref PubMed Scopus (574) Google Scholar, 27Vogalis F. Vincent T. Qureshi I. Schmalz F. Ward M.W. Sanders K.M. Horowitz B. Am. J. Physiol. 1996; 271: G629-G639PubMed Google Scholar, 28Dworetzky S.I. Trojnacki J.T. Gribkoff V.K. Brain Res. 1994; 27: 189-193Google Scholar). These studies indicate that BKCa isoforms belong to the voltage-gated K+ (KV) channel superfamily. The primary sequence of the S1-S6 segment of BKCa channels is homologous to the corresponding regions in KV channels. The long carboxyl terminus is the region of Ca2+-sensing (29Wei A. Solaro C. Lingle C. Salkoff L. Neuron. 1994; 13: 671-681Abstract Full Text PDF PubMed Scopus (230) Google Scholar, 30Schreiber M. Salkoff L. Biophys. J. 1997; 73: 1355-1363Abstract Full Text PDF PubMed Scopus (341) Google Scholar), and cslo-α contains a single high affinity phosphorylation site for PKG at Ser-1072 (3Toro L. Wallner M. Merra P. Tanaka Y. News Physiol. Sci. 1998; 13: 112-117PubMed Google Scholar). However additional putative PKG phosphorylation sites have been identified in other splice variants (31Saito M. Nelson C. Salkoff L. Lingle C.J. J. Biol. Chem. 1997; 272: 11710-11717Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Expression of theslo channel in Xenopus oocytes or mammalian cells gives rise to voltage-gated, Ca2+-sensitive currents with electrophysiological and pharmacological features similar to those of native BKCa (32Adelman J.P. Shen K.Z. Kavanaugh M.P. Warren R.A. Wu Y.N. Lagrutta A. Bond C.T. North R.A. Neuron. 1992; 9: 209-216Abstract Full Text PDF PubMed Scopus (432) Google Scholar, 33Perez G. Lagrutta A. Adelman J.P. Toro L. Biophys. J. 1994; 66: 1022-1027Abstract Full Text PDF PubMed Scopus (51) Google Scholar, 34Esguerra M. Wang J. Foster C.D. Adelman J.P. North R.A. Levitan I.B. Nature. 1994; 369: 563-565Crossref PubMed Scopus (97) Google Scholar). However, although many studies of native cells suggest that BKCa channel activity is also modulated by various protein kinases (35Lechleiter J.D. Dartt D.A. Brehm P. Neuron. 1988; 1: 227-235Abstract Full Text PDF PubMed Scopus (46) Google Scholar, 36Yamakage M. Hirshman C.A. Croxton T.L. Am. J. Physiol. 1996; 270: L338-L345PubMed Google Scholar, 37Minami K. Fukuzawa K. Nakaya Y. Biochem. Biophys. Res. Commun. 1993; 190: 263-269Crossref PubMed Scopus (82) Google Scholar, 38Prevarskaya N.B. Skryma R.N. Vacher P. Daniel N. Djiane J. Dufy B. J. Biol. Chem. 1995; 270: 24292-24299Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), this property has been difficult to reproduce in cloned channels. Two studies in whichslo channels have been expressed in either oocytes (27Vogalis F. Vincent T. Qureshi I. Schmalz F. Ward M.W. Sanders K.M. Horowitz B. Am. J. Physiol. 1996; 271: G629-G639PubMed Google Scholar) or Chinese hamster ovary cells (39Zhou X.B. Schlossmann J. Hofmann F. Ruth P. Korth M. Pflügers Arch. 1998; 436: 725-734Crossref PubMed Scopus (41) Google Scholar) have reported that PKG was without effect on slo channel activity. In contrast, Perez et al. (33Perez G. Lagrutta A. Adelman J.P. Toro L. Biophys. J. 1994; 66: 1022-1027Abstract Full Text PDF PubMed Scopus (51) Google Scholar) reported that an endogenous cAMP-dependent protein kinase-like activity activated dslo-α channels expressed in Xenopus oocytes. A recent study showed that PKG-Iα phosphorylated hslo channels reconstituted into lipid bilayers but had no effect on channel activity in inside-out patches expressed in Xenopus oocytes (24Alioua A. Tanaka Y. Wallner M. Hofmann F. Ruth P. Meera P.F. Toro L. J. Biol. Chem. 1998; 273: 32950-32956Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). The purpose of this study was to examine PKG-induced modulation of cloned BKCa channels and determine whether direct phosphorylation of the channel was involved. The α-subunit ofcslo, a BKCa channel α-subunit cloned from canine colon, was expressed in HEK293 cells, and currents were measured using both the whole cell mode as well as cell-attached and detached patches. Evidence was obtained suggesting that the activity of cloned BKCa channels is enhanced by the NO/PKG pathway and that stimulation is mediated by direct phosphorylation of Ser-1072 ofcslo-α by PKG. The cDNA encoding the α-subunit of the BKCa channel (cslo-α) was previously cloned from canine colonic smooth muscle using reverse transcription and a polymerase chain reaction (GenBank™ accession number U-41001). Northern blot analysis showed that thecslo-α transcripts are expressed in the muscles of the canine gastrointestinal tract and blood vessels (27Vogalis F. Vincent T. Qureshi I. Schmalz F. Ward M.W. Sanders K.M. Horowitz B. Am. J. Physiol. 1996; 271: G629-G639PubMed Google Scholar). Thecslo-α construct was subcloned into the mammalian expression vector pZeoSV (Invitrogen, CA). The S1072A mutation of cslo-α was created by recombinant mutagenesis (40Higuchi R. Innis M.A. Gelfand D.H. Sninsky J.J. White T.J. PCR Protocols: A Guide to Methods and Applications. Academic Press Inc., San Diego, CA1990: 177-183Google Scholar). Briefly, linearized cslo-α plasmid was modified and amplified simultaneously by PCR in two separate reactions. Two primer pairs were used for PCR. In the first amplification reaction, one-half of the plasmid was amplified using a forward primer containing the S1072A mutation, spanning nucleotides 3194 to 3233 (5′-AGTCCTCCAGCAAGAAGAGCGCCTCCGTGCACTCCATCCC-3′) and a reverse primer complementary to the plasmid sequence (5′-GAACGGCACTGGTCAACTTGGCCATGGTGGCCCTC-3′). The second half of the reaction amplified the remaining half of the plasmid using the reverse-mutating primer (5′-AGTCCTCCAGCAAGAAGAGCGCCTCCGTGCACTCCATCCC-3′) and the forward plasmid-specific primer (5′-ATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTCAC-3′). Both the mutating and plasmid primers were designed to contain a ≥24-base pair homology at their 5′ ends, which generated an overlap between the ends of the two PCR products. The homologous ends of the PCR products undergo recombination in vivo following transformation of RecAEscherichia coli cells. PCR amplification was performed in 50-μl reactions containing 1× PCR buffer (50 mm KCl, 10 mm Tris-Cl, 1.5 mm MgCl2 (pH 8.3); Invitrogen), 200 μm each dNTP (Invitrogen), 25 pmol of primer, 2 ng of plasmid template, 10% Me2SO, and 2. 5 units ofTaq polymerase (Promega, Madison, WI). Reactants underwent an initial denaturation (94 °C × 1 min), 30 amplification cycles (94 °C × 30 s, 50 °C × 30 s, and 72 °C × 3 min) and a final extension of 72 °C × 10 min. PCR products were gel-purified, and 2.5 μl of each PCR reaction were mixed and transformed directly into 50 μl of Max CompetentTM DH5α E. coli (Life Technologies, Inc) and selected on low salt LB zeocin plates (25 μg/ml). Plasmid DNA was prepared from overnight cultures using the QIAprep Miniprep kit (Qiagen, CA). Plasmid DNA of the correct size was sequenced using the ABI Prism cycle sequencing kit (Perkin-Elmer, CA) and analyzed on a Perkin-Elmer 310 Genetic Analyzer. HEK293 cells were obtained from ATCC (cell line number CRL-1573, Manassas, VA) and maintained in modified RPMI medium (Life Technologies, Inc.) supplemented with 10% heat-inactivated horse serum (Summit Biotechnology, Fort Collins, CO) and 1% glutamine (Life Technologies, Inc.) in a humidified 5% CO2 incubator at 37 °C. Cells were subcultured twice a week by treatment with trypsin-EDTA (Life Technologies, Inc.). The cslo-α DNA was transfected into HEK cells by electroporation. Electroporation was performed as follows. After harvesting the HEK cells by trypsin-EDTA, the cells were washed twice with phosphate buffer solution and resuspended in ice-cold phosphate buffer solution at a density of 5 × 106 cells/ml in the cuvette for electroporation. Each cuvette was supplemented with appropriate combinations of CD8 (a lymphocyte cell-surface antigen) in the πH3-CD8 plasmid construct as a marker for transfection (4 μg), cslo-α, or S1072Acslo-α in the pZeoSV vector (20 μg). After a 10-min incubation on ice, electroporation was done by applying a 330-V pulse using a pulse generator (Electroporator II, invitrogen, CA). HEK cells expressing cslo-α were subcultured on glass coverslips for electrophysiological recording. Current recording was performed 1 to 4 days after the electroporation procedure. Transfected cells were identified by their binding to CD8-coated beads (Dyna-beads M-450 CD8; Great Neck, NY) (41Jurman M.E. Boland L.M. Liu Y. Yellen G. Biotechniques. 1994; 17: 876-881PubMed Google Scholar). The patch-clamp technique was used to measure membrane currents in whole cell and single cell configuration. Patch pipettes were made from borosilicate grass capillaries pulled with a three-stage micropipette puller (P.80/PC, Sutter, CA) and heat-polished with a microforge (MF-83, Narishige, Japan). The pipettes had tip resistances of 2 to 5 megaohm for whole cell recordings and 8 to 10 megaohm for single-channel recordings. Coverslips containing HEK cells were placed in a recording chamber (volume 1.0 ml) mounted on the stage of an Olympus inverted microscope and superfused with bath solution at a rate of 1.0 ml/min. Standard gigaohm seal patch-clamp recording techniques were used to measure the currents of whole cell, cell-attached, and excised inside-out configurations. An Axopatch 200A patch-clamp amplifier (Axon Instruments, CA) was used to measure whole cell and single-channel recordings. Capacitance and series resistance compensation were performed. The output signals were filtered at 1 kHz with an 8-pole Bessel filter, digitized at a sampling rate of 3 kHz, and stored on the hard disk of a computer for off-line analysis. Data acquisition and analysis were performed with pClamp software (version 6.0.4., Axon Instruments). Channel open probability (NPo) in patches was determined from recordings of more than 3 min by fitting the sum of Gaussian functions to an all-points histogram plot at each potential. Single channel conductance was determined from all-point amplitude histograms using Fechan and Pstat programs (Axon Instruments). For whole cell recordings of HEK cells, the bath solution contained 135 mm NaCl, 5 mmKCl, 1.8 mm CaCl2, 1 mmMgCl2, 10 mm HEPES, 10 mm glucose (pH 7.4), and the pipette solution contained 50 mm KCl, 70 mm l-aspartic acid monopotassium, 8 mm NaCl, 0.826 mm CaCl2, 1 mmMgCl2, 2 mm MgATP, 0.3 mm NaGTP, 10 mm HEPES, 1 mm N-(2-hydroxyethyl)ethylenediaminetriacetic acid (pH 7.2). For single channel recordings in the inside-out mode, the bath solution contained 140 mm KCl, 1 mmMgCl2, 10 mm HEPES, 1 mm N-(2-hydroxyethyl)ethylenediaminetriacetic acid (pH 7.2). The concentration of free Ca2+ in the bath solution was changed from 10−8m to 10−4m to determine the Ca2+ sensitivity of BKCa channels. Ca2+ concentration was estimated by a computer program (42Bers D. Patton C. Nuccitelli R. Methods in Cell Biology: A Practical Guide to the Study of Ca2+in Living Cells. 40. Academic Press, Inc., San Diego, CA1994Google Scholar), and the appropriate amounts of CaCl2 were added. The ionized Ca2+concentration was confirmed using a Ca2+-sensitive electrode. The pipette solution contained 140 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2, 10 mm HEPES (pH 7.4). For single channel recordings in the cell-attached mode, the bath solution contained 140 mm KCl, 1.8 mm CaCl2, 1 mmMgCl2, 10 mm HEPES, 10 mm glucose (pH 7.4), and the pipette solution contained 140 mm KCl, 1.8 mm CaCl2, 1 mmMgCl2, 10 mm HEPES (pH 7.4). All patch-clamp experiments were performed at room temperature (22 °C). PKG-Iα and KT5823 were purchased from Calbiochem. Diethylenetriamine/nitric oxide (DETA/NO) was from RBI (Natick, MA) and other drugs were from Sigma. Data are expressed as mean ±S.E. Statistical significance was determined using Student's t test for paired observations. Membrane currents of nontransfected native HEK cells were measured in whole cell voltage clamp configuration. Depolarizing steps triggered an outward current in native HEK cells. The current showed little or no inactivation during the pulse. The steady-state current at +50 mV was 0.29 ± 0.04 nA (n = 10). This current was suppressed by the K+ channel blocker 4-aminopyridine (0.09 ± 0.03 nA at 1 mm; n = 4) but was unaffected by the specific BKCa channel inhibitor iberiotoxin (IBTX 100 nm; n = 3). Expression of CD8 DNA (marker plasmid) in HEK cells had no effect on the current (n = 6). The amplitude of outward current in HEK cells expressingcslo-α was considerably larger than in native HEK cells. Mean current amplitude obtained under steady-state conditions at +50 mV in cells expressing cslo-α was 4.56 ± 0.42 nA (n = 10). Representative whole cell currents obtained in transfected and native cells are shown in Fig.1 A. The current-voltage relationships of transfected and native cells are shown in Fig.1 B. Membrane conductance plotted as a function of voltage in HEK cells expressing cslo-α is shown in Fig.1 C. Discernible conductance was apparent at potentials positive to −40 mV, and maximum conductance was reached at approximately +60 mV. The V 0.5 was +20.3 mV, and the slope was 15.1. BKCa channels are specifically blocked by IBTX purified from venom of the scorpion (43Galvez A. Gimenez-Gallego G. Reuben J.P. Roy-Contancin L. Feigenbaum P. Kaczorowski G.J. Garcia M.L. J. Biol. Chem. 1990; 265: 11083-11090Abstract Full Text PDF PubMed Google Scholar). Experiments were therefore undertaken to determine whether outward currents recorded incslo-α cells were blocked by IBTX. The addition of IBTX (100 nm) to the bathing solution produced a marked reduction in outward current at all voltages tested as seen in Fig.2, A and B. In 5 cells expressing cslo-α, IBTX significantly (p < 0.01) reduced current amplitude by greater than 90% (Fig. 2 C). The current remaining in the presence of IBTX was not different from that of native currents recorded in HEK cells (p > 0.05). To further examine the properties of outward currents in cells transfected withcslo-α, single channel activity was recorded in inside-out patches in a symmetrical KCl solution (140 mm KCl). Channel openings could be detected at membrane potentials from −60 mV to +60 mV (Fig. 3 A). The activity of these channels was voltage-dependent, i.e. NPo increased from 0.064 ± 0.030 at −60 mV to 0.942 ± 0.192 at +60 mV (n = 5). The current voltage relationship of channels was linear between −60 mV to +60 mV (Fig. 3 B) with a mean slope conductance of 253 ± 9.7 pS (n = 8) and a reversal potential of ≈0 mV. When the Ca2+concentration on the cytosolic side of the membrane patch was increased, ranging from 10−8 to 10−4m, channel activity increased dramatically (holding potential = +40 mV, see Fig. 3 C). The relationship between Ca2+ concentration and Po of the cslo-α channel at +40 mV is shown in Fig. 3 D. The NO donor sodium nitroprusside (SNP) increased the activity of native BKCa channels in smooth muscle (13Mistry D.K. Garland C.J. Br. J. Pharmacol. 1998; 124: 1131-1140Crossref PubMed Scopus (178) Google Scholar, 14Fujino K. Nakaya S. Wakatsuki T. Miyoshi Y. Nakaya Y. Mori H. Inoue I. J. Pharmacol. Exp. Ther. 1991; 256: 371-377PubMed Google Scholar, 15Archer S.L. Huang J.M. Hampl V. Nelson D.P. Shultz P.J. Weir E.K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7583-7587Crossref PubMed Scopus (746) Google Scholar, 16Khan S.A. Mathews W.R. Meisheri K.D. J. Pharmacol. Exp. Ther. 1993; 267: 1327-1335PubMed Google Scholar, 17Bychkov R. Gollasch M. Steinke T. Ried C. Luft F.C. Haller H. J. Pharmacol. Exp. Ther. 1998; 285: 293-298PubMed Google Scholar, 18Jiang F. Li C.G. Rand M.J. Br. J. Pharmacol. 1998; 123: 106-112Crossref PubMed Scopus (12) Google Scholar). Experiments were therefore undertaken to determine the action of SNP on cells expressingcslo-α using the whole cell patch-clamp mode. The addition of SNP (10−4m) to the bathing solution led to a significant increase in whole cell outward current (Fig.4, A and B). In 8 cells tested, SNP significantly (p < 0.01) increased outward current amplitude 2.3-fold at + 50 mV (Fig. 4 C). When SNP was removed from the bathing solution, current amplitude returned to the prestimulus amplitude. Cytosolic Ca2+concentration was buffered at 10−5m withN-(2-hydroxyethyl)ethylenediaminetriacetic acid in these experiments. Because SNP significantly enhances whole cell cslo-α current amplitude, additional experiments were performed to determine whether changes also occur in single channel activity recorded in cell-attached patches. The addition of SNP (10−4m) to the bathing solution led to a marked increase in cslo-α channel activity, which returned to near control levels 10–15 min after wash-out (Fig. 5, A andC). In Fig. 5 B, the time course of changes incslo-α activity after the addition of SNP is shown. SNP increased NPo from 0.092 to 0.656 in this cell. In 8 cells, SNP significantly (p < 0.01) increased NPo 3.3-fold (holding potential = +40 mV; see Fig. 5 C) but had no effect on unitary current amplitude (control 253 ± 11.3, SNP 262 ± 13.1, wash-out 258 ± 8.8 pS, n = 8,p > 0.05). SNP had no significant effect oncslo-α activity in the presence of the PKG-specific inhibitor KT5823 (10−6m) in cell-attached patches (Fig. 5 D). SNP had no effect on cslo-α channels in inside-out patches (NPo; control 0.318 ± 0.123, SNP 0.370 ± 0.184, n = 8, p > 0.05). Additional experiments were performed with the NO donor, DETA/NO (300 μm), because there is evidence that different donors may have differing effects upon potassium channels (44Goyal R.K. He X.D. Am. J. Physiol. 1998; 275: G1185-G1192PubMed Google Scholar). In contrast to SNP, DETA/NO increased cslo-α channel activity in the absence (NPo: control, 0.036 ± 0.023; DETA/NO, 0.072 ± 0.029, n = 6, p < 0.05) and in the presence (NPo: control, 0.152 ± 0.121, DETA/NO, 0.249 ± 0.117, n = 8, p < 0.05) of KT5823 in cell-attached patches. Our experiments with SNP suggest that cslo-α channels are activated by the cGMP/PKG pathway. To provide more direct evidence for PKG-induced modulation of cslo-α, we examined the effects of PKG-Iα on cslo-α channel activity in inside-out patches. Application of PKG-Iα to the cytosolic side of the membrane did not modify cslo-α currents (Fig.6, B and C). The effect of ATP (1 mm) on cslo-α channel was variable (11 of 21 increased, 7 of 21 decreased, 3 of 21 showed no change), so that overall, no significant change was observed in the pooled data (Fig. 6 C). ATP (1 mm) plus cGMP (0.1 mm) also had no effect on cslo-α current (Fig.6, A and C). However, when PKG-Iα was added to the bath solution in the presence of ATP (1 mm) plus cGMP (0.1 mm), cslo-α channel activity significantly (p < 0.05) increased (Fig. 6,A and C). Wash out of PKG led to a return of channel activity to the control level (Fig. 6 C). PKG had no effect on the unitary conductance of cslo-α channels (control 253 ± 9.7; PKG + ATP + cGMP, 252 ± 8.3; wash-out, 248 ± 8.6 pS, n = 10, p > 0.05). Activation of PKG may indirectly activate BKCa channels through other kinases or related proteins. To clarify this point, we made a point mutation on the cslo-α channel. The amino acid sequence of cslo-α has only one optimal consensus sequence for PKG phosphorylation at Ser-1072. Thus a point mutation of cslo-α was created in which Ser-1072 was replaced by Ala. The general characteristics of mutatedcslo-α channels is shown in Fig.7, A–C. The mutated cslo-α channel was activated by membrane depolarization, and its conductance (247.2 ± 13 pS,n = 10) was not different (p > 0.05) from that of wild-type cslo-α channels. Increases in Ca2+ concentration at the cytosolic surface activated the channel in a concentration-dependent manner. These characteristics of the mutated cslo-α channel were comparable with wild-type cslo-α channels. However, application of PKG-Iα in the presence of ATP (1 mm) plus cGMP (0.1 mm) was without effect on mutatedcslo-α channel activity (n = 10; Fig. 7,D and E). The single channel conductance of thecslo-α channel was also unchanged by PKG (n = 10; Fig. 7 F). SNP also had no effect on mutated cslo-α channel activity in cell-attached patches (NPo: control, 0.049 ± 0.042; SNP, 0.054 ± 0.051,n = 6, p > 0.05). This study provides direct evidence that cloned BKCachannels expressed in HEK cells can be activated by cGMP-dependent protein kinase. Activation required ATP and cGMP, suggesting that PKG stimulates BKCa channels through phosphorylation. Mutation of cslo-α at Ser-1072, the only optimal consensus phosphorylation site for PKG on cslo-α, abolished the stimulatory effect of PKG on cslo-α channels. These results indicate that PKG activates cslo-α channels through direct phosphorylation at Ser-1072. Native outward currents in HEK cells were small and inhibited by 4-aminopyridine but not by IBTX, suggesting that these currents were largely because of delayed rectifier-type K+ channels with little contribution from BKCa channels. This result agrees with another recent study of these cells (45Yu S.P. Kerchner G.A. J. Neurosci. Res. 1998; 52: 612-617Crossref PubMed Scopus (111) Google Scholar). Several kinds of Cl− channels also contribute to native HEK cell currents (46Zhu G. Zhang Y. Xu H. Jiang C. J. Neurosci. Methods. 1998; 81: 73-83Crossref PubMed Scopus (87) Google Scholar). However, under the conditions of our experiments, both delayed rectifier and chloride currents were minimal compared with currents recorded in cells transfected with cslo-α. Whole cell outward currents recorded in transfected cells were blocked by IBTX and exhibited a voltage dependence comparable with native BKCachannel currents. cslo-α channel activity recorded in single channel mode was enhanced by membrane depolarization and by increases in Ca2+ concentration at the cytosolic surface. Half-maximal activation of cslo-α for Ca2+ at +40 mV was 10−5m. This Ca2+sensitivity is comparable with the sensitivity observed by others when only slo-α is expressed (3Toro L. Wallner M. Merra P. Tanaka Y. News Physiol. Sci. 1998; 13: 112-117PubMed Google Scholar, 27Vogalis F. Vincent T. Qureshi I. Schmalz F. Ward M.W. Sanders K.M. Horowitz B. Am. J. Physiol. 1996; 271: G629-G639PubMed Google Scholar, 39Zhou X.B. Schlossmann J. Hofmann F. Ruth P. Korth M. Pflügers Arch. 1998; 436: 725-734Crossref PubMed Scopus (41) Google Scholar) but is 10 to 20 times less than the Ca2+ sensitivity observed whenslo-α is co-expressed with the β-subunit (27Vogalis F. Vincent T. Qureshi I. Schmalz F. Ward M.W. Sanders K.M. Horowitz B. Am. J. Physiol. 1996; 271: G629-G639PubMed Google Scholar) or when native BKCa channel currents are recorded (47Carl A. Sanders K.M. Am. J. Physiol. 1989; 257: C470-C480Crossref PubMed Google Scholar). In addition, the single channel conductance of cslo-α (253 pS) was similar to that of native BKCa channels. These results indicate that cslo-α current expressed in HEK cells exhibits functional features of native BKCa channels in smooth muscle cells. In this study, SNP activated whole cell BKCa channel currents and increased NPo of cslo-α channels in cell-attached patches without a change in single channel conductance. There are three possible mechanisms by which SNP could activate thecslo-α channel. First, NO derived from SNP may directly activate the cslo-α channel (12Bolotina V.M. Najibi S. Palacino J.J. Pagano P.J. Cohen R.A. Nature. 1994; 368: 850-853Crossref PubMed Scopus (1514) Google Scholar, 39Zhou X.B. Schlossmann J. Hofmann F. Ruth P. Korth M. Pflügers Arch. 1998; 436: 725-734Crossref PubMed Scopus (41) Google Scholar). Second, NO may activate PKG, which then leads to direct phosphorylation of thecslo-α channel. Third, activation of PKG by NO may lead to stimulation of a phosphatase (possibly phosphoprotein phosphatase 2A), which dephosphorylates the channel (39Zhou X.B. Schlossmann J. Hofmann F. Ruth P. Korth M. Pflügers Arch. 1998; 436: 725-734Crossref PubMed Scopus (41) Google Scholar, 48White R.E. Lee A.B. Shcherbatko A.D. Lincoln T.M. Schonbrunn A. Armstrong D.L. Nature. 1993; 361: 263-266Crossref PubMed Scopus (226) Google Scholar). Our results suggest that the mechanism involved in activation of cslo is dependent upon the NO donor used. In the case of SNP, activation ofcslo appears to be because of a PKG-dependent mechanism without an appreciable contribution from the direct activation of channels by NO. This conclusion was reached because 1) SNP was without affect when applied to the cytosolic surface of the membrane in inside-out patches, 2) the stimulatory effect of SNP was blocked by the PKG inhibitor KT5823 in cell-attached patch recordings, and 3) SNP was without effect on mutated cslo-α channel activity. Further support for this conclusion comes from studies by other laboratories suggesting that the cGMP-PKG pathway is functional in HEK cells (49Dinerman J.L. Steiner J.P. Dawson T.M. Dawson V. Snyder S.H. Neuropharmacology. 1994; 33: 1245-1251Crossref PubMed Scopus (122) Google Scholar, 50Bischof G. Serwold T.F. Machen T.E. Cell Calcium. 1997; 21: 135-142Crossref PubMed Scopus (22) Google Scholar, 51Jockers R. Petit L. Lacroix I. de Coppet P. Barrett P. Morgan P.J. Guardiola B. Delagrange P. Marullo S. Strosberg A.D. Mol. Endocrinol. 1997; 11: 1070-1081PubMed Google Scholar, 52Ramamoorthy S. Giovanetti E. Qian Y. Blakely R.D. J. Biol. Chem. 1998; 273: 2458-2466Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). In contrast to SNP, the effect of DETA/NO was not blocked by the PKG inhibitor KT 5823, suggesting that this NO-donor may have direct effects upon the cslo-α channel. This conclusion is in good agreement with studies by Zhou et al.(39Zhou X.B. Schlossmann J. Hofmann F. Ruth P. Korth M. Pflügers Arch. 1998; 436: 725-734Crossref PubMed Scopus (41) Google Scholar) who recently reported that hslo-α channels expressed in Chinese hamster ovary cells are directly activated by NO derived from diethylamine/NO via S-nitrosylation. The differences between SNP and DETA/NO may be because of the differences in the redox state of NO generated by these NO-donors as suggested by others (44Goyal R.K. He X.D. Am. J. Physiol. 1998; 275: G1185-G1192PubMed Google Scholar). Additional experiments were undertaken to distinguish between direct PKG-mediated phosphorylation of the channel versus more indirect effects of PKG. Exposure of the cytosolic surface of inside-out patches to PKG-Iα in the presence of ATP plus cGMP increased NPo of cslo-α channels as previously reported for native BK channels (10Robertson B.E. Schubert R. Hescheler J. Nelson M.T. Am. J. Physiol. 1993; 265: C299-C303Crossref PubMed Google Scholar, 23Alioua A. Huggins J.P. Rousseau E. Am. J. Physiol. 1995; 268: L1057-L1063PubMed Google Scholar). This suggests that the action of PKG involves a phosphorylation event. In a previous study by our laboratory using cslo-α (27Vogalis F. Vincent T. Qureshi I. Schmalz F. Ward M.W. Sanders K.M. Horowitz B. Am. J. Physiol. 1996; 271: G629-G639PubMed Google Scholar) and a study using hslo-α channels (24Alioua A. Tanaka Y. Wallner M. Hofmann F. Ruth P. Meera P.F. Toro L. J. Biol. Chem. 1998; 273: 32950-32956Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar) expressed in oocytes, PKG did not activate slochannels, although, interestingly, PKG-induced regulation was observed when the hslo-channels were reconstituted in a lipid bilayer (24Alioua A. Tanaka Y. Wallner M. Hofmann F. Ruth P. Meera P.F. Toro L. J. Biol. Chem. 1998; 273: 32950-32956Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). In our studies, neither ATP alone nor ATP plus cGMP increased the activity of channels, suggesting that significant quantities of PKG are not bound to the cytoplasmic surface of the isolated patch. This result differs from a study by Fujino et al. (14Fujino K. Nakaya S. Wakatsuki T. Miyoshi Y. Nakaya Y. Mori H. Inoue I. J. Pharmacol. Exp. Ther. 1991; 256: 371-377PubMed Google Scholar), who reported that cGMP plus ATP increased BKCa channel activity in isolated patches of porcine coronary artery myocytes. However, it is in agreement with studies of vascular (7Taniguchi J. Furukawa K.I. Shigekawa M. Pflügers Arch. 1993; 423: 167-172Crossref PubMed Scopus (169) Google Scholar, 10Robertson B.E. Schubert R. Hescheler J. Nelson M.T. Am. J. Physiol. 1993; 265: C299-C303Crossref PubMed Google Scholar) and tracheal (36Yamakage M. Hirshman C.A. Croxton T.L. Am. J. Physiol. 1996; 270: L338-L345PubMed Google Scholar) smooth muscles in which cGMP plus ATP were without affect in isolated patches. These disparate results suggest that mammalian expression systems and different smooth muscle preparations may contain differing amounts of bound PKG. PKG has been reported to phosphorylate many proteins that regulate smooth muscle tone (21Butt E. Geiger J. Jarchau T. Lohmann S.M. Walter U. Neurochem. Res. 1993; 18: 27-42Crossref PubMed Scopus (124) Google Scholar, 22Lincoln T.M. Cornwell T.L. Blood Vessels. 1991; 28: 129-137PubMed Google Scholar), and it is possible that PKG could regulate BKCa channel activity indirectly by phosphorylating a protein that then regulates channel activity. A particularly intriguing target in this regard are phosphatases that could regulate channel activity through dephosphorylation (39Zhou X.B. Schlossmann J. Hofmann F. Ruth P. Korth M. Pflügers Arch. 1998; 436: 725-734Crossref PubMed Scopus (41) Google Scholar, 48White R.E. Lee A.B. Shcherbatko A.D. Lincoln T.M. Schonbrunn A. Armstrong D.L. Nature. 1993; 361: 263-266Crossref PubMed Scopus (226) Google Scholar, 53Zhou X.B. Ruth P. Schlossmann J. Hofmann F. Korth M. J. Biol. Chem. 1996; 271: 19760-19767Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). To investigate whether PKG directly acts on the channel or, alternatively, requires some intermediary protein, we mutated the single optimal consensus sequence for PKG phosphorylation in the carboxyl-terminal region of thecslo-α channel (i.e. KKSS at 1069–1072). Mutation of Ser-1072 abolished PKG-induced modulation of channel activity but did not change the electrophysiological characteristics of the channel. The mutated cslo-α channels exhibited all of the features described for wild-type channels, i.e. they were activated by membrane depolarization and by elevation of Ca2+ on the cytosolic side of the membrane and had the same single channel conductance as the wild-type cslo-α channel. Thus, the lack of effect of PKG on mutated channels could not be attributed to general channel dysfunction. These mutation experiments suggest that PKG enhances channel activity through direct phosphorylation of the channel rather than requiring the actions of a phosphatase. In studies of reconstituted hslo-α channels, it was also concluded that activation of channels by PKG involved phosphorylation rather than dephosphorylation (24Alioua A. Tanaka Y. Wallner M. Hofmann F. Ruth P. Meera P.F. Toro L. J. Biol. Chem. 1998; 273: 32950-32956Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Furthermore, the cloned human slo channel hslo-α has the same optimal consensus phosphorylation site as cslo-α, and this channel has been reported to be directly phosphorylated by PKG-Iα (24Alioua A. Tanaka Y. Wallner M. Hofmann F. Ruth P. Meera P.F. Toro L. J. Biol. Chem. 1998; 273: 32950-32956Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). In summary, we have found that the cslo-α channel activity recorded in whole cell and single channel configuration is increased by the NO donor SNP, presumably through activation of PKG. Direct application of PKG-Iα also activated cslo-α channels but only in the presence of ATP and cGMP. A point mutation at the only optimal consensus phosphorylation site for PKG on cslo-α abolished the stimulatory effects of PKG. From these results we conclude that PKG activates cslo-α channel by direct phosphorylation at serine 1072. We are grateful to N. Horowitz for help with the cell cultures. We also thank L. Toro for providing their paper in press.

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