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Reconstitution of β-Adrenergic Modulation of Large Conductance, Calcium-activated Potassium (Maxi-K) Channels in Xenopus Oocytes

1998; Elsevier BV; Volume: 273; Issue: 24 Linguagem: Inglês

10.1074/jbc.273.24.14920

1083-351X

Masayuki Nara, Prasad Dhulipala, Yong-Xiao Wang, Michael I. Kotlikoff,

Receptor Mechanisms and Signaling

The human large conductance, calcium-activated potassium (maxi-K) channel (α and β subunits) and β2-adrenergic receptor genes were coexpressed inXenopus oocytes in order to study the mechanism of β-adrenergic modulation of channel function. Isoproterenol and forskolin increased maxi-K potassium channel currents in voltage-clamped oocytes expressing the receptor and both channel subunits by 33 ± 5% and 35 ± 8%, respectively, without affecting current activation or inactivation. The percentage of stimulation by isoproterenol and forskolin was not different in oocytes coexpressing the α and β subunits versus those expressing the only the α subunit, suggesting that the α subunit is the target for regulation. The stimulatory effect of isoproterenol was almost completely blocked by intracellular injection of the cyclic AMP dependent protein kinase (cAMP-PK) regulatory subunit, whereas injection of a cyclic GMP dependent protein kinase inhibitory peptide had little effect, indicating that cellular coupling of β2-adrenergic receptors to maxi-K channels involves endogenous cAMP-PK. Mutation of one of several potential consensus cAMP-PK phosphorylation sites (serine 869) on the α subunit almost completely inhibited β-adrenergic receptor/channel stimulatory coupling, whereas forskolin still stimulated currents moderately (16 ± 4%). These data demonstrate that physiological coupling between β2 receptors and maxi-K channels occurs by the cAMP-PK mediated phosphorylation of serine 869 on the α subunit on the channel. The human large conductance, calcium-activated potassium (maxi-K) channel (α and β subunits) and β2-adrenergic receptor genes were coexpressed inXenopus oocytes in order to study the mechanism of β-adrenergic modulation of channel function. Isoproterenol and forskolin increased maxi-K potassium channel currents in voltage-clamped oocytes expressing the receptor and both channel subunits by 33 ± 5% and 35 ± 8%, respectively, without affecting current activation or inactivation. The percentage of stimulation by isoproterenol and forskolin was not different in oocytes coexpressing the α and β subunits versus those expressing the only the α subunit, suggesting that the α subunit is the target for regulation. The stimulatory effect of isoproterenol was almost completely blocked by intracellular injection of the cyclic AMP dependent protein kinase (cAMP-PK) regulatory subunit, whereas injection of a cyclic GMP dependent protein kinase inhibitory peptide had little effect, indicating that cellular coupling of β2-adrenergic receptors to maxi-K channels involves endogenous cAMP-PK. Mutation of one of several potential consensus cAMP-PK phosphorylation sites (serine 869) on the α subunit almost completely inhibited β-adrenergic receptor/channel stimulatory coupling, whereas forskolin still stimulated currents moderately (16 ± 4%). These data demonstrate that physiological coupling between β2 receptors and maxi-K channels occurs by the cAMP-PK mediated phosphorylation of serine 869 on the α subunit on the channel. Hormones and neurotransmitters alter cellular excitability in part through the modulation of plasmalemmal ion channel function. Two prominent mechanisms by which hormone/neurotransmitter receptor occupation results in the modulation of membrane ion channels are the phosphorylation of one or more residues of the target channel protein(s) (for review see Ref. 1Levitan I.B. Annu. Rev. Physiol. 1994; 56: 193-212Crossref PubMed Scopus (479) Google Scholar), and the binding of a heterotrimeric G protein subunit to a modulatory channel domain (for review see Ref.2Wickman K. Clapham D.E. Physiol. Rev. 1995; 75: 865-885Crossref PubMed Scopus (344) Google Scholar). Stimulation of β2 receptors relaxes smooth muscle by modulating the activity of several protein targets (3Bulbring E. Tomita T. Pharmacol. Rev. 1987; 39: 49-96PubMed Google Scholar). One prominent target of β2 adrenergic signaling in smooth muscle is the large conductance, calcium-activated potassium (maxi-K) 1The abbreviations used are: maxi-K, large conductance, calcium-activated potassium; cAMP-PK, cAMP-dependent protein kinase; cGMP-PK, cGMP-dependent protein kinase; PKA, cAMP-dependent protein kinase; β2AR, β2-adrenergic receptor; ISO, isoproterenol. 1The abbreviations used are: maxi-K, large conductance, calcium-activated potassium; cAMP-PK, cAMP-dependent protein kinase; cGMP-PK, cGMP-dependent protein kinase; PKA, cAMP-dependent protein kinase; β2AR, β2-adrenergic receptor; ISO, isoproterenol. channel, the activity of which is markedly increased following receptor binding (4Sadoshima J. Akaike N. Kanaide H. Nakamura M. Am. J. Physiol. 1988; 255: H754-H759Crossref PubMed Google Scholar, 5Kume H. Takai A. Tokuno H. Tomita T. Nature. 1989; 341: 152-154Crossref PubMed Scopus (301) Google Scholar, 6Kume H. Hall I.P. Washabau R.J. Takagi K. Kotlikoff M.I. J. Clin. Invest. 1994; 93: 371-379Crossref PubMed Scopus (220) Google Scholar, 7Song Y. Simard J.M. Pflugers Arch. Eur. J. Physiol. 1995; 430: 984-993Crossref PubMed Scopus (69) Google Scholar). Modulation of maxi-K channel activity appears to be a functionally important component of β-adrenergic relaxation of smooth muscle, since charybdotoxin and iberiotoxin, selective peptide inhibitors of maxi-K channels, markedly inhibit the relaxant ability of isoproterenol and other β-adrenergic agents (8Jones T.R. Charette L. Garcia M.L. Kaczorowski G.J. J. Pharmacol. Exp. Ther. 1990; 255: 697-706PubMed Google Scholar, 9Jones T.R. Charette L. Garcia M.L. Kaczorowski G.J. J. Appl. Physiol. 1993; 74: 1879-1884Crossref PubMed Scopus (108) Google Scholar, 10Miura M. Belvisi M.G. Stretton D. Yacoub M.H. Barnes P.J. Am. Rev. Respir. Dis. 1992; 146: 132-136Crossref PubMed Scopus (114) Google Scholar). The molecular mechanism by which channel modulation occurs is unclear, however, since studies have indicated that single maxi-K channels are regulated by phosphorylation and by phosphorylation-independent G protein interactions (5Kume H. Takai A. Tokuno H. Tomita T. Nature. 1989; 341: 152-154Crossref PubMed Scopus (301) Google Scholar, 6Kume H. Hall I.P. Washabau R.J. Takagi K. Kotlikoff M.I. J. Clin. Invest. 1994; 93: 371-379Crossref PubMed Scopus (220) Google Scholar, 11Carl A. Kenyon J.L. Uemura D. Fusetani N. Sanders K.M. Am. J. Physiol. 1991; 261: C387-C392Crossref PubMed Google Scholar, 12Kume H. Graziano M.P. Kotlikoff M.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11051-11055Crossref PubMed Scopus (148) Google Scholar, 13Scornik F.S. Codina J. Birnbaumer L. Toro L. Am. J. Physiol. 1993; 265: H1460-H1465PubMed Google Scholar, 14Lee M.Y. Bang H.W. Lim I.J. Uhm D.Y. Rhee S.D. Pflugers Arch. Eur. J. Physiol. 1994; 429: 150-152Crossref PubMed Scopus (22) Google Scholar). Further, with respect to channel phosphorylation, maxi-K channel stimulation has been attributed to channel phosphorylation by cAMP-dependent protein kinase (5Kume H. Takai A. Tokuno H. Tomita T. Nature. 1989; 341: 152-154Crossref PubMed Scopus (301) Google Scholar, 6Kume H. Hall I.P. Washabau R.J. Takagi K. Kotlikoff M.I. J. Clin. Invest. 1994; 93: 371-379Crossref PubMed Scopus (220) Google Scholar, 11Carl A. Kenyon J.L. Uemura D. Fusetani N. Sanders K.M. Am. J. Physiol. 1991; 261: C387-C392Crossref PubMed Google Scholar, 15Savaria D. Lanoue C. Cadieux A. Rousseau E. Am. J. Physiol. 1992; 262: L327-L336PubMed Google Scholar, 16Meera P. Anwer K. Monga M. Oberti C. Stefani E. Toro L. Sanborn B.M. Am. J. Physiol. 1995; 269: C312-C317Crossref PubMed Google Scholar), by cGMP-dependent protein kinase (17Taniguchi J. Furukawa K.I. Shigekawa M. Pflugers Arch. Eur. J. Physiol. 1993; 423: 167-172Crossref PubMed Scopus (169) Google Scholar, 18Robertson B.E. Schubert R. Hescheler J. Nelson M.T. Am. J. Physiol. 1993; 265: C299-C303Crossref PubMed Google Scholar, 19White R.E. Lee A.B. Shcherbatko A.D. Lincoln T.M. Schonbrunn A. Armstrong D.L. Nature. 1993; 362: 263-266Crossref Scopus (225) Google Scholar, 20White R.E. Darkow D.J. Lang J.L. Circ. Res. 1995; 77: 936-942Crossref PubMed Scopus (337) Google Scholar, 21George M.J. Shibata E.F. J. Invest. Med. 1995; 43: 451-458PubMed Google Scholar), and by channel dephosphorylation (19White R.E. Lee A.B. Shcherbatko A.D. Lincoln T.M. Schonbrunn A. Armstrong D.L. Nature. 1993; 362: 263-266Crossref Scopus (225) Google Scholar, 22White R.E. Schonbrunn A. Armstrong D.L. Nature. 1991; 351: 570-573Crossref PubMed Scopus (228) Google Scholar, 23Zhou X.B. Ruth P. Schlossmann J. Hofmann F. Korth M. J. Biol. Chem. 1996; 271: 19760-19767Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar).Maxi-K channels are composed of at least two dissimilar subunits: the α subunit, which forms the channel pore (24Atkinson N.S. Robertson G.A. Ganetzky B. Science. 1991; 253: 551-553Crossref PubMed Scopus (534) Google Scholar, 25Adelman 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 (429) Google Scholar, 26Butler A. Tsunoda S. McCobb D.P. Wei A. Salkoff L. Science. 1993; 261: 221-224Crossref PubMed Scopus (567) Google Scholar, 27Pallanck L. Ganetzky B. Hum. Mol. Genet. 1994; 3: 1239-1243Crossref PubMed Scopus (156) Google Scholar), and the β subunit (28Garcia-Calvo M. Knaus H.-G. McManus O.B. Giangiacomo K.M. Kaczorowski G.J. Garcia M.L. J. Biol. Chem. 1994; 269: 676-682Abstract Full Text PDF PubMed Google Scholar, 29Knaus H.G. Folander K. Garcia-Calvo M. Garcia M.L. Kaczorowski G.J. Smith M. Swanson R. J. Biol. Chem. 1994; 269: 17274-17278Abstract Full Text PDF PubMed Google Scholar, 30Tseng-Crank J. Godinot N. Johansen T.E. Ahring P.K. Strobaek D. Mertz R. Foster C.D. Olesen S.-P. Reinhart P.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9200-9205Crossref PubMed Scopus (139) Google Scholar), which modifies the voltage and calcium sensitivity of the pore-forming subunit (31McManus O.B. Helms L.M.H. Pallanck L. Ganetzky B. Swanson R. Leonard R.J. Neuron. 1995; 14: 645-650Abstract Full Text PDF PubMed Scopus (415) Google Scholar). Both the human α (hSlo) and β (hKv,Caβ) subunit genes encode several consensus cAMP-PK, although the site(s) associated with physiological regulation have not been determined. We examined β-adrenergic receptor/maxi K coupling by coexpression of hSlo and hKv,Caβ and the human β2-adrenergic receptor gene (hβ2AR) inXenopus laevis oocytes using the two-electrode voltage clamp technique. We demonstrate that isoproterenol and forskolin increase maxi-K currents in voltage-clamped oocytes, that this action is mediated by PKA phosphorylation of the α subunit, and that mutation of Ser-869 almost completely eliminates β2receptor/channel coupling.DISCUSSIONThe present study demonstrates the reconstitution of β2-adrenergic receptor stimulatory coupling to maxi-K channels by heterologous expression of receptor and channel RNAs inX. laevis oocytes. Isoproterenol and forskolin increased maxi-K currents in oocytes expressing either the pore-forming α subunit alone, or co-expressing the α and regulatory β subunits (Figs. 1 and 2, Table I). The stimulated currents were not endogenous oocyte currents since there was no augmentation of outward current in oocytes expressing β2 receptors but not maxi-K channels, and since forskolin, but not ISO, stimulated currents in oocytes expressing maxi-K channels, but not β2 receptors (TableI). Moreover, virtually all of the outward current was inhibited by iberiotoxin (Figs. 1 and 2), a selective peptidyl inhibitor of maxi-K channels (36Galvez A. Gimenez-Gallego G. Reuben J.P. Roy-Contancin L. Geigenbaum P. Kaczorowski G.J. Garcia M.L. J. Biol. Chem. 1990; 265: 11083-11090Abstract Full Text PDF PubMed Google Scholar). Since heterologous expression avoids complications associated with expression of multiple receptor or channel subtypes in target tissues, the present results clearly establish stimulatory coupling between human β2 receptors and maxi-K channels. Further, since stimulation was achieved with expression of only the β2 receptor and the α subunit and the degree of stimulation was similar with or without the regulatory β subunit (Table I), these studies localize the modulatory site to the channel α subunit. It should be noted that the degree of stimulation observed (30–35%) is somewhat less than reported in mammalian cells. In smooth muscle cells, current stimulation varies from approximately 50% to 100%, and is somewhat dependent on the step voltage and [Ca2+]i (7Song Y. Simard J.M. Pflugers Arch. Eur. J. Physiol. 1995; 430: 984-993Crossref PubMed Scopus (69) Google Scholar, 38Anwer K. Toro L. Oberti C. Stefani E. Sanborn B.M. Am. J. Physiol. 1992; 263: C1049-C1056Crossref PubMed Google Scholar, 39Fan S.-F. Wang S. Kao C.Y. J. Gen. Physiol. 1993; 102: 257-275Crossref PubMed Scopus (16) Google Scholar).Considerable controversy exists with respect to the mechanism of stimulatory coupling to maxi-K channels. Stimulatory mechanisms that have been proposed include cAMP-dependent phosphorylation (5Kume H. Takai A. Tokuno H. Tomita T. Nature. 1989; 341: 152-154Crossref PubMed Scopus (301) Google Scholar, 6Kume H. Hall I.P. Washabau R.J. Takagi K. Kotlikoff M.I. J. Clin. Invest. 1994; 93: 371-379Crossref PubMed Scopus (220) Google Scholar, 11Carl A. Kenyon J.L. Uemura D. Fusetani N. Sanders K.M. Am. J. Physiol. 1991; 261: C387-C392Crossref PubMed Google Scholar, 15Savaria D. Lanoue C. Cadieux A. Rousseau E. Am. J. Physiol. 1992; 262: L327-L336PubMed Google Scholar, 16Meera P. Anwer K. Monga M. Oberti C. Stefani E. Toro L. Sanborn B.M. Am. J. Physiol. 1995; 269: C312-C317Crossref PubMed Google Scholar), cGMP-dependent phosphorylation (17Taniguchi J. Furukawa K.I. Shigekawa M. Pflugers Arch. Eur. J. Physiol. 1993; 423: 167-172Crossref PubMed Scopus (169) Google Scholar, 18Robertson B.E. Schubert R. Hescheler J. Nelson M.T. Am. J. Physiol. 1993; 265: C299-C303Crossref PubMed Google Scholar,20White R.E. Darkow D.J. Lang J.L. Circ. Res. 1995; 77: 936-942Crossref PubMed Scopus (337) Google Scholar, 21George M.J. Shibata E.F. J. Invest. Med. 1995; 43: 451-458PubMed Google Scholar, 40Carrier G.O. Fuchs L.C. Winecoff A.P. Giulumian A.D. White R.E. Am. J. Physiol. 1997; 273: H76-H84PubMed Google Scholar), cGMP-dependent dephosphorylation (19White R.E. Lee A.B. Shcherbatko A.D. Lincoln T.M. Schonbrunn A. Armstrong D.L. Nature. 1993; 362: 263-266Crossref Scopus (225) Google Scholar, 22White R.E. Schonbrunn A. Armstrong D.L. Nature. 1991; 351: 570-573Crossref PubMed Scopus (228) Google Scholar, 23Zhou X.B. Ruth P. Schlossmann J. Hofmann F. Korth M. J. Biol. Chem. 1996; 271: 19760-19767Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), by G protein subunits (12Kume H. Graziano M.P. Kotlikoff M.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11051-11055Crossref PubMed Scopus (148) Google Scholar, 13Scornik F.S. Codina J. Birnbaumer L. Toro L. Am. J. Physiol. 1993; 265: H1460-H1465PubMed Google Scholar), and direct stimulation by NO (41Bolotina V.M. Najibi S. Palacino J.J. Pagano P.J. Cohen R.A. Nature. 1994; 368: 850-853Crossref PubMed Scopus (1505) Google Scholar). We used the heterologous expression system and site-directed mutagenesis to identify the mechanism of β adrenergic stimulatory coupling to maxi-K channels and to identify the relevant channel modulatory site. Our data clearly implicate cAMP-PK in β2 receptor/channel stimulatory coupling since injection of the regulatory subunit of cAMP-PK disrupts β2 receptor/maxi-K channel stimulatory coupling (Fig. 3). In addition to disrupting stimulatory coupling, injection of oocytes with the cAMP-PK regulatory subunit also consistently reduced the amplitude of the basal current before application of isoproterenol (Fig. 3 B ), suggesting that phosphorylation by cAMP-PK regulates maxi-K channel activity under resting conditions. Conversely, a semi-selective (6-fold) cGMP-PK inhibitory peptide only slightly reduced ISO current stimulation, consistent with the predicted inhibition of cAMP-PK. Further evidence suggesting functional modulation of maxi-K channels by cAMP-PK was the finding that heterologously expressed maxi-K channels are stimulated by forskolin, and that this stimulation occurs with or without expression of the β2 receptor (Fig. 2, Table I).dSlo, the Drosophila maxi-K channel gene, contains a single optimal consensus sequence for PKA phosphorylation in the C-terminal region of the protein (RRGS at 959–962; Ref. 24Atkinson N.S. Robertson G.A. Ganetzky B. Science. 1991; 253: 551-553Crossref PubMed Scopus (534) Google Scholar), and mutations at this site were later found to prevent channel activation by exogenous PKA in inside-out patches (42Chung S.K. Reinhart P.H. Martin B.L. Brautigan D. Levitan I.B. Science. 1991; 253: 560-562Crossref PubMed Scopus (152) Google Scholar). The mammalian homologues of dSlo contain a less cAMP-PK -specific phosphorylation site at approximately the same position (RQPS at 866–869, e.g. GenBank accession no. U11058), which is conserved in mammalian genes, and which occupies a similar position upstream of a highly conserved region, aspartate-rich region that is a likely calcium-binding site (43Schreiber M. Salkoff L. Biophys. J. 1997; 73: 1355-1363Abstract Full Text PDF PubMed Scopus (336) Google Scholar). However, as many as 10 other potential cAMP-PK consensus phosphorylation sites exist throughout the coding region of the α subunit (37Kennelly P.J. Krebs E.G. J. Biol. Chem. 1997; 266: 15555-15558Abstract Full Text PDF Google Scholar). To ascertain the physiologically relevant phosphorylation site, we mutated serine 869 to alanine, and coexpressed the mutant α subunit with the wild type β subunit and the β2receptor. This single mutation markedly reduced, but did not abolish, β2 receptor stimulation of the current (from 33.3 ± 5.1% to 5.5 ± 1.3%, Table I), strongly implicating serine 869 in receptor/effector coupling. The low degree of current stimulation consistently observed in the mutant may relate to a slight role for additional cAMP phosphorylation sites, coupling through other kinases, or direct G protein interactions. Moreover, forskolin stimulation was only decreased by about half in mutant channels, suggesting that additional sites of channel modulation exist. It is possible that the dominant mechanism of stimulatory coupling observed in our experiments relates to factors unique to the Xenopus oocyte, such as cell size or endogenous G proteins, and that other regulatory mechanisms may be more prominent in mammalian cells. However, our results are consistent with the major mechanism of β2receptor/maxi-K channel stimulatory coupling occurring by cAMP-PK phosphorylation of serine 869.Finally, the stimulatory effect of both the β2 receptor agonist ISO and forskolin was an increase in current amplitude at all voltages, without a change in current kinetics, similar to the modulatory action of β2 receptor stimulation on cardiac sodium channel α subunits heterologously expressed inXenopus oocytes (44Schreibmayer W. Frohnwieser B. Dascal N. Platzer D. Spreitzer B. Zechner R. Kallen R.G. Lester H.A. Receptors Channels. 1994; 2: 339-350PubMed Google Scholar). In that study, mutation of all consensus cAMP-PK sites on the sodium channel failed to remove channel stimulation, leading to the suggestion that modulation might occur by phosphorylation of an unrelated protein resulting in a redistribution of channels to the plasma membrane (44Schreibmayer W. Frohnwieser B. Dascal N. Platzer D. Spreitzer B. Zechner R. Kallen R.G. Lester H.A. Receptors Channels. 1994; 2: 339-350PubMed Google Scholar). ISO stimulation of sodium currents was quite slow (approximately 10 min to maximum stimulation), which could be consistent with a redistribution of membranes within the oocyte. In the present study, however, stimulatory coupling occurred quite rapidly (approximately 1 min to maximum), which would be consistent with a direct effect on channel gating.In summary, we have used a heterologous expression system to demonstrated β2-adrenergic receptor/maxi-K channel stimulatory coupling. Coupling involves a cAMP-PK-dependent phosphorylation of the channel α subunit; mutation of one consensus cAMP-PK phosphorylation site (serine 869) almost eliminates β2-adrenergic modulation of the maxi-K current. These findings do not rule out other modulatory mechanisms, but do confine the physiologically relevant molecular mechanisms associated with β2 receptor/maxi-K channel coupling. Hormones and neurotransmitters alter cellular excitability in part through the modulation of plasmalemmal ion channel function. Two prominent mechanisms by which hormone/neurotransmitter receptor occupation results in the modulation of membrane ion channels are the phosphorylation of one or more residues of the target channel protein(s) (for review see Ref. 1Levitan I.B. Annu. Rev. Physiol. 1994; 56: 193-212Crossref PubMed Scopus (479) Google Scholar), and the binding of a heterotrimeric G protein subunit to a modulatory channel domain (for review see Ref.2Wickman K. Clapham D.E. Physiol. Rev. 1995; 75: 865-885Crossref PubMed Scopus (344) Google Scholar). Stimulation of β2 receptors relaxes smooth muscle by modulating the activity of several protein targets (3Bulbring E. Tomita T. Pharmacol. Rev. 1987; 39: 49-96PubMed Google Scholar). One prominent target of β2 adrenergic signaling in smooth muscle is the large conductance, calcium-activated potassium (maxi-K) 1The abbreviations used are: maxi-K, large conductance, calcium-activated potassium; cAMP-PK, cAMP-dependent protein kinase; cGMP-PK, cGMP-dependent protein kinase; PKA, cAMP-dependent protein kinase; β2AR, β2-adrenergic receptor; ISO, isoproterenol. 1The abbreviations used are: maxi-K, large conductance, calcium-activated potassium; cAMP-PK, cAMP-dependent protein kinase; cGMP-PK, cGMP-dependent protein kinase; PKA, cAMP-dependent protein kinase; β2AR, β2-adrenergic receptor; ISO, isoproterenol. channel, the activity of which is markedly increased following receptor binding (4Sadoshima J. Akaike N. Kanaide H. Nakamura M. Am. J. Physiol. 1988; 255: H754-H759Crossref PubMed Google Scholar, 5Kume H. Takai A. Tokuno H. Tomita T. Nature. 1989; 341: 152-154Crossref PubMed Scopus (301) Google Scholar, 6Kume H. Hall I.P. Washabau R.J. Takagi K. Kotlikoff M.I. J. Clin. Invest. 1994; 93: 371-379Crossref PubMed Scopus (220) Google Scholar, 7Song Y. Simard J.M. Pflugers Arch. Eur. J. Physiol. 1995; 430: 984-993Crossref PubMed Scopus (69) Google Scholar). Modulation of maxi-K channel activity appears to be a functionally important component of β-adrenergic relaxation of smooth muscle, since charybdotoxin and iberiotoxin, selective peptide inhibitors of maxi-K channels, markedly inhibit the relaxant ability of isoproterenol and other β-adrenergic agents (8Jones T.R. Charette L. Garcia M.L. Kaczorowski G.J. J. Pharmacol. Exp. Ther. 1990; 255: 697-706PubMed Google Scholar, 9Jones T.R. Charette L. Garcia M.L. Kaczorowski G.J. J. Appl. Physiol. 1993; 74: 1879-1884Crossref PubMed Scopus (108) Google Scholar, 10Miura M. Belvisi M.G. Stretton D. Yacoub M.H. Barnes P.J. Am. Rev. Respir. Dis. 1992; 146: 132-136Crossref PubMed Scopus (114) Google Scholar). The molecular mechanism by which channel modulation occurs is unclear, however, since studies have indicated that single maxi-K channels are regulated by phosphorylation and by phosphorylation-independent G protein interactions (5Kume H. Takai A. Tokuno H. Tomita T. Nature. 1989; 341: 152-154Crossref PubMed Scopus (301) Google Scholar, 6Kume H. Hall I.P. Washabau R.J. Takagi K. Kotlikoff M.I. J. Clin. Invest. 1994; 93: 371-379Crossref PubMed Scopus (220) Google Scholar, 11Carl A. Kenyon J.L. Uemura D. Fusetani N. Sanders K.M. Am. J. Physiol. 1991; 261: C387-C392Crossref PubMed Google Scholar, 12Kume H. Graziano M.P. Kotlikoff M.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11051-11055Crossref PubMed Scopus (148) Google Scholar, 13Scornik F.S. Codina J. Birnbaumer L. Toro L. Am. J. Physiol. 1993; 265: H1460-H1465PubMed Google Scholar, 14Lee M.Y. Bang H.W. Lim I.J. Uhm D.Y. Rhee S.D. Pflugers Arch. Eur. J. Physiol. 1994; 429: 150-152Crossref PubMed Scopus (22) Google Scholar). Further, with respect to channel phosphorylation, maxi-K channel stimulation has been attributed to channel phosphorylation by cAMP-dependent protein kinase (5Kume H. Takai A. Tokuno H. Tomita T. Nature. 1989; 341: 152-154Crossref PubMed Scopus (301) Google Scholar, 6Kume H. Hall I.P. Washabau R.J. Takagi K. Kotlikoff M.I. J. Clin. Invest. 1994; 93: 371-379Crossref PubMed Scopus (220) Google Scholar, 11Carl A. Kenyon J.L. Uemura D. Fusetani N. Sanders K.M. Am. J. Physiol. 1991; 261: C387-C392Crossref PubMed Google Scholar, 15Savaria D. Lanoue C. Cadieux A. Rousseau E. Am. J. Physiol. 1992; 262: L327-L336PubMed Google Scholar, 16Meera P. Anwer K. Monga M. Oberti C. Stefani E. Toro L. Sanborn B.M. Am. J. Physiol. 1995; 269: C312-C317Crossref PubMed Google Scholar), by cGMP-dependent protein kinase (17Taniguchi J. Furukawa K.I. Shigekawa M. Pflugers Arch. Eur. J. Physiol. 1993; 423: 167-172Crossref PubMed Scopus (169) Google Scholar, 18Robertson B.E. Schubert R. Hescheler J. Nelson M.T. Am. J. Physiol. 1993; 265: C299-C303Crossref PubMed Google Scholar, 19White R.E. Lee A.B. Shcherbatko A.D. Lincoln T.M. Schonbrunn A. Armstrong D.L. Nature. 1993; 362: 263-266Crossref Scopus (225) Google Scholar, 20White R.E. Darkow D.J. Lang J.L. Circ. Res. 1995; 77: 936-942Crossref PubMed Scopus (337) Google Scholar, 21George M.J. Shibata E.F. J. Invest. Med. 1995; 43: 451-458PubMed Google Scholar), and by channel dephosphorylation (19White R.E. Lee A.B. Shcherbatko A.D. Lincoln T.M. Schonbrunn A. Armstrong D.L. Nature. 1993; 362: 263-266Crossref Scopus (225) Google Scholar, 22White R.E. Schonbrunn A. Armstrong D.L. Nature. 1991; 351: 570-573Crossref PubMed Scopus (228) Google Scholar, 23Zhou X.B. Ruth P. Schlossmann J. Hofmann F. Korth M. J. Biol. Chem. 1996; 271: 19760-19767Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Maxi-K channels are composed of at least two dissimilar subunits: the α subunit, which forms the channel pore (24Atkinson N.S. Robertson G.A. Ganetzky B. Science. 1991; 253: 551-553Crossref PubMed Scopus (534) Google Scholar, 25Adelman 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 (429) Google Scholar, 26Butler A. Tsunoda S. McCobb D.P. Wei A. Salkoff L. Science. 1993; 261: 221-224Crossref PubMed Scopus (567) Google Scholar, 27Pallanck L. Ganetzky B. Hum. Mol. Genet. 1994; 3: 1239-1243Crossref PubMed Scopus (156) Google Scholar), and the β subunit (28Garcia-Calvo M. Knaus H.-G. McManus O.B. Giangiacomo K.M. Kaczorowski G.J. Garcia M.L. J. Biol. Chem. 1994; 269: 676-682Abstract Full Text PDF PubMed Google Scholar, 29Knaus H.G. Folander K. Garcia-Calvo M. Garcia M.L. Kaczorowski G.J. Smith M. Swanson R. J. Biol. Chem. 1994; 269: 17274-17278Abstract Full Text PDF PubMed Google Scholar, 30Tseng-Crank J. Godinot N. Johansen T.E. Ahring P.K. Strobaek D. Mertz R. Foster C.D. Olesen S.-P. Reinhart P.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9200-9205Crossref PubMed Scopus (139) Google Scholar), which modifies the voltage and calcium sensitivity of the pore-forming subunit (31McManus O.B. Helms L.M.H. Pallanck L. Ganetzky B. Swanson R. Leonard R.J. Neuron. 1995; 14: 645-650Abstract Full Text PDF PubMed Scopus (415) Google Scholar). Both the human α (hSlo) and β (hKv,Caβ) subunit genes encode several consensus cAMP-PK, although the site(s) associated with physiological regulation have not been determined. We examined β-adrenergic receptor/maxi K coupling by coexpression of hSlo and hKv,Caβ and the human β2-adrenergic receptor gene (hβ2AR) inXenopus laevis oocytes using the two-electrode voltage clamp technique. We demonstrate that isoproterenol and forskolin increase maxi-K currents in voltage-clamped oocytes, that this action is mediated by PKA phosphorylation of the α subunit, and that mutation of Ser-869 almost completely eliminates β2receptor/channel coupling. DISCUSSIONThe present study demonstrates the reconstitution of β2-adrenergic receptor stimulatory coupling to maxi-K channels by heterologous expression of receptor and channel RNAs inX. laevis oocytes. Isoproterenol and forskolin increased maxi-K currents in oocytes expressing either the pore-forming α subunit alone, or co-expressing the α and regulatory β subunits (Figs. 1 and 2, Table I). The stimulated currents were not endogenous oocyte currents since there was no augmentation of outward current in oocytes expressing β2 receptors but not maxi-K channels, and since forskolin, but not ISO, stimulated currents in oocytes expressing maxi-K channels, but not β2 receptors (TableI). Moreover, virtually all of the outward current was inhibited by iberiotoxin (Figs. 1 and 2), a selective peptidyl inhibitor of maxi-K channels (36Galvez A. Gimenez-Gallego G. Reuben J.P. Roy-Contancin L. Geigenbaum P. Kaczorowski G.J. Garcia M.L. J. Biol. Chem. 1990; 265: 11083-11090Abstract Full Text PDF PubMed Google Scholar). Since heterologous expression avoids complications associated with expression of multiple receptor or channel subtypes in target tissues, the present results clearly establish stimulatory coupling between human β2 receptors and maxi-K channels. Further, since stimulation was achieved with expression of only the β2 receptor and the α subunit and the degree of stimulation was similar with or without the regulatory β subunit (Table I), these studies localize the modulatory site to the channel α subunit. It should be noted that the degree of stimulation observed (30–35%) is somewhat less than reported in mammalian cells. In smooth muscle cells, current stimulation varies from approximately 50% to 100%, and is somewhat dependent on the step voltage and [Ca2+]i (7Song Y. Simard J.M. Pflugers Arch. Eur. J. Physiol. 1995; 430: 984-993Crossref PubMed Scopus (69) Google Scholar, 38Anwer K. Toro L. Oberti C. Stefani E. Sanborn B.M. Am. J. Physiol. 1992; 263: C1049-C1056Crossref PubMed Google Scholar, 39Fan S.-F. Wang S. Kao C.Y. J. Gen. Physiol. 1993; 102: 257-275Crossref PubMed Scopus (16) Google Scholar).Considerable controversy exists with respect to the mechanism of stimulatory coupling to maxi-K channels. Stimulatory mechanisms that have been proposed include cAMP-dependent phosphorylation (5Kume H. Takai A. Tokuno H. Tomita T. Nature. 1989; 341: 152-154Crossref PubMed Scopus (301) Google Scholar, 6Kume H. Hall I.P. Washabau R.J. Takagi K. Kotlikoff M.I. J. Clin. Invest. 1994; 93: 371-379Crossref PubMed Scopus (220) Google Scholar, 11Carl A. Kenyon J.L. Uemura D. Fusetani N. Sanders K.M. Am. J. Physiol. 1991; 261: C387-C392Crossref PubMed Google Scholar, 15Savaria D. Lanoue C. Cadieux A. Rousseau E. Am. J. Physiol. 1992; 262: L327-L336PubMed Google Scholar, 16Meera P. Anwer K. Monga M. Oberti C. Stefani E. Toro L. Sanborn B.M. Am. J. Physiol. 1995; 269: C312-C317Crossref PubMed Google Scholar), cGMP-dependent phosphorylation (17Taniguchi J. Furukawa K.I. Shigekawa M. Pflugers Arch. Eur. J. Physiol. 1993; 423: 167-172Crossref PubMed Scopus (169) Google Scholar, 18Robertson B.E. Schubert R. Hescheler J. Nelson M.T. Am. J. Physiol. 1993; 265: C299-C303Crossref PubMed Google Scholar,20White R.E. Darkow D.J. Lang J.L. Circ. Res. 1995; 77: 936-942Crossref PubMed Scopus (337) Google Scholar, 21George M.J. Shibata E.F. J. Invest. Med. 1995; 43: 451-458PubMed Google Scholar, 40Carrier G.O. Fuchs L.C. Winecoff A.P. Giulumian A.D. White R.E. Am. J. Physiol. 1997; 273: H76-H84PubMed Google Scholar), cGMP-dependent dephosphorylation (19White R.E. Lee A.B. Shcherbatko A.D. Lincoln T.M. Schonbrunn A. Armstrong D.L. Nature. 1993; 362: 263-266Crossref Scopus (225) Google Scholar, 22White R.E. Schonbrunn A. Armstrong D.L. Nature. 1991; 351: 570-573Crossref PubMed Scopus (228) Google Scholar, 23Zhou X.B. Ruth P. Schlossmann J. Hofmann F. Korth M. J. Biol. Chem. 1996; 271: 19760-19767Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), by G protein subunits (12Kume H. Graziano M.P. Kotlikoff M.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11051-11055Crossref PubMed Scopus (148) Google Scholar, 13Scornik F.S. Codina J. Birnbaumer L. Toro L. Am. J. Physiol. 1993; 265: H1460-H1465PubMed Google Scholar), and direct stimulation by NO (41Bolotina V.M. Najibi S. Palacino J.J. Pagano P.J. Cohen R.A. Nature. 1994; 368: 850-853Crossref PubMed Scopus (1505) Google Scholar). We used the heterologous expression system and site-directed mutagenesis to identify the mechanism of β adrenergic stimulatory coupling to maxi-K channels and to identify the relevant channel modulatory site. Our data clearly implicate cAMP-PK in β2 receptor/channel stimulatory coupling since injection of the regulatory subunit of cAMP-PK disrupts β2 receptor/maxi-K channel stimulatory coupling (Fig. 3). In addition to disrupting stimulatory coupling, injection of oocytes with the cAMP-PK regulatory subunit also consistently reduced the amplitude of the basal current before application of isoproterenol (Fig. 3 B ), suggesting that phosphorylation by cAMP-PK regulates maxi-K channel activity under resting conditions. Conversely, a semi-selective (6-fold) cGMP-PK inhibitory peptide only slightly reduced ISO current stimulation, consistent with the predicted inhibition of cAMP-PK. Further evidence suggesting functional modulation of maxi-K channels by cAMP-PK was the finding that heterologously expressed maxi-K channels are stimulated by forskolin, and that this stimulation occurs with or without expression of the β2 receptor (Fig. 2, Table I).dSlo, the Drosophila maxi-K channel gene, contains a single optimal consensus sequence for PKA phosphorylation in the C-terminal region of the protein (RRGS at 959–962; Ref. 24Atkinson N.S. Robertson G.A. Ganetzky B. Science. 1991; 253: 551-553Crossref PubMed Scopus (534) Google Scholar), and mutations at this site were later found to prevent channel activation by exogenous PKA in inside-out patches (42Chung S.K. Reinhart P.H. Martin B.L. Brautigan D. Levitan I.B. Science. 1991; 253: 560-562Crossref PubMed Scopus (152) Google Scholar). The mammalian homologues of dSlo contain a less cAMP-PK -specific phosphorylation site at approximately the same position (RQPS at 866–869, e.g. GenBank accession no. U11058), which is conserved in mammalian genes, and which occupies a similar position upstream of a highly conserved region, aspartate-rich region that is a likely calcium-binding site (43Schreiber M. Salkoff L. Biophys. J. 1997; 73: 1355-1363Abstract Full Text PDF PubMed Scopus (336) Google Scholar). However, as many as 10 other potential cAMP-PK consensus phosphorylation sites exist throughout the coding region of the α subunit (37Kennelly P.J. Krebs E.G. J. Biol. Chem. 1997; 266: 15555-15558Abstract Full Text PDF Google Scholar). To ascertain the physiologically relevant phosphorylation site, we mutated serine 869 to alanine, and coexpressed the mutant α subunit with the wild type β subunit and the β2receptor. This single mutation markedly reduced, but did not abolish, β2 receptor stimulation of the current (from 33.3 ± 5.1% to 5.5 ± 1.3%, Table I), strongly implicating serine 869 in receptor/effector coupling. The low degree of current stimulation consistently observed in the mutant may relate to a slight role for additional cAMP phosphorylation sites, coupling through other kinases, or direct G protein interactions. Moreover, forskolin stimulation was only decreased by about half in mutant channels, suggesting that additional sites of channel modulation exist. It is possible that the dominant mechanism of stimulatory coupling observed in our experiments relates to factors unique to the Xenopus oocyte, such as cell size or endogenous G proteins, and that other regulatory mechanisms may be more prominent in mammalian cells. However, our results are consistent with the major mechanism of β2receptor/maxi-K channel stimulatory coupling occurring by cAMP-PK phosphorylation of serine 869.Finally, the stimulatory effect of both the β2 receptor agonist ISO and forskolin was an increase in current amplitude at all voltages, without a change in current kinetics, similar to the modulatory action of β2 receptor stimulation on cardiac sodium channel α subunits heterologously expressed inXenopus oocytes (44Schreibmayer W. Frohnwieser B. Dascal N. Platzer D. Spreitzer B. Zechner R. Kallen R.G. Lester H.A. Receptors Channels. 1994; 2: 339-350PubMed Google Scholar). In that study, mutation of all consensus cAMP-PK sites on the sodium channel failed to remove channel stimulation, leading to the suggestion that modulation might occur by phosphorylation of an unrelated protein resulting in a redistribution of channels to the plasma membrane (44Schreibmayer W. Frohnwieser B. Dascal N. Platzer D. Spreitzer B. Zechner R. Kallen R.G. Lester H.A. Receptors Channels. 1994; 2: 339-350PubMed Google Scholar). ISO stimulation of sodium currents was quite slow (approximately 10 min to maximum stimulation), which could be consistent with a redistribution of membranes within the oocyte. In the present study, however, stimulatory coupling occurred quite rapidly (approximately 1 min to maximum), which would be consistent with a direct effect on channel gating.In summary, we have used a heterologous expression system to demonstrated β2-adrenergic receptor/maxi-K channel stimulatory coupling. Coupling involves a cAMP-PK-dependent phosphorylation of the channel α subunit; mutation of one consensus cAMP-PK phosphorylation site (serine 869) almost eliminates β2-adrenergic modulation of the maxi-K current. These findings do not rule out other modulatory mechanisms, but do confine the physiologically relevant molecular mechanisms associated with β2 receptor/maxi-K channel coupling. The present study demonstrates the reconstitution of β2-adrenergic receptor stimulatory coupling to maxi-K channels by heterologous expression of receptor and channel RNAs inX. laevis oocytes. Isoproterenol and forskolin increased maxi-K currents in oocytes expressing either the pore-forming α subunit alone, or co-expressing the α and regulatory β subunits (Figs. 1 and 2, Table I). The stimulated currents were not endogenous oocyte currents since there was no augmentation of outward current in oocytes expressing β2 receptors but not maxi-K channels, and since forskolin, but not ISO, stimulated currents in oocytes expressing maxi-K channels, but not β2 receptors (TableI). Moreover, virtually all of the outward current was inhibited by iberiotoxin (Figs. 1 and 2), a selective peptidyl inhibitor of maxi-K channels (36Galvez A. Gimenez-Gallego G. Reuben J.P. Roy-Contancin L. Geigenbaum P. Kaczorowski G.J. Garcia M.L. J. Biol. Chem. 1990; 265: 11083-11090Abstract Full Text PDF PubMed Google Scholar). Since heterologous expression avoids complications associated with expression of multiple receptor or channel subtypes in target tissues, the present results clearly establish stimulatory coupling between human β2 receptors and maxi-K channels. Further, since stimulation was achieved with expression of only the β2 receptor and the α subunit and the degree of stimulation was similar with or without the regulatory β subunit (Table I), these studies localize the modulatory site to the channel α subunit. It should be noted that the degree of stimulation observed (30–35%) is somewhat less than reported in mammalian cells. In smooth muscle cells, current stimulation varies from approximately 50% to 100%, and is somewhat dependent on the step voltage and [Ca2+]i (7Song Y. Simard J.M. Pflugers Arch. Eur. J. Physiol. 1995; 430: 984-993Crossref PubMed Scopus (69) Google Scholar, 38Anwer K. Toro L. Oberti C. Stefani E. Sanborn B.M. Am. J. Physiol. 1992; 263: C1049-C1056Crossref PubMed Google Scholar, 39Fan S.-F. Wang S. Kao C.Y. J. Gen. Physiol. 1993; 102: 257-275Crossref PubMed Scopus (16) Google Scholar). Considerable controversy exists with respect to the mechanism of stimulatory coupling to maxi-K channels. Stimulatory mechanisms that have been proposed include cAMP-dependent phosphorylation (5Kume H. Takai A. Tokuno H. Tomita T. Nature. 1989; 341: 152-154Crossref PubMed Scopus (301) Google Scholar, 6Kume H. Hall I.P. Washabau R.J. Takagi K. Kotlikoff M.I. J. Clin. Invest. 1994; 93: 371-379Crossref PubMed Scopus (220) Google Scholar, 11Carl A. Kenyon J.L. Uemura D. Fusetani N. Sanders K.M. Am. J. Physiol. 1991; 261: C387-C392Crossref PubMed Google Scholar, 15Savaria D. Lanoue C. Cadieux A. Rousseau E. Am. J. Physiol. 1992; 262: L327-L336PubMed Google Scholar, 16Meera P. Anwer K. Monga M. Oberti C. Stefani E. Toro L. Sanborn B.M. Am. J. Physiol. 1995; 269: C312-C317Crossref PubMed Google Scholar), cGMP-dependent phosphorylation (17Taniguchi J. Furukawa K.I. Shigekawa M. Pflugers Arch. Eur. J. Physiol. 1993; 423: 167-172Crossref PubMed Scopus (169) Google Scholar, 18Robertson B.E. Schubert R. Hescheler J. Nelson M.T. Am. J. Physiol. 1993; 265: C299-C303Crossref PubMed Google Scholar,20White R.E. Darkow D.J. Lang J.L. Circ. Res. 1995; 77: 936-942Crossref PubMed Scopus (337) Google Scholar, 21George M.J. Shibata E.F. J. Invest. Med. 1995; 43: 451-458PubMed Google Scholar, 40Carrier G.O. Fuchs L.C. Winecoff A.P. Giulumian A.D. White R.E. Am. J. Physiol. 1997; 273: H76-H84PubMed Google Scholar), cGMP-dependent dephosphorylation (19White R.E. Lee A.B. Shcherbatko A.D. Lincoln T.M. Schonbrunn A. Armstrong D.L. Nature. 1993; 362: 263-266Crossref Scopus (225) Google Scholar, 22White R.E. Schonbrunn A. Armstrong D.L. Nature. 1991; 351: 570-573Crossref PubMed Scopus (228) Google Scholar, 23Zhou X.B. Ruth P. Schlossmann J. Hofmann F. Korth M. J. Biol. Chem. 1996; 271: 19760-19767Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), by G protein subunits (12Kume H. Graziano M.P. Kotlikoff M.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11051-11055Crossref PubMed Scopus (148) Google Scholar, 13Scornik F.S. Codina J. Birnbaumer L. Toro L. Am. J. Physiol. 1993; 265: H1460-H1465PubMed Google Scholar), and direct stimulation by NO (41Bolotina V.M. Najibi S. Palacino J.J. Pagano P.J. Cohen R.A. Nature. 1994; 368: 850-853Crossref PubMed Scopus (1505) Google Scholar). We used the heterologous expression system and site-directed mutagenesis to identify the mechanism of β adrenergic stimulatory coupling to maxi-K channels and to identify the relevant channel modulatory site. Our data clearly implicate cAMP-PK in β2 receptor/channel stimulatory coupling since injection of the regulatory subunit of cAMP-PK disrupts β2 receptor/maxi-K channel stimulatory coupling (Fig. 3). In addition to disrupting stimulatory coupling, injection of oocytes with the cAMP-PK regulatory subunit also consistently reduced the amplitude of the basal current before application of isoproterenol (Fig. 3 B ), suggesting that phosphorylation by cAMP-PK regulates maxi-K channel activity under resting conditions. Conversely, a semi-selective (6-fold) cGMP-PK inhibitory peptide only slightly reduced ISO current stimulation, consistent with the predicted inhibition of cAMP-PK. Further evidence suggesting functional modulation of maxi-K channels by cAMP-PK was the finding that heterologously expressed maxi-K channels are stimulated by forskolin, and that this stimulation occurs with or without expression of the β2 receptor (Fig. 2, Table I). dSlo, the Drosophila maxi-K channel gene, contains a single optimal consensus sequence for PKA phosphorylation in the C-terminal region of the protein (RRGS at 959–962; Ref. 24Atkinson N.S. Robertson G.A. Ganetzky B. Science. 1991; 253: 551-553Crossref PubMed Scopus (534) Google Scholar), and mutations at this site were later found to prevent channel activation by exogenous PKA in inside-out patches (42Chung S.K. Reinhart P.H. Martin B.L. Brautigan D. Levitan I.B. Science. 1991; 253: 560-562Crossref PubMed Scopus (152) Google Scholar). The mammalian homologues of dSlo contain a less cAMP-PK -specific phosphorylation site at approximately the same position (RQPS at 866–869, e.g. GenBank accession no. U11058), which is conserved in mammalian genes, and which occupies a similar position upstream of a highly conserved region, aspartate-rich region that is a likely calcium-binding site (43Schreiber M. Salkoff L. Biophys. J. 1997; 73: 1355-1363Abstract Full Text PDF PubMed Scopus (336) Google Scholar). However, as many as 10 other potential cAMP-PK consensus phosphorylation sites exist throughout the coding region of the α subunit (37Kennelly P.J. Krebs E.G. J. Biol. Chem. 1997; 266: 15555-15558Abstract Full Text PDF Google Scholar). To ascertain the physiologically relevant phosphorylation site, we mutated serine 869 to alanine, and coexpressed the mutant α subunit with the wild type β subunit and the β2receptor. This single mutation markedly reduced, but did not abolish, β2 receptor stimulation of the current (from 33.3 ± 5.1% to 5.5 ± 1.3%, Table I), strongly implicating serine 869 in receptor/effector coupling. The low degree of current stimulation consistently observed in the mutant may relate to a slight role for additional cAMP phosphorylation sites, coupling through other kinases, or direct G protein interactions. Moreover, forskolin stimulation was only decreased by about half in mutant channels, suggesting that additional sites of channel modulation exist. It is possible that the dominant mechanism of stimulatory coupling observed in our experiments relates to factors unique to the Xenopus oocyte, such as cell size or endogenous G proteins, and that other regulatory mechanisms may be more prominent in mammalian cells. However, our results are consistent with the major mechanism of β2receptor/maxi-K channel stimulatory coupling occurring by cAMP-PK phosphorylation of serine 869. Finally, the stimulatory effect of both the β2 receptor agonist ISO and forskolin was an increase in current amplitude at all voltages, without a change in current kinetics, similar to the modulatory action of β2 receptor stimulation on cardiac sodium channel α subunits heterologously expressed inXenopus oocytes (44Schreibmayer W. Frohnwieser B. Dascal N. Platzer D. Spreitzer B. Zechner R. Kallen R.G. Lester H.A. Receptors Channels. 1994; 2: 339-350PubMed Google Scholar). In that study, mutation of all consensus cAMP-PK sites on the sodium channel failed to remove channel stimulation, leading to the suggestion that modulation might occur by phosphorylation of an unrelated protein resulting in a redistribution of channels to the plasma membrane (44Schreibmayer W. Frohnwieser B. Dascal N. Platzer D. Spreitzer B. Zechner R. Kallen R.G. Lester H.A. Receptors Channels. 1994; 2: 339-350PubMed Google Scholar). ISO stimulation of sodium currents was quite slow (approximately 10 min to maximum stimulation), which could be consistent with a redistribution of membranes within the oocyte. In the present study, however, stimulatory coupling occurred quite rapidly (approximately 1 min to maximum), which would be consistent with a direct effect on channel gating. In summary, we have used a heterologous expression system to demonstrated β2-adrenergic receptor/maxi-K channel stimulatory coupling. Coupling involves a cAMP-PK-dependent phosphorylation of the channel α subunit; mutation of one consensus cAMP-PK phosphorylation site (serine 869) almost eliminates β2-adrenergic modulation of the maxi-K current. These findings do not rule out other modulatory mechanisms, but do confine the physiologically relevant molecular mechanisms associated with β2 receptor/maxi-K channel coupling. We thank Laura Lynch for technical assistance; Drs. L. Toro, E. Stefani, M. Wallner, L. Salkoff, and J. Benovic for supplying cDNAs; and Dr. P. Drain for assistance with oocyte collection.