Guanylyl cyclase stimulatory coupling to KCa channels

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Am J Physiol Cell Physiol 279: C1938–C1945, 2000.

Guanylyl cyclase stimulatory coupling to KCa channels M. NARA,1 P. D. K. DHULIPALA,1 G. J. JI,1 U. R. KAMASANI,1 Y.-X. WANG,1 S. MATALON,2 AND M. I. KOTLIKOFF1 1 Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6046; and 2Department of Anesthesiology and Comparative Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35249 Received 21 October 1999; accepted in final form 21 August 2000

Nara, M., P. D. K. Dhulipala, G. J. Ji, U. R. Kamasani, Y.-X. Wang, S. Matalon, and M. I. Kotlikoff. Guanylyl cyclase stimulatory coupling to KCa channels. Am J Physiol Cell Physiol 279: C1938–C1945, 2000.—We coexpressed the human large-conductance, calcium-activated K (KCa) channel (␣- and ␤-subunits) and rat atrial natriuretic peptide (ANP) receptor genes in Xenopus oocytes to examine the mechanism of guanylyl cyclase stimulatory coupling to the channel. Exposure of oocytes to ANP stimulated whole cell KCa currents by 21 ⫾ 3% (at 60 mV), without altering current kinetics. Similarly, spermine NONOate, a nitric oxide donor, increased KCa currents (20 ⫾ 4% at 60 mV) in oocytes expressing the channel subunits alone. Stimulation of KCa currents by ANP was inhibited in a concentration-dependent manner by a peptide inhibitor of cGMP-dependent protein kinase (PKG). Receptor/channel stimulatory coupling was not completely abolished by mutating the cAMP-dependent protein kinase phosphorylation site on the ␣-subunit (S869; Nars M, Dhulipals PD, Wang YX, and Kotlikoff MI. J Biol Chem 273: 14920–14924, 1998) or by mutating a neighboring consensus PKG site (S855), but mutation of both residues virtually abolished coupling. Spermine NONOate also failed to stimulate channels expressed from the double mutant cRNAs. These data indicate that nitric oxide donors stimulate KCa channels through cGMP-dependent phosphorylation and that two serine residues (855 and 869) underlie this stimulatory coupling. ion channel; calcium-activated potassium channels; mutagenesis; smooth muscle; cGMP-dependent protein kinase

LARGE-CONDUCTANCE, calcium-activated K (KCa) channels are expressed in muscle, neurons, and other cell types (26). Channel phosphorlyation appears to be a major mechanism by which G protein-coupled receptors (GPCRs) modulate channel activity (24, 25, 28). After stimulation of specific GPCRs, KCa channels have been reported to be stimulated due to phosphorylation by cAMP-dependent protein kinase (PKA; see Refs. 24, 25, 30, 33) and by cGMP-dependent protein kinase (PKG; see Refs. 1, 4, 7, 34, 37, 39, 45) as well as by channel dephosphorylation (43, 44, 47). Recent studies evaluating the mechanism of action of cGMP-dependent channel modulation in reconstituted or heterologously expressed channels indicate that the channel is

Address for reprint requests and other correspondence: M. I. Kotlikoff, Dept. of Biomedical Sciences, T4 018 VRT, Box 11, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853-6401 (E-mail: [email protected]). C1938

a substrate for phosphorylation by purified protein kinase G (1, 2). We have recently reported that ␤2-adrenergic receptor/KCa channel stimulatory coupling requires PKA phosphorylation of the channel ␣-subunit at S869 in experiments in which the channel subunits and receptor were heterologously expressed in Xenopus laevis oocytes (33). Numerous studies have indicated that cGMP-dependent phosphorylation also mediates channel stimulatory coupling (1, 4, 7, 34, 37, 40, 45). Soluble and particulate guanylyl cyclases (GC) catalyze the formation of cGMP, which in turn activates PKG, resulting in the phosphorylation of target proteins. Soluble GC is a heterodimer that is activated by nitric oxide (NO) and other free radicals, whereas particulate GC possesses an extracellular ligand-binding domain, a single transmembrane domain, and an intracellular domain containing protein kinase-like and cyclase catalytic domains (3, 14, 46). GC-A is a member of this latter category, comprising the functional atrial natriuretic peptide (ANP; see Refs. 3, 8, 14, 22, 46). Here we reconstitute cGMP-dependent KCa channel stimulatory coupling in Xenopus oocytes expressing rat GC-A and human KCa channel (hSlo and hKV,Ca␤) RNAs. We also show that the NO donor NONOate (17, 29) stimulates KCa currents in oocytes expressing the channel genes. Mutational analysis indicates that full stimulatory coupling by ANP (particulate GC) and by NO (soluble GC) involves phosphorlyation of the channel ␣-subunit on S855 and S869. EXPERIMENTAL PROCEDURES

Expression of KCa channel and ANP receptor cRNAs in X. laevis oocytes. Expression of hSlo and hKV,Ca␤ cRNAs has been described previously (33). The rat GC-A clone was obtained from Dr. Michael Chinkers, and the 5⬘-untranslated portion of the clone was eliminated by amplifying the coding region using initiation (5⬘-ATGCCGGGCTCCCGACGCGTC3⬘) and termination (5⬘-TCAGCCTCGAGTGCTACATCC-3⬘) primers. The PCR fragment was subcloned in a TA 2.1 PCR vector (Invitrogen), sequenced, and used for cRNA synthesis. Site-directed mutagenesis of hSlo cDNA was carried out using the pAlter mutation kit (Promega). The full-length

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PKG STIMULATION OF KCA CHANNELS

cDNA was subcloned in the pAlter-1 vector at the Sma I site after being filled with Klenow DNA polymerase. The mutation primers were 2596–2617 (5⬘-CGTCAACCATCCATCAGGAGGGA-3⬘) and 2549–2575 (5⬘-ATGGATAGATCCGCTCCAGATAACAGC-3⬘). These primers were annealed to denatured DNA individually or together to mutated residues 855, or 855 and 869, from Ser to Ala. The DNA containing annealed mutagenic oligos were used to carry out site-specific mutagenesis using the manufacturer’s protocol, and the cDNAs were confirmed by sequence analysis. The human KCa {hSlo, hKV,Ca␤, hSlo(⌬S855A), hSlo(⌬S869A), and hSlo[⌬S(855/869)A]} and rat GC-A cDNA were linearized with appropriate restriction enzymes, and cRNAs were synthesized using T7 RNA polymerase (mMesssage mMachine Kit; Ambion). CRNA quality was confirmed on ethidium bromide-stained agarose gels, and concentrations were estimated by spectrophotometry using Pharmacia Genequant-II. Oocytes from X. laevis were prepared for injection as previously described (16). hSlo cRNA was injected at a concentration of 0.8–1.0 ␮g/␮l, hKV,Ca␤ was injected at a 2:1 molar ratio with hSlo to maximize regulatory subunit interaction (31), and GC-A was injected at 0.7–0.9 ␮g/␮l; ⬃50 nl of these solutions were injected per oocyte. Oocytes were incubated for 3–5 days in ND-96 (96 mM NaCl, 2.0 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, and 5.0 mM HEPES, pH 7.5) with

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sodium pyruvate (2.5 mM), penicillin (100 U/ml), and streptomycin (100 ␮g/ml) at 18°C until recorded. Electrophysiology. Two-electrode voltage-clamp recordings were made at room temperature in ND-96 as previously described (33). Currents were amplified (OC-725C; Warner Instrument, Hamden, CT), filtered at 200 Hz (⫺3 dB, 8-pole low-pass filter; Frequency Devices, Haverhill, MA), digitized at 1 kHz, and stored on computer disk (Digidata 1200 and pCLAMP software; Axon Instruments, Foster City, CA). KCa currents were monitored by holding the membrane potential constant at ⫺60 mV. Every 20 s, a 500-ms test pulse to ⫹60 mV was applied. A 5-min equilibration period was allowed following voltage clamp; oocytes that showed unstable currents over this period were discarded. Statistical significance was determined using the Student’s t-test for paired data or by one-way ANOVA for multiple comparisons. Chemicals. Rat ANP, PKG inhibitory peptide (H-R-K-R-AR-K-E-OH; see Ref. 15), and PKA inhibitory peptide (H-T-TY-A-D-F-I-A-S-G-R-T-G-R-R-N-A-I-H-D-OH) were obtained from Calbiochem (La Jolla, CA). Frog ANP was from Sigma (St. Louis, MO). Spermine NONOate was purchased from Alexis (San Diego, CA), and iberiotoxin was from Peptide Institute (Osaka, Japan). A 50 mM solution of spermine NONOate was prepared at pH 8.5 before each experiment, as described (17).

Fig. 1. Stimulatory coupling between heterologously expressed atrial natriuretic peptide (ANP) receptors and large-conductance, calcium-activated (KCa) channels. A: traces of currents from voltage-clamp steps to ⫹60 mV in oocytes coexpressing KCa channel ␣- and ␤-subunits and ANP receptor [guanylyl cyclase (GC)-A] genes. ANP (100 nM) increased the amplitude of the current in a reversible manner. Traces are shown at 2-min intervals. Dotted line shows the level of peak current before stimulation with ANP. Currents were elicited by 500-ms step depolarization from a holding potential of ⫺60 mV. B: superimposed whole cell current traces before and after stimulation with 100 nM ANP. Traces are from each experimental condition at the indicated points (* and E, respectively) in A. ANP increased the current amplitude without altering current activation or inactivation kinetics. The experimental protocol was the same as described for A. C: comparison of %stimulation of currents by ANP from oocytes expressing KCa channel and ANP receptor with KCa channel alone. Application of rat ANP to oocytes expressing KCa channel subunits and the ANP receptor resulted in a significant increase in current amplitude, whereas frog ANP did not have significant effect on oocytes expressing only ␣- and ␤-subunits of KCa channel (without ANP receptor). ***Significant stimulation over control current amplitude (P ⬍ 0.001). **Significant difference between percent stimulation of current from hSlo ⫹ ANP receptor and that from hSlo alone (P ⬍ 0.01). Error bars indicate SE; n, no. of experiments.

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PKG STIMULATION OF KCA CHANNELS

RESULTS

Hormone-stimulated GC activates KCa channels. Oocytes injected with cRNA encoding the human KCa channel subunits (hSlo and hKV,Ca␤) and the rat ANP receptor expressed large, voltage-dependent K currents that were completely blocked by iberiotoxin (100 nM), a specific peptidyl inhibitor of KCa channels (13). Moreover, functional stimulatory coupling between the ANP receptor and KCa channels was observed. As shown in Fig. 1, when oocytes expressing the CG-A receptor and both KCa subunits were perfused with rat ANP, the KCa current was consistently stimulated, with no effect on current activation or inactivation kinetics. Virtually all of the voltage-dependent K current was abolished in the presence of iberiotoxin, indicating that both the basal and stimulated current resulted from KCa channel activity. The mean current stimulation by 100 nM ANP was 20.5 ⫾ 2.5% (at 60 mV). Current stimulatory coupling resulted from activation of the heterologously expressed rat ANP receptor, because frog ANP (100 nM) failed to stimulate currents in noninjected oocytes (data not shown) or in oocytes injected with the KCa channel subunits but not the ANP receptor cRNA (2.1 ⫾ 3.2%; Fig. 1C). Stimulatory coupling requires activation of PKG. GC-A encodes a particulate GC whose activity is stimulated following the binding of ANP (3, 8, 14, 22, 46). To determine the mechanism of ANP-KCa channel coupling, we first used a pseudosubstrate peptide inhibitor of PKG (15) to test whether the stimulation of KCa currents by ANP requires kinase activation. Intracellular injection of the inhibitory peptide decreased stimulatory coupling in a concentration-dependent manner, whereas stimulatory coupling was not disrupted in sham-injected oocytes (Fig. 2). To use individual oocytes as their own controls, cells were exposed to ANP before and after either sham injection or injection of the PKG inhibitory peptide (80 or 750 ␮M estimated final concentration). Before injection of the inhibitor, ANP was washed out of the bath and the stimulated currents were allowed to return to basal levels. With an estimated final concentration of 80 ␮M inhibitory peptide, stimulation of the current by ANP was reduced by 43% relative to stimulation in the same oocyte after sham injection (22.4 ⫾ 5.5 before sham injection vs. 12.8 ⫾ 3.1% after peptide injection, n ⫽ 5). At 750 ␮M estimated final concentration, the stimulation was reduced by 82% (17.1 ⫾ 4.6 vs. 3.1 ⫾ 3.1%, n ⫽ 3). This dose dependency is consistent with an effect on PKG [inhibitory constant (Ki) ⫽ 86 ␮M] but not with an effect of the peptide on PKA (Ki ⫽ 550 ␮M; see Ref. 15). Moreover, analogous experiments with a PKA peptide inhibitor confirmed this result. Injection of the peptide to a final estimated concentration of 50 ␮M (Ki ⫽ 2.3 nM) did not inhibit ANP-induced channel stimulation (Fig. 2C). The mean stimulation before and after injection was 23.7 ⫾ 1.5 and 25.4 ⫾ 1.8%, respectively, in five experiments. Thus these data suggest that PKG plays a principal role in ANP receptor/KCa channel stimulatory coupling.

Fig. 2. ANP receptor/KCa channel stimulatory coupling is dependent on cGMP-dependent protein kinase (PKG) and independent of cAMP-dependent protein kinase (PKA) activity. A: repeated application of ANP (100 nM) stimulated KCa currents to a similar extent. The oocyte was microinjected with water between additions of ANP. B: in matched experiments, after an initial exposure to ANP, oocytes were injected with a PKG-specific pseudosubstrate inhibitory peptide (inhib pep) to a final concentration of 750 ␮M, and ANP was reapplied. Stimulatory coupling was markedly decreased in the presence of the peptide. Voltage-clamp steps to ⫹60 mV for 500 ms were imposed at 20-s intervals. C: after an initial exposure to 100 nM ANP, PKA inhibitory peptide was injected to an approximate final concentration of 50 ␮M before a second exposure to ANP. In 5 similar experiments, ANP stimulation was equivalent in the presence of the peptide inhibitor.

Mutation of KCa␣-subunit consensus phosphorylation sites. Stimulatory coupling between the adenylyl cyclase-linked ␤2-receptor and KCa channels occurs at serine 869 on the channel ␣-subunit (33). The RQPS sequence at this site (866–869, e.g., GenBank acces-

PKG STIMULATION OF KCA CHANNELS

sion no. U11058) is conserved in mammalian genes, although this position appears to be a less optimal PKA phosphorylation site than the analogous site in the Drosophila channel ␣-subunit (dSlo; see Ref. 9). Because consensus PKA and PKG phosphoacceptor sequences overlap considerably and PKG sites preferentially contain at least one arginine residue amino terminal to the phosphoacceptor serine (21), we examined whether phosphorylation of serine 869 was required for ANP/KCa stimulatory coupling. Oocytes expressing a mutant ␣-subunit in which serine 869 was replaced by alanine [hSlo(⌬S869A)], as well as the wild-type ␤-subunit and ANP receptor, were examined for stimulatory coupling. As shown in Fig. 3, A and E, ANP increased the hSlo(⌬S869A)/␤-current, although the degree of stimulation was less (15.0 ⫾ 2.4%; n ⫽ 7). We next examined the RSS sequence (853–855), which lies close to the PKA site and contains a 5⬘-arginine, by making a similar mutant ␣-subunit [hSlo(⌬S855A)] and conducting equivalent experiments. In these experiments, ANP increased the KCa current by 10.4 ⫾ 0.4% (n ⫽ 4) in oocytes coexpressing hSlo(⌬S855A), hKV,Ca␤, and ANP receptor cRNA (Fig. 3, B and E). Because stimulatory coupling was diminished in experiments expressing ⌬S869A or ⌬S855A ␣-subunits, we made and tested the double mutant construct

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⌬S(855/869)A. The amplitude and kinetics of the KCa currents were not affected by the double mutation (Fig. 3B), whereas, as shown in Fig. 3, C and E, ANP receptor/KCa stimulatory coupling was absent in the double mutant (3.1 ⫾ 1.7%; n ⫽ 5), suggesting that phosphorylation of the KCa channel by PKG occurs at both serine 869 and serine 855. PKG has recently been reported to stimulate KCa channels in excised patches through phosphorylation of a carboxy terminal serine residue (S107; see Ref. 12). To resolve this discrepancy, we made the ⌬S(1072)A mutation and examined ANP stimulatory coupling. As shown in Fig. 3D, application of ANP to oocytes expressing the carboxy terminal mutant ␣-subunit (and coexpressing the ␤-subunit) resulted in current stimulation. The average stimulation in eight experiments was 25.8 ⫾ 10.1%, not significantly different from that observed in oocytes expressing the wild-type ␣-subunit. NO stimulates KCa channel activity. Our results indicate that a receptor GC mediates stimulatory coupling of KCa channels through the action of PKG. Because soluble GC also catalyzes the production of cGMP and activates PKG and because such action has been reported to underlie physiologically relevant channel stimulation by NO (5), we sought to determine whether the NO stimulation of endogenous soluble GC

Fig. 3. Mutational analysis of PKG phosphorylation site mediating ANP stimulatory coupling. A: ANP (100 nM) stimulatory coupling is blunted in oocytes expressing hSlo(⌬S869A), along with hKV␤ and the ANP receptor; stimulatory coupling was ⬃15%. Data points show peak current evoked by a 500-ms test depolarization from ⫺60 to ⫹60 mV at 20-s intervals. IbTX, iberiotoxin. B: similarly weak stimulation in an oocyte expressing hSlo(⌬S855A). ANP (100 nM) increased the KCa current by ⬃10%. The experimental protocol was the same as described for A. C: ANP stimulatory coupling is absent in an oocyte expressing the double mutation [⌬S(855/ 869)A], along with hKV␤ and GC-A. The experimental protocol was the same as described for A. D: stimulation of KCa current in an oocyte expressing the ⌬S1072A, carboxy terminal mutation of hSlo. E: mean ANP stimulation in the 4 groups in A–D. In all experiments, the ␣-subunit was coexpressed with hKV␤ and GC-A, and oocytes were stimulated with 100 nM ANP. ANP significantly increased wild-type hSlo, hSlo(⌬S869A), hSlo(⌬S855A), and hSlo(⌬S1072A) currents, whereas it failed to stimulate hSlo[⌬S(855/869)A] current. Stimulation of the single 855 and 869 mutations was weaker than in wild type or the 1072 mutant, which were not significantly different. Note that ANP stimulation of the single S855 or S869 mutant channels was significantly greater than the double mutant. *P ⬍ 0.05, **P ⬍ 0.01, and ***P ⬍ 0.001. Error bars indicate SE.

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PKG STIMULATION OF KCA CHANNELS

NO has been reported to stimulate KCa channel activity by direct (5, 18) and PKG-dependent actions (1, 4, 34, 37, 40, 45). To determine whether stimulatory coupling by the NO donor results from channel phosphorylation at the sites associated with PKG (S855 and S869), we perfused spermine NONOate (200 ␮A) in oocytes coexpressing the hSlo[⌬A(855/869)S] and ␤-subunits. As shown in Fig. 5, spermine NONOate failed to stimulate hSlo[⌬A(855/869)S]/␤-currents or alter channel kinetics. hSlo[⌬A(855/869)S]/␤-current amplitude after exposure to spermine NONOate was 0.4 ⫾ 4.7% of control (n ⫽ 6), whereas wild-type hSlo/

Fig. 4. Stimulation of heterologously expressed KCa channels by nitric oxide (NO). A: plot of peak KCa current following addition of spermine NONOate (200 ␮M). The NO donor increased the amplitude of the current from oocytes expressing both KCa channel subunits (␣ ⫹ ␤). The current was abolished by IbTX (100 nM), and spermine NONOate did not stimulate the current in the presence of IbTX. B: superimposed whole cell current recordings before and after stimulation with 200 ␮M spermine NONOate. Traces are from the experiment shown in A at the indicated points (* and E, respectively). The experimental protocol was the same as described for Fig. 3A.

could enhance KCa activity. Oocytes expressing both ␣and ␤-channel subunits were perfused with spermine NONOate (200 ␮M), an NO donor (17, 29). As shown in Fig. 4, application of spermine NONOate resulted in an increase in amplitude of KCa currents, although the stimulatory effect was poorly reversible. Iberiotoxin (100 nM) abolished the current almost completely, and spermine NONOate failed to stimulate a current in the presence of the KCa channel blocker (Fig. 4A). Moreover, similar to the action of heterologously expressed particulate GC (GC-A), the spermine NONOate produced a scaled increase in the control current without altering activation or inactivation kinetics (Fig. 4B). In control experiments, the 200 ␮M spermine NONOate solution was incubated for 48 h at room temperature before experiments to eliminate NO (half-life ⬃2 h at 25°C). In five experiments, there was no increase in current amplitude when oocytes were exposed to this solution (data not shown).

Fig. 5. Mutation of serine 855 and serine 869 abolishes stimulation of KCa channels by NO. A: spermine NONOate (200 ␮M) failed to stimulate the current from an oocyte coexpressing hSlo[⌬S(855/ 869)A] and the wild-type ␤-subunits. The experimental protocol was the same as described for Fig. 3A. B: whole cell current traces before and after stimulation with 200 ␮M spermine NONOate. Traces are from each experimental condition at the indicated points (* and E, respectively) in A. The experimental protocol was the same as described for Fig. 3A; scale bars are 0.5 ␮A and 100 ms. C: comparison of percent stimulation of current with spermine NONOate (200 ␮M). NONOate significantly increased wild-type hSlo currents but failed to stimulate currents in oocytes expressing hSlo[⌬S(855/869)A] and the wild-type ␤-subunits. **Significant difference between before and after application of ANP (P ⬍ 0.05). ***Significant difference between percent stimulation of wild-type hSlo current and that of hSlo[⌬S(855/869)A] currents (P ⬍ 0.05). Error bars indicate SE.

PKG STIMULATION OF KCA CHANNELS

␤-current amplitude was augmented by 20.1 ⫾ 4.1% (n ⫽ 4; Fig. 5C). These findings suggest that NO/KCa channel stimulatory coupling is phosphorylation dependent, requiring phosphorylation of the 855 and 869 serine residues on hSlo. DISCUSSION

The relaxant effect of ANP (11, 32, 38) and NO (5, 6, 18, 20, 36) on smooth muscle has been reported to involve the activation of KCa channels. We have shown that the heterologous expression of a receptor GC (GC-A) and KCa channels in Xenopus oocytes reconstitutes stimulatory receptor-effector coupling and have used this system to determine the coupling mechanism underlying KCa stimulation following the activation of particulate (ANP) and soluble (NO) GC. Our results indicate that this coupling is mediated by PKG (Fig. 2) and requires phosphorylation of two phosphoacceptor sites near a putative calcium-binding region of the ␣-subunit (35). Moreover, we show that this stimulatory coupling mechanism appears to underlie the stimulatory action of soluble NO on KCa channels. PKG activates KCa channel proteins reconstituted into lipid bilayers (1) and phosphorylates hSlo proteins expressed in Xenopus oocyte membranes (2), suggesting a direct phosphorylation of the channel despite evidence indicating channel modulation by other mechanisms (5, 23, 47). The specific phosphorylation site(s) underlying modulation by the kinase remains uncertain, however. The results of Fukao et al. (12) indicate that exposure of inside-out patches to PKG stimulates channel activity, an effect that is abolished in patches pulled from HEK cells expressing channels in which a putative cGMP-dependent phosphorylation sequence at the carboxy terminus of hSlo (S1072) is mutated. This sequence (KKSS) was identified as a likely phosphorylation site by Toro et al. (41), despite the lack of an amino terminal arginine residue previously identified as a universal feature in a survey of PKG phosphorylation sites (21). Our results indicate that stimulatory coupling persists in oocytes expressing ⌬S1072A ␣-subunit mutation reported to eliminate PKG-dependent stimulation (12). The major differences between the present studies and those of Fukao et al. are the different expressed cDNAs (hSlo and hKV␤ vs. cslo-␣) and expression systems (X. laevis oocytes vs. HEK293 cells) and the reliance on endogenous kinase in whole cell experiments vs. the use of exogenous PKG-I␣ applied to excised patches in the latter studies. It is interesting to note that, despite the fact that cSlo is 94% homologous (DNA) with hSlo, there is a key difference at the putative phosphorylation site reported in this study. Whereas the hSlo sequence is RSS, containing the requisite arginine, the canine sequence is KSS, and it is tempting to speculate that this difference, which is 1 of only 22 differences (1115 residues for the proteins, neglecting the extreme carboxy terminus), is significant. This may also be consistent with previous findings indicating that puri-

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fied PKG failed to activate cSlo in excised patches (42). It is also possible that the use of purified kinase vs. stimulation of endogenous kinase in the oocyte could explain the results. Although the expression of exogenous PKG in the Xenopus oocyte has been questioned based on lack of reactivity to mammalian anti-PKG antibodies (2), several groups have reported functional kinase activation (10, 19, 27). The investigation of stimulatory coupling resulting from activation of a receptor-mediated GC (CG-A) or a soluble GC (NO donor) avoids potential nonphysiological effects related to application of exogenous kinase. Current stimulation by a maximal concentration of ANP (100 nM ANP) was 20.5 ⫾ 2.5%, whereas the level of stimulation was 33% in similar experiments examining heterologously expressed ␤2-receptors signaling through PKA (33). Moreover, the relative signaling strength was mimicked when the respective kinases were activated by postreceptor mechanisms (NO, 20.1 ⫾ 4.1% and forskolin, 35 ⫾ 8; see Ref. 33). These data may indicate a more pronounced effect resulting from the specific phosphorylation at serine 869. In summary, we have demonstrated ANP receptor/ KCa channel stimulatory coupling by coexpression of KCa channel (␣- ⫹ ␤-subunits) and ANP receptor genes in X. laevis oocytes. The coupling of the ANP receptor to KCa channels involves channel phosphorylation by PKG, further supporting work indicating that the ␣-subunit of the KCa channel complex contains a phosphoacceptor site PKG-mediated phosphorylation. Our results indicate that physiological phosphoryation occurs following activation of particulate or soluble GC and that the major phosphorylation sites are serine 855 and serine 869. We thank Mario Brenes and Laura Lynch for technical assistance; Drs. L. Toro, E. Stefani, M. Wallner, L. Salkoff, and M. Chinkers for supplying cDNAs; and Dr. P. Drain for assistance with oocyte collection. This work was supported by National Heart, Lung, and Blood Institute Grants HL-41084 and HL-45239 (M. I. Kotlikoff) and HL31197 and HL-51173 (S. Matalon). REFERENCES 1. Alioua A, Huggins JP, and Rousseau E. PKG-I␣ phosphorylates the ␣-subunit and upregulates reconstituted GKCa channels from tracheal smooth muscle. Am J Physiol Lung Cell Mol Physiol 268: L1057–L1063, 1995. 2. Alioua A, Tanaka Y, Wallner M, Hofmann F, Ruth P, Meera P, and Toro L. The large conductance, voltage-dependent, and calcium-sensitive K⫹ channel, Hslo, is a target of cGMP-dependent protein kinase phosphorylation in vivo. J Biol Chem 273: 32950–32956, 1998. 3. Anand-Srivastava MB and Trachte GJ. Atrial natriuretic factor receptors and signal transduction mechanisms. Pharmacol Rev 45: 455–497, 1993. 4. Archer SL, Huang JM, Hampl V, Nelson DP, Shultz PJ, and Weir EK. Nitric oxide and cGMP cause vasorelaxation by activation of a charybdotoxin-sensitive K channel by cGMPdependent protein kinase. Proc Natl Acad Sci USA 91: 7583– 7587, 1994. 5. Bolotina VM, Najibi S, Palacino JJ, Pagano PJ, and Cohen RA. Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle. Nature 368: 850–853, 1994.

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