1 Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6046; and 2 Department of Anesthesiology and Comparative Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35249
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ABSTRACT |
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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
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INTRODUCTION |
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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 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.
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EXPERIMENTAL PROCEDURES |
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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'-ATGCCGGGCTCCCGACGCGTC-3') 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 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.
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-A-R-K-E-OH; see Ref. 15), and PKA inhibitory peptide (H-T-T-Y-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).
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RESULTS |
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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).
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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 µM 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 µM 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 µM 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 µM] but not with an effect of
the peptide on PKA (Ki = 550 µM; 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 µM (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.
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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 accession 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), 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
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.
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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 could enhance KCa activity. Oocytes expressing both - and
-channel
subunits were perfused with spermine NONOate (200 µM), 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 µM 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).
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DISCUSSION |
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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 purified 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-41084 and HL-45239 (M. I. Kotlikoff) and HL-31197 and HL-51173 (S. Matalon).
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: mik7{at}cornell.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 21 October 1999; accepted in final form 21 August 2000.
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