From the Department of Animal Biology, School of Veterinary
Medicine, University of Pennsylvania,
Philadelphia, Pennsylvania 19104-6046
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INTRODUCTION |
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. 1), and the binding of a heterotrimeric
G protein subunit to a modulatory channel domain (for review see Ref.
2). Stimulation of
2 receptors relaxes smooth muscle by
modulating the activity of several protein targets (3). One prominent
target of
2 adrenergic signaling in smooth muscle is the
large conductance, calcium-activated potassium (maxi-K)1 channel, the
activity of which is markedly increased following receptor binding
(4-7). Modulation of maxi-K channel activity appears to be a
functionally important component of
-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
-adrenergic agents (8-10). 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 (5, 6, 11-14). Further, with respect to channel
phosphorylation, maxi-K channel stimulation has been attributed to
channel phosphorylation by cAMP-dependent protein kinase
(5, 6, 11, 15, 16), by cGMP-dependent protein kinase
(17-21), and by channel dephosphorylation (19, 22, 23).
Maxi-K channels are composed of at least two dissimilar subunits: the
subunit, which forms the channel pore (24-27), and the
subunit
(28-30), which modifies the voltage and calcium sensitivity of the
pore-forming subunit (31). Both the human
(hSlo) and
(hKv,Ca
) subunit genes encode several consensus cAMP-PK,
although the site(s) associated with physiological regulation have not been determined. We examined
-adrenergic receptor/maxi K coupling by
coexpression of hSlo and hKv,Ca
and the human
2-adrenergic receptor gene (h
2AR) in
Xenopus 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
subunit, and that mutation
of Ser-869 almost completely eliminates
2
receptor/channel coupling.
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EXPERIMENTAL PROCEDURES |
Expression of Maxi-K and
2 Receptor cRNAs in
Xenopus Oocytes--
hSlo was obtained from Drs. Ligia Toro, Enrico
Stefani, and Martin Walner (Department of Anesthesiology, UCLA, Los
Angeles, CA) and from Dr. Lawrence Salkoff (Department of Genetics,
Washington University, St. Louis, MO), and the H
2AR
cDNA clone was obtained from Dr. Jeffery Benovic (Kimmel Cancer
Center, Thomas Jefferson University, Philadelphia, PA). We cloned
hKV,Ca
(32) by reverse transcription and polymerase
chain reaction. Double-stranded cDNA was synthesized from human
tracheal RNA (CLONTECH) using avian myeloblastosis
virus-reverse transcriptase (Boehringer Mannheim); the cDNA was
amplified with hKV,Ca
-specific sense
(5'-ATGGTGAAGCTGGTGATGGCC-3') and antisense
(5'-CTACTTCTGGGCCGCCAGGAGGGA-3') primers, and the polymerase chain
reaction fragment cloned in TA 2.1 cloning vector (Invitrogen) and
sequenced.
Mutagenesis of the hSlo cDNA was carried out using the pAlter
mutation kit (Promega). The full-length cDNA was subcloned in pAlter-1 vector at SmaI site after blunt ending with Klenow
enzyme. The plasmid DNA was denatured with 2 M NaOH, 2 mM EDTA solution. The mutation primer, spanning from
nucleotide 2596 to 2617 (5'-CGTCAACCATCCATCAGGAGGGA-3'), was annealed
to the denatured DNA to mutate amino acid 869 from Ser to Ala. The
annealed DNA was used to carry out site-specific mutagenesis using the
manufacturer's protocol; mutated cDNA (hSlo(
S869A)) was
confirmed by sequencing (33).
cRNA was prepared from the human maxi-K cDNAs (hSlo,
hKV,Ca
, hSlo(
S869A)) and h
2AR cDNA
using the mMessage mMachine kit (Ambion). The plasmid DNAs were
linearized with appropriate restriction enzymes and the cRNAs were
synthesized using T7 (hSlo, hKV,Ca
) and Sp6
(h
2AR receptor) RNA polymerase enzymes (34). The
integrity of the cRNAs was tested on ethidium bromide-stained agarose
gels, and cRNA concentrations were estimated by spectrophotometric
measurements.
Oocytes from X. laevis were prepared for injection as
described previously (35). Channel and receptor cRNAs were mixed at final concentrations of 0.6-0.9 µg/µl and 0.35-0.45 µg/µl,
respectively; the final concentration of hKV,Ca
cRNA was
adjusted to achieve a 1:2 molar ratio to hSlo, so that all expressed
channel
subunits would likely interact with
subunits (32).
Approximately 50 nl of these solutions were injected per oocyte, and
oocytes were then incubated for 3-6 days in ND96 (96 mM
NaCl, 2.0 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, 5.0 mM HEPES, pH 7.5)
with sodium pyruvate (5.0 mM), penicillin (100 units/ml),
and streptomycin (100 µg/ml) at 18 °C.
Electrophysiology--
Two-electrode voltage clamp measurements
were made at room temperature in ND96, using pipettes of 0.3-1.0
megohms resistance filled with 3 M KCl. Currents were
amplified (OC-725C, Warner Instrument, Hamden, CT), filtered at 200 Hz
(
3 dB; 8-pole Bessel filter, model 902, Frequency Devices), digitized
at 1 kHz (Digidata, Axon Instruments), monitored (pClamp, Axon
Instruments), and simultaneously stored on disk. Maxi-K currents were
monitored by holding the membrane potential constant at
60 mV and
imposing a 500-ms test pulse to 60 mV, every 20 s. Following
voltage clamp initiation, currents were monitored for a 5-min
equilibration period; oocytes in which currents spontaneously varied in
amplitude over this period were discarded. All currents are shown after
leak subtraction. Results are expressed as means ± S.E.
Statistical significance was determined using Student's t
test for paired observations.
Chemicals--
Isoproterenol and forskolin were obtained from
Sigma. Iberiotoxin was purchased from Peptide Institute (Osaka, Japan),
and the PKA regulatory subunit was obtained from Promega (Madison, WI).
50 nl (3.125 units) of PKA regulatory subunit peptide was injected for
kinase inhibition. The peptide PKG inhibitor used was the 7-residue
peptide corresponding to histone H2B (residues 29-35), obtained from
Calbiochem.
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RESULTS |
Reconstitution of
2-Adrenergic Stimulation of Maxi-K
Channels--
Xenopus oocytes expressing the human
2-adrenergic receptor (
2AR) and either
the pore-forming (
) subunit, or the pore-forming and regulatory (
+
) subunits of the human maxi-K channel were examined in
two-electrode voltage-clamp experiments. Functional expression of
channel proteins was determined by activation of iberiotoxin sensitive
outward currents. As reported previously (30, 31), average currents
recorded from oocytes injected with both channel subunit cRNAs were
larger at all voltages than those recorded from oocytes injected with
hSlo alone (3225.8 ± 256.9 nA (n = 25)
versus 2671.9 ± 365.5 nA (n = 14) at
60 mV). As shown in Fig. 1, application
of the
-adrenergic receptor agonist isoproterenol (ISO) to oocytes
injected with hSlo, hKV,Ca
, and h
2AR
cRNAs resulted in a rapid stimulation of maxi-K current monitored with
a clamp step to 60 mV. The current was maximally stimulated 78 ± 11 s after ISO addition (n = 17; current examined at 20-s intervals), and fell to prestimulation levels upon removal of
the agonist. Iberiotoxin, a highly specific peptidyl inhibitor of
maxi-K channels (36), blocked almost all of the
voltage-dependent current, and isoproterenol failed to
activate a current in the presence of the maxi-K antagonist (Fig.
1A). As shown in Table I, the
mean current stimulation by 10 µM ISO was 33.3 ± 5.1% (60 mV), whereas ISO had no effect on oocytes injected with cRNA encoding only the channel subunits (without h
2AR), or on
oocytes injected with only h
2AR (without maxi-K channel
genes). Qualitatively similar results were observed in oocytes injected
with only hSlo and h
2AR cRNAs (Fig. 1B),
i.e. ISO stimulated
subunit maxi-K currents to a degree
similar to that for
/
currents (25.8 ± 5.3 versus 33.3 ± 5.1%; Table I). In both cases, ISO
stimulation was not associated with an alteration in current activation
or decay kinetics; rather, stimulated
/
or
maxi-K currents
were scaled increases of control currents. Current stimulation was observed over the entire voltage range examined, resulting in a marked
shift in the conductance/voltage relationship toward more negative
potentials (Fig. 1C).

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Fig. 1.
Stimulatory coupling between heterologously
expressed 2 receptors and maxi-K channels. A,
ISO (10 µM) stimulates currents recorded from a
voltage-clamped Xenopus oocyte coexpressing human
2-adrenergic receptor and maxi-K channel ( and subunits) genes. Data points show peak current at voltage clamp steps
from 60 to +60 mV at 20-s intervals. ISO application resulted in an
increase in the current amplitude within 1 min. Outward current was
almost completely abolished by 100 nM iberiotoxin
(IBTX), after which no current stimulation was observed.
B, representative traces before and after stimulation with
ISO (10 µM). Traces at left were obtained in
an experiment in which both channel subunits were expressed, whereas
traces at right were from the channel subunit alone. ISO
resulted in a scaled increase in the maxi-K current. Currents were
elicited by a 500-ms step depolarization from 60 mV to +60 mV;
scale bars are 1 µA and 100 ms. C, ISO
stimulation resulted in an increase in the maxi-K conductance at all
potentials. Experiment shows the mean conductance from eight
experiments in which a current/voltage analysis was performed.
Solid lines are Boltzmann fits to the data; error
bars indicate standard error.
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Table I
Effect of isoproterenol (ISO) and forskolin on currents from
oocytes expressing various maxi-K channel subunits and
2-receptor combinations
Data were obtained by averaging five currents at peak stimulation and
calculating the percent stimulation above the average of five currents
immediately before drug application. Diagrams at left refer to injected
cRNAs; 2 refers to the human 2 adrenergic
receptor, to the human maxi-K pore-forming subunit, and to the
human maxi-K regulatory subunit. *, p < 0.05; **,
p < 0.01; ***, p < 0.001.
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Mechanism of
2AR/Maxi-K Channel Coupling--
To
determine whether
2AR/maxi-K channel coupling occurs via
stimulation of adenylyl cyclase and activation of cAMP-PK, we first
examined the stimulation of maxi-K currents by forskolin in oocytes
expressing
/
or
channel subunits. As shown in Fig. 2A, forskolin (10 µM) stimulated maxi-K currents to a degree that was
similar to that observed with ISO (34.9 ± 7.8% stimulation at 60 mV), although the time to peak was substantially slower than observed
following exposure to ISO (466 ± 35 s; n = 13). As with ISO, forskolin application did not result in an alteration of current kinetics, but in a scaled increase in the current at all
voltages (Fig. 2B), and stimulated both
/
and
maxi-K currents to a similar degree (Table I). Taken together with
results obtained with ISO, these data indicate that the regulatory
target associated with
2AR receptor stimulation is the
channel
subunit.

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Fig. 2.
Stimulation of heterologously expressed
maxi-K channels by forskolin. A, application of forskolin
(10 µM) stimulated heterologously expressed maxi-K
currents ( and subunits), as shown by the plot of peak current.
The maximum stimulatory effect was observed after 10 min and was poorly
reversible. No current augmentation was observed in the presence of
iberiotoxin (100 nM). Currents were obtained by voltage
clamp steps from 60 to +60 mV at 20 s. B, current
traces from similar experiments show current augmentation before and
after peak forskolin effect for expression of both channel subunits or
the subunit alone. As with ISO, stimulated currents were scaled
increases in the basal current. Currents shown were from 500 ms clamp
steps to +60 mV; scale bars indicate 1 µA and 100 ms.
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It has been suggested that
2AR-mediated stimulation of
maxi-K currents is associated with stimulation of
cGMP-dependent protein kinase (cGMP-PK) rather than cAMP-PK
(23), and that stimulation may occur via a direct interaction between G
protein subunits and the channels (12, 13). We directly examined the
role of cAMP-PK in
2AR/maxi-K coupling by intracellular
injection cAMP-PK and cGMP-PK regulatory peptides. As shown in Fig.
3, ISO stimulation was quite similar
before and after sham injection of oocytes, whereas injection of the
PKA regulatory subunit markedly reduced ISO stimulation. In three of
four experiments, injection of the regulatory subunit inhibited the
previous ISO stimulation by 57% (28.2 ± 9.6 versus
12.2 ± 2.8% stimulation before and after injection of peptide),
clearly indicating a role for cAMP-PK in receptor/channel coupling.
Conversely, ISO stimulated maxi-K currents in oocytes injected with
cGMP-PK inhibitory peptide (final concentration 80 µM) by
23.9 ± 6.2% (n = 6, data not shown). This level
of stimulation was less than observed with ISO alone; however, the
degree of current stimulation was consistent with the
Ki of the peptide for cAMP-PK (predicted to inhibit
the kinase by about 13%). To determine whether the mode of
receptor/channel coupling was sensitive to the amount of expressed
receptor, we performed experiments in the amount of injected
2 receptor cRNA was reduced 10-fold (2.5 ng); however,
no current stimulation was observed under these conditions (-1.6 ± 0.4%, n = 6; data not shown).

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Fig. 3.
Inhibition of cAMP-PK markedly decreases
2AR/maxi-K stimulatory coupling. A, in
control stimulatory experiments, ISO (10 µM) application
repeatedly augmented maxi-K currents. B, following initial
ISO stimulation, oocyte was injected with 3.125 units of cAMP-PK
regulatory subunit. Subsequent applications of ISO were markedly less
effective in stimulation maxi-K currents. All currents were obtained by
step depolarizations from 60 to +60 mV at 20-s intervals.
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The mammalian homologues of dSlo do not share the optimal cAMP-PK
phosphorylation site of dSlo (24, 37), but share a conserved, less
cAMP-PK-specific phosphorylation site at approximately the same
position (e.g. 866-869, GenBank accession no. U11058), as
well as several other potential phosphorylation sites. To determine the
relevant phosphorylation site associated with physiological
2AR stimulation, we used site-directed mutagenesis to
replace serine 869 with alanine (hSlo(
S869A)), and coexpressed the
mutant channel subunit with the
subunit and the
2AR.
As shown in Fig. 4, hSlo(
S869A)/
currents were almost completely insensitive to modulation by ISO; ISO
increased the current amplitude by 5.5 ± 1.3% (n = 9), whereas exposure to forskolin still produced a significant
increase in current magnitude (16.0 ± 3.9%; n = 9), although substantially less than in the wild type channels (Table I). The amplitude and kinetics of hSlo(
S869A)/
currents were not
different from hSlo(wt)/
currents (data not shown). These data
indicate that cAMP-dependent phosphorylation occurs at
Ser-869.

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Fig. 4.
Mutation of serine 869 abolishes
2AR/maxi-K stimulatory coupling. ISO application to
an oocyte injected with wild type 2AR and maxi-K subunit genes, and a mutant maxi-K subunit gene ( S869A). Whereas
ISO (10 µM) failed to stimulate the current, forskolin
(10 µM) produced a modest increase in current magnitude
with kinetics similar to that observed in wild type experiments. The
experimental protocol was the same as described for Fig. 1.
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DISCUSSION |
The present study demonstrates the reconstitution of
2-adrenergic receptor stimulatory coupling to maxi-K
channels by heterologous expression of receptor and channel RNAs in
X. laevis oocytes. Isoproterenol and forskolin increased
maxi-K currents in oocytes expressing either the pore-forming
subunit alone, or co-expressing the
and regulatory
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
2 receptors but not maxi-K channels,
and since forskolin, but not ISO, stimulated currents in oocytes
expressing maxi-K channels, but not
2 receptors (Table
I). Moreover, virtually all of the outward current was inhibited by
iberiotoxin (Figs. 1 and 2), a selective peptidyl inhibitor of maxi-K
channels (36). 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
2 receptors and maxi-K channels.
Further, since stimulation was achieved with expression of only the
2 receptor and the
subunit and the degree of
stimulation was similar with or without the regulatory
subunit
(Table I), these studies localize the modulatory site to the channel
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 (7, 38, 39).
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 (5, 6, 11, 15, 16), cGMP-dependent phosphorylation (17, 18,
20, 21, 40), cGMP-dependent dephosphorylation (19, 22, 23),
by G protein subunits (12, 13), and direct stimulation by NO (41). We
used the heterologous expression system and site-directed mutagenesis
to identify the mechanism of
adrenergic stimulatory coupling to
maxi-K channels and to identify the relevant channel modulatory site.
Our data clearly implicate cAMP-PK in
2 receptor/channel
stimulatory coupling since injection of the regulatory subunit of
cAMP-PK disrupts
2 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. 3B), 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
2 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. 24), and mutations at
this site were later found to prevent channel activation by exogenous
PKA in inside-out patches (42). 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 (43).
However, as many as 10 other potential cAMP-PK consensus phosphorylation sites exist throughout the coding region of the
subunit (37). To ascertain the physiologically relevant phosphorylation site, we mutated serine 869 to alanine, and coexpressed the mutant
subunit with the wild type
subunit and the
2
receptor. This single mutation markedly reduced, but did not abolish,
2 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
2
receptor/maxi-K channel stimulatory coupling occurring by cAMP-PK
phosphorylation of serine 869.
Finally, the stimulatory effect of both the
2 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
2 receptor stimulation on cardiac
sodium channel
subunits heterologously expressed in
Xenopus oocytes (44). 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 (44). 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
2-adrenergic receptor/maxi-K channel
stimulatory coupling. Coupling involves a cAMP-PK-dependent
phosphorylation of the channel
subunit; mutation of one consensus
cAMP-PK phosphorylation site (serine 869) almost eliminates
2-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
2 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.