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The Journal of Neuroscience, April 15, 1998, 18(8):2834-2841
Protein Kinase C Disrupts Cannabinoid Actions by Phosphorylation
of the CB1 Cannabinoid Receptor
D. E.
Garcia1, 3,
S.
Brown2,
B.
Hille1, and
K.
Mackie1, 2
Departments of 1 Physiology and Biophysics and
2 Anesthesiology, University of Washington, Seattle,
Washington 98195, and 3 Departamento de Fisiologia,
Facultad de Medicina, Universidad Nacional Autonoma de Mexico, CP 04510 Mexico DF, Mexico
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ABSTRACT |
We have found that phosphorylation of a G-protein-coupled receptor
by protein kinase C (PKC) disrupts modulation of ion channels by the
receptor. In AtT-20 cells transfected with rat cannabinoid receptor
(CB1), the activation of an inwardly rectifying potassium current
(Kir current) and depression of P/Q-type calcium channels by cannabinoids were prevented by stimulation of protein kinase C by
100 nM phorbol 12-myristate 13-acetate (PMA). In contrast, activation of Kir current by somatostatin was unaffected,
and inhibition of calcium channels was only modestly attenuated. The possibility that PKC acted by phosphorylating CB1 receptors was confirmed by demonstrating that PKC phosphorylated a single serine (S317) of a fusion protein incorporating the third intracellular loop
of CB1. Mutating this serine to alanine did not affect the ability of
CB1 to modulate currents, but it eliminated disruption by PMA,
demonstrating that PKC can disrupt ion channel modulation by receptor
phosphorylation.
Key words:
cannabinoid; G-protein-coupled receptor; calcium channel; inwardly rectifying potassium channel; protein kinase C; phosphorylation
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INTRODUCTION |
Neurons are continuously exposed to
neuromodulators that bind G-protein-coupled receptors (GPCRs) and alter
neuronal excitability (Hille, 1994 ). Many modulators decrease
electrical excitability by activating inwardly rectifying potassium
currents (Kir currents) and inhibiting voltage-dependent
calcium currents (ICa), often via
pertussis toxin (PTX)-sensitive G-proteins (North, 1989). Other
modulators stimulate phospholipase C, generating diacylglycerol and
IP3, which increase intracellular calcium levels and
activate protein kinase C (PKC) (Huang and Huang, 1993). Activation of PKC often enhances neurotransmission (Malenka et al., 1986; Capogna et
al., 1995). The mechanism and the interaction and balance between neuronal stimulation and inhibition by these two classes of receptors can now be studied directly.
Activation of protein kinase C attenuates the modulation of N- and
P/Q-type calcium currents and of Kir currents by
G-protein-coupled receptors. For all three channels, the fast
modulation is mediated by direct binding of G-protein   subunits
to the channel itself (Reuveny et al., 1994; Krapivinsky et al., 1995;
Herlitze et al., 1996; Ikeda, 1996; De Waard et al., 1997; Zamponi et
al., 1997). The attenuation by PKC could therefore involve
phosphorylation of the receptor, the G-protein, or the channel. Much
work has analyzed attenuation of the modulation of N-type (class B)
calcium current by PKC (Golard et al., 1993; Swartz, 1993; Zhu and
Ikeda, 1994; Shapiro et al., 1996; Zamponi et al., 1997), which is
thought to be attributable to phosphorylation of the linker between
domains I and II of the  1 channel subunit (Zamponi et
al., 1997). Much less is known about attenuation of modulation of
identified P/Q-type (class A) calcium currents. Results from oocyte
experiments raise the possibility that effects of PKC on P/Q-type
calcium channels are not mediated by phosphorylation of their
1 subunit (Stea et al., 1995). Protein kinase C can also
enhance neuronal excitability by inhibiting potassium current
Kir currents or their activation (Takano et al., 1995;
Velimirovic et al., 1995). One possible explanation for these varied
actions of PKC is that it disrupts a signaling step proximal to ion
channels.
Cannabinoids produce their behavioral effects as a consequence of
binding to a G-protein-coupled receptor, the CB1 cannabinoid receptor
(Matsuda et al., 1990; Pertwee, 1993). The abundance of these receptors
and the discovery of several endogenous ligands (Devane et al., 1992;
Stella et al., 1997) suggests that cannabinoid neuromodulatory systems
play important physiological roles (Di Marzo et al., 1994). Indeed,
endogenous cannabinoids are released during chronic painful
inflammation and also play a role in some types of memory (Terranova et
al., 1996; Richardson et al., 1997). Cellular consequences specifically
linked to CB1 receptor activation include inhibition of adenylyl
cyclase and modulation of ion channels (Pertwee, 1993). It is not known
whether PKC activation disrupts cannabinoid modulation of ion currents.
In this study we used AtT-20 cells, which express P/Q-type calcium
current (Mackie et al., 1995), two species of Kir channel
protein, GIRK1 and GIRK2 (J. Redell, B. L. Tempel, and K. Mackie,
unpublished results), and modest N-type calcium currents (Mackie et
al., 1995). We compared the effects of PKC activation on modulation of
P/Q-type calcium channels and of Kir current in cells
stably transfected with rat CB1 cannabinoid receptor and endogenously
expressing somatostatin receptors. Activation of protein kinase C
strongly suppressed modulation of these currents by cannabinoids but
only weakly suppressed their modulation by somatostatin. The effect of
protein kinase C appears to be a result of phosphorylation of a serine
in the third intracellular loop of CB1.
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MATERIALS AND METHODS |
Cell culture. AtT-20 cells stably transfected with
rat CB1 (Mackie et al., 1995) or CB1-S317A were plated on
poly-L-lysine-coated coverslips and grown in DMEM, 10%
heat-inactivated horse serum, 1:200 penicillin/streptomycin, and 400 µg/ml G418 in a humidified environment with 5% CO2 at
35°C. Cells were passaged with 0.05 mg/ml trypsin in PBS and used
within 15 passages after the initial clones were isolated.
Electrophysiological recording. Currents were recorded using
the whole-cell voltage-clamp technique (Hamill et al., 1980). Pipettes
were pulled from microhematocrit glass (VWR Scientific) and were
fire-polished. For recording, a coverslip containing cells was
transferred to a 200 µl chamber that was constantly perfused (1-2
ml/min) with the appropriate external solution. Solution reservoirs
were selected by means of a series of solenoid valves, and solution
changes were accomplished in <30 sec. Voltage protocols were
generated, and data were digitized, recorded, and analyzed using
BASIC-FASTLAB (Indec Systems, Capitola, CA). Liquid junction potentials
were uncorrected.
For measuring potassium currents, the pipette solution contained (in
mM): 120 KCl, 10 HEPES, 5 EGTA, 3 MgCl2,
3 Na2ATP, 0.3 GTP, and 0.1 leupeptin, pH 7.2, with KOH,
whereas the external solution contained (in mM): 40 KCl,
110 N-methylglucamine, 1 CaCl2, 25 HEPES,
and 10 glucose, pH 7.35, with NaOH. Fatty acid-free bovine serum
albumin (BSA; 3 µM) was added to decrease adsorption of
cannabinoids to surfaces. The Kir current was defined as
that component of the current sensitive to 1 mM
Ba2+ elicited during the final 150 msec of a 250 msec hyperpolarizing pulse to  100 mV from a holding potential of 45
mV. Currents were sampled at 1 kHz. Because the magnitude of the
Kir current was dependent on cell size, aggregate current
data are presented as current densities normalized to cell
capacitance.
For measuring barium currents, the pipette solution contained (in
mM): 100 CsCl, 40 HEPES, 10 EGTA, 5 MgCl2, 3 Na2ATP, 0.3 GTP, and 0.1 leupeptin, pH 7.2, with CsOH, whereas the external solution contained
(in mM): 140 NaCl, 10 BaCl2, 5 CsCl, 1 MgCl2, 10 HEPES, and 10 glucose, pH 7.3, with NaOH.
Tetrodotoxin (200 nM) was added to block voltage-dependent
sodium currents, 2 µM nifedipine was added to block
L-type calcium currents, and BSA was added to decrease adsorption of
the cannabinoids. IBa was measured near the end
of a 25 msec depolarizing pulse to 0 mV from a holding potential of
90 mV and was defined as the component of current sensitive to 100 µM CdCl2. Currents were sampled at 4 kHz.
To control for possible variations of response with passage number and
to avoid one source of systematic bias, experimental and control
measurements were alternated whenever possible, and concurrent controls
were always performed. Where appropriate, data are expressed as
mean ± SE.
Fusion protein generation, purification, and
phosphorylation. All fusion proteins were generated in a similar
manner using PCR. As an example, the procedure used to generate the IC3
fusion protein follows. An amplicon incorporating the third
intracellular loop of the rat CB1 receptor was generated
with the following sense
(5'-CGGGATCCTGGAAGGCTCACAGCCAC) and
antisense (5'-GCGAATTCGGTTTTGGCCAGGCTAAT) primers and
digested with EcoRI and BamHI. After ligation
into pGEX-3X (Pharmacia, Piscataway, NJ) and transformation, colonies were screened for expression of the appropriate length fusion protein.
Mutations were made by direct substitution in the sense primer (S304A)
or by the PCR overlap technique (S317A and S323A) (Ho et al., 1989) and
were verified by sequencing. Glutathione S-transferase (GST)
fusion proteins were purified from bacterial lysates using
glutathione-Sepharose (Pharmacia).
Phosphorylation of purified GST proteins was performed as described
previously, with 50 ng of purified rat brain PKC (a mixture of ,
, and isoforms). Phosphorylated proteins were separated on 10%
polyacrylamide gels and identified by autoradiography. Phosphoamino
acid analysis was performed using standard techniques (Mackie et al.,
1989).
Materials. Tissue culture reagents were from Life
Technologies (Gaithersburg, MD); BSA and staurosporine were from Sigma
(St. Louis, MO); leupeptin was from Bachem; somatostatin was from
Peninsula Labs; and bisindolylmaleimide I (HCl salt),
bisindolylmaleimide V, TTX, PMA, 4-phorbol, and GTP were from
Calbiochem (La Jolla, CA). WIN 55,212-2 was a gift from Sterling
Research Group.
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RESULTS |
Stimulation of PKC attenuates cannabinoid activation of
Kir current
Our electrophysiological experiments were designed to examine
effects of PKC on receptor-mediated modulation of ion channels. To
activate PKC, we exposed cells to a bath solution containing 100 nM PMA at 20-22°C for 10 min. Because the actions of PMA
generally do not reverse in tens of minutes, it was not necessary to
include PMA in any of the subsequent solutions. Control experiments
were done with cells similarly exposed to 100 nM
4-phorbol, an inactive analog. In both cases, cells were
patch-clamped within 5 min of ending the PMA or 4-phorbol
incubation.
In cells preincubated with 4-phorbol, 100 nM WIN
55,212-2 activated Kir current in the usual manner in
AtT-20 cells stably expressing rat CB1 cannabinoid receptor (Figs.
1A,
2) (cf. Mackie et al., 1995). However, in
cells treated with PMA, WIN 55,212-2 activated Kir current
only minimally (Figs. 1B, 2). Incubation of the cells
for 5 min with a 1 µM concentration of the protein kinase
inhibitor staurosporine before incubation with PMA prevented the PMA
effect, providing further evidence that PMA was activating a protein
kinase (Fig. 2). A briefer stimulation of protein kinase C by applying
phorbol ester for 2.5 min after establishing the whole-cell recording
configuration produced similar but weaker effects. In the cells treated
with 4-phorbol, a 100 nM concentration of the
cannabinoid agonist WIN 55,212-2 increased the normalized Kir current by 5.2 ± 0.9 pA/pF (n = 23), whereas in cells treated similarly with PMA, 100 nM
WIN 55,212-2 increased the Kir current by only 2.2 ± 0.5 pA/pF (n = 16) (data not shown). PMA applied in
this manner had no effect on the Kir current before
application of WIN 55,212-2 (data not shown).
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Figure 1.
Stimulation of protein kinase C attenuates
activation of Kir current by cannabinoids but not by
somatostatin. Cells were exposed to 100 nM 4-phorbol or
100 nM PMA for 10 min at room temperature and transferred
to the recording chamber, and potassium currents were recorded.
Kir current was defined as the component of the current
inhibited by 1 mM Ba2+. A,
Left, Mean current activated by a 250 msec hyperpolarization to
100 mV from a holding potential of 45 mV is plotted versus time in
a cell preincubated with 100 nM 4-phorbol for 10 min.
Bath application of 100 nM of the cannabimimetic WIN
55,212-2 (WIN) for the indicated time greatly
increases the component of the current sensitive to 1 mM
Ba2+. The increase in inwardly rectifying potassium
current is defined as indicated to the left
(Kir current). Right, Individual current
traces taken at the times indicated. B, Left, In a cell
pretreated with 100 nM PMA (see Results), 100 nM WIN 55,212-2 slightly increases the barium-sensitive
current. Right, Individual current traces taken at the
times indicated. C, Bath application of 10 nM somatostatin (SOM) to a cell
pretreated with 100 nM 4-phorbol for 10 min elicits a
large increase in the barium-sensitive inward current.
D, In a cell pretreated with 100 nM PMA, 10 nM somatostatin still results in a large increase in the
barium-sensitive current.
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Figure 2.
Protein kinase C stimulation markedly reduces the
activation of Kir current by cannabinoids but not by
somatostatin. Summary of the experiments illustrated in Figure 1. The
Kir current activated by 100 nM WIN 55,212-2 (WIN) and 10 nM somatostatin
(SOM) was normalized to cell capacitance and
compared for the following conditions: 100 nM 4-phorbol,
100 nM PMA (PMA), and 1 µM
staurosporine (STAU). The number of cells tested
for each condition is in parentheses.
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Somatostatin also activates a Kir current in AtT-20 cells
(Pennefather et al., 1988). More current is activated in
these cells than with cannabinoid or muscarinic agonists, despite the
greater density of cannabinoid compared with somatostatin receptors
(Felder et al., 1995). This observation suggests that the coupling of endogenous somatostatin receptors to Kir current in these
cells differs from the coupling of transfected CB1 or endogenous
m4 receptors to these channels. Thus it was of interest to
investigate the effect of protein kinase C stimulation on activation of
Kir current by somatostatin. Surprisingly, preincubation
with PMA had little effect on activation of Kir current by
10 nM (Figs. 1D, 2) or by 250 nM somatostatin [10.1 ± 1.6 pA/pF; n = 12 (4-phorbol); vs 9.3 ± 1.4 pA/pF; n = 10 (PMA); data not shown].
Stimulation of PKC attenuates modulation of P/Q-type calcium
currents by cannabinoids
AtT-20 cells also express prominent L-, P/Q-, and R-type
high-voltage-activated calcium currents (cf. Mackie et al., 1995). Of
these, the P/Q-type current is the major current inhibited by
cannabinoids and somatostatin (Mackie et al., 1995). Of note, N-type
calcium current, defined as inhibited by 1 µM
 -conotoxin GVIA (-CgTX GVIA), is a minor (<10%) component of
the high-voltage-activated calcium current in these cells (Mackie et
al., 1995). Using barium as the charge carrier through calcium channels
and nifedipine to block current through L-type calcium channels, we
determined whether cannabinoid inhibition of P/Q-type calcium channels
was blunted by protein kinase C stimulation. Protein kinase C was stimulated using the same preincubation protocols as in the
Kir current experiments. In cells pretreated with
4-phorbol, 100 nM WIN 55,212-2 inhibited
IBa (Figs.
3A,
4), as expected from previous studies
(Mackie et al., 1995). However, pretreatment with PMA markedly
attenuated the inhibition by WIN 55,212-2 (Figs. 3B, 4).
Further evidence that the attenuation was mediated by protein kinase C
included the findings that it was prevented by previous treatment with
1 µM staurosporine or a 100 nM concentration of the more specific protein kinase C inhibitor bisindolylmaleimide I
(Fig. 4). The inactive analog bisindolylmaleimide V did not inhibit the
PMA effect (Fig. 4).
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Figure 3.
Activation of protein kinase C greatly reduces
inhibition of P/Q-type calcium currents by cannabinoids but not by
somatostatin. Cells were exposed to 100 nM 4-phorbol or
100 nM PMA for 10 min at room temperature and transferred
to the recording chamber, and barium currents flowing through calcium
channels were recorded. A, Left, Mean current activated
by a 25 msec depolarization to 0 mV from a holding potential of 80 mV
is plotted against time in a cell preincubated for 10 min with 100 nM 4-phorbol. Bath application of 100 nM of
the cannabimimetic WIN 55,212-2 (WIN) for the
indicated time inhibits ~40% of the barium current [defined as the
fraction of the current sensitive to 100 µM
CdCl2 (Cd)]. Right,
Individual current traces taken at the times indicated. B,
Left, In a cell pretreated with 100 nM PMA (see
text), 100 nM WIN 55,212-2 has a minimal effect on the
barium current. Right, Individual current traces taken
at the times indicated. C, Bath application of 10 nM somatostatin (SOM) to a cell
pretreated with 100 nM 4-phorbol for 10 min inhibits
~50% of the barium current. D, In a cell pretreated
with 100 nM PMA, 10 nM somatostatin still
inhibits a significant fraction of the barium current.
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Figure 4.
Protein kinase C stimulation greatly reduces the
inhibition of barium current by cannabinoids but not by somatostatin.
Summary of the experiments illustrated in Figure 3. The barium current
activated by 100 nM WIN 55,212-2 (WIN) and 10 nM somatostatin
(SOM) was compared for the following conditions:
100 nM 4-phorbol, 100 nM PMA
(PMA), 1 µM staurosporine
(STAU), 100 nM bisindolylmaleimide I
(BIS), and 100 nM bisindolylmaleimide V
(IBIS). The number of cells tested for each condition is
indicated in parentheses.
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We repeated the same protocols with somatostatin as the agonist.
Protein kinase C activation decreased inhibition of the barium current
by somatostatin less effectively than it decreased inhibition by
cannabinoid (Fig. 4). Preincubation with PMA reduced barium current
inhibition by 10 nM somatostatin from 45 ± 3%
(n = 18) to 28 ± 3% (n = 17)
(p < 0.0005) (Figs. 3D, 4). Because
N-type calcium current comprises only 10% of the
high-voltage-activated calcium current, it is likely that the reduction
in modulation by PMA reflects decreased inhibition of both P/Q- and
N-type calcium channels. Again, further evidence for involvement of PKC
comes from the observation that 1 µM staurosporine and
100 nM bisindolylmaleimide I prevented the PMA effect,
whereas bisindolylmaleimide V was ineffective (Fig. 4).
Protein kinase C phosphorylates a fusion protein containing the
third intracellular loop of the CB1 receptor
Because attenuation by PKC was much greater for
cannabinoid-mediated actions than for somatostatin-mediated ones, we
reasoned that CB1 receptors might be directly phosphorylated by PKC. We undertook a biochemical approach to test this possibility by
constructing glutathione S-transferase fusion proteins for
each of the intracellular domains of the rat CB1 receptor. The fusion
proteins for intracellular loops I and III (IC3) could be
phosphorylated by purified protein kinase C, whereas fusion proteins
for the second intracellular loop and C terminus could not (data not
shown). Because the third intracellular loop is important for signaling
in many G-protein-coupled receptors, further efforts focused on this
loop. Phosphoamino acid analysis demonstrated that the IC3 fusion
protein was phosphorylated on serine (data not shown). IC3 contains
three serines, so three fusion proteins were constructed; in each a
different serine was mutated to alanine (S304A, S317A, S323A). Whereas
the mutant S317A protein was a poor substrate for PKC, the S304A and
S323A proteins were phosphorylated as effectively as the wild-type IC3
fusion protein (Fig. 5). The low amounts
of phosphorylation seen in the S317A mutant suggest that PKC can
phosphorylate at least one of the two other serines in IC3, but to a
lesser degree. These results suggested that S317 is the best PKC site
in the third intracellular loop and that its phosphorylation might be
responsible for the PKC-mediated disruption of CB1 signaling seen in
the electrophysiological experiments. To test this possibility, we
mutated S317 to alanine in the full-length rat CB1 and stably expressed
it in AtT-20 cells.
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Figure 5.
Protein kinase C phosphorylates GST-IC3 fusion
proteins on the serine corresponding to serine 317 in rat CB1. Fusion
proteins were phosphorylated by purified rat brain PKC (50 ng) for 30 min and separated by SDS-PAGE. Phosphorylated proteins were identified
by autoradiography of the gel. Wild-type third intracellular loop,
S304A, and S323A fusion proteins were phosphorylated to a similar
extent, whereas the S317A fusion protein was minimally phosphorylated.
Arrowhead, GST-IC3 fusion protein.
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Mutation of serine 317 in rat CB1 abolishes PKC-mediated disruption
of CB1 signaling
We first tested the coupling of CB1-S317A to Kir
current. Figure 6, A and
C, shows that WIN 55,212-2 could activate Kir
current in AtT-20 cells expressing the CB1-S317A mutant as effectively as in cells expressing wild-type CB1 (6.3 ± 0.4 pA/pF;
n = 6; vs 6.7 ± 0.7 pA/pF; n = 5). Furthermore, whereas preincubation with 100 nM PMA
strongly reduced Kir current activation by WIN 55,212-2 in
cells expressing the wild-type CB1 receptor, it had no effect on cells
expressing CB1-S317A (1.7 ± 0.2 pA/pF; n = 8; vs
6.3 ± 0.4 pA/pF; n = 10) (Fig.
6B,C). Thus PKC appears to disrupt
activation of Kir current by the CB1 receptor by
phosphorylating the receptor on serine 317.
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Figure 6.
The CB1-S317A mutant receptor activation of
Kir current is not attenuated by protein kinase C. A, Time course of Kir current activation by
100 nM WIN 55,212-2 in a CB1-S317A cell after preincubation
with 100 nM 4-phorbol (Con).
Inset, Current traces from the indicated times.
B, Time course of Kir current activation by
100 nM WIN 55,212-2 after preincubation with 100 nM PMA to stimulate protein kinase C. Inset,
Current traces from the indicated times. C, Comparison
of Kir current activation in cells expressing CB1 or
CB1-S317A. The number of cells for each condition is in
parentheses.
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We next tested the coupling of CB1-S317A to calcium channels. Figure
7, A and C, shows
that cannabinoid agonist inhibited calcium channels in AtT-20 cells
expressing the CB1-S317A mutant to the same extent as in cells
expressing wild-type CB1 (30.8 ± 1.5%; n = 5; vs
31.1 ± 1.1%; n = 5). However, whereas
preincubation with 100 nM PMA strongly reduced inhibition
of the calcium channels by WIN 55,212-2 in cells expressing wild-type
CB1, the same treatment had a much smaller effect on cells expressing
CB1-S317A (7.2 ± 3.6%; n = 7; vs 24.6 ± 2.3%; n = 11) (Fig. 7B,C). Evidently PKC strongly disrupts the inhibition of calcium channels by the CB1 receptor when it phosphorylates the CB1 receptor on serine 317.
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Figure 7.
The CB1-S317A mutant receptor inhibition of
calcium channels is resistant to PKC. A, Time course of
barium current inhibition by 100 nM WIN 55,212-2 in a
CB1-S317A cell after preincubation with 100 nM 4-phorbol
(Con). Inset, Current traces from the
indicated times. B, Time course of barium current
inhibition by 100 nM WIN 55,212-2 after preincubation with
100 nM PMA to stimulate protein kinase C. Inset, Current traces from the indicated times.
C, Comparison of barium current inhibition
(Inh) in cells expressing CB1 or CB1-S317A. The small
residual PMA effect in the S317A mutant was abolished by -conotoxin
GVIA (GVIA). The number of cells for each condition is
in parentheses.
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Although most of the attenuation of CB1 inhibition of total barium
current was prevented by the S317A mutation, a small effect of PMA
remained (Fig. 7C). We hypothesized that this might reflect CB1 modulation of the small number of N-type calcium channels present
in these cells (Mackie et al., 1995). PKC attenuation of this kind of
modulation is likely to be a consequence of phosphorylation of the
1B channel subunit and thus would not be rescued by
mutation of the CB1 receptor. To test this possibility we blocked the
N-type current by 1 µM -CgTX GVIA. Figure
7C shows that in the -CgTX GVIA-treated CB1-S317A cells,
WIN inhibited the barium current to a similar extent in the
4-phorbol- and PMA-treated cells. These results suggest that the
modest reduction by PMA of barium current inhibition by cannabinoids in
the S317A mutant is attributable to action on N-type calcium channels,
consistent with the hypothesis that by phosphorylating the linker
between domains I and II of 1B (Stea et al., 1995;
Zamponi et al., 1997), PKC weakens G-protein subunit binding to
the channel.
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DISCUSSION |
We have discovered a potent physiological consequence of
phosphorylating cannabinoid receptors by protein kinase C. Modulation of Kir current and P/Q-type calcium channels by
cannabinoids is exquisitely sensitive to inhibition by protein kinase
C, and the inhibition is largely attributable to the phosphorylation of
the CB1 receptor on a single serine in the third intracellular
loop.
This sensitivity provides a mechanism for neuromodulators that activate
protein kinase C (such as substance P or glutamate acting at
metabotropic glutamate receptors) to attenuate the effects of
cannabinoids on neuronal excitability. The CB1 receptor mediates inhibition of adenylyl cyclase (Howlett, 1985) and calcium currents (Mackie and Hille, 1992; Mackie et al., 1995) and activation of potassium currents (Henry and Chavkin, 1995; Mackie et al., 1995). Because most of these actions tend to decrease the excitability of a
neuron and synaptic transmission, stimulation of PKC provides a
mechanism whereby neurotransmitters coupled with protein kinase C can
restore neuronal excitability and synaptic strength when endogenous
cannabinoid levels are high. Whereas the CB1 receptor was the subject
of these studies, our results probably apply to other G-protein-coupled
receptors. It will be interesting to learn how widespread this kind of
crosstalk is.
It is well established that modulation of N- and P/Q-type calcium
channels by G-protein-coupled receptors can be disrupted by activation
of protein kinase C (Golard et al., 1993; Swartz, 1993; Zhu and Ikeda,
1994; Stea et al., 1995; Shapiro et al., 1996). For 1B
(N-type) calcium channels, the suppression has been attributed to
direct phosphorylation of the channel. PKC phosphorylates the domain
I-II linker of the 1B subunit at a site (or sites)
possibly involved in binding of G-protein subunits (Stea et al.,
1995; Zamponi et al., 1997). This covalent modification of the target
channel would be expected to suppress actions of free G- subunits
regardless of which agonist and receptor had generated them. However,
with P/Q-type channels, we have found that the effects of cannabinoids
are very sensitive to PKC activation, whereas those of somatostatin are
affected slightly. [Because N-type current is only a minor (<10%)
component of the calcium current in these cells, and somatostatin
inhibits >40% of the calcium current, most of the current that
somatostatin inhibits must be P/Q type.] The receptor-specific nature
of actions on P/Q current modulation suggests that activation of PKC
does not materially reduce the sensitivity of P/Q channels to G-
subunits. This conclusion is in accord with earlier findings that PKC
activation does not change the amplitude of currents from expressed
1A subunits, whereas it does change the amplitudes of
those from 1B subunits (Stea et al., 1995; Zamponi et
al., 1997). When interpreting our results, it must be kept in mind that
there are alternatively spliced forms of 1A (e.g.,
Sakurai et al., 1995) and that these may be affected differently from
the form(s) expressed in AtT-20 cells. The insensitivity of P/Q
channels in these cells to a direct action of PKC makes them an
effector of choice in future studies to characterize receptor-specific
actions of PKC.
Our results also provide insight into the role PKC has in inhibiting
activation of Kir currents. They are the first
demonstration that phosphorylation of a G-protein-coupled receptor can
disrupt its activation of a Kir current. In some studies,
activation of Kir currents by G-protein-coupled receptors
is found to be disrupted by PKC stimulation, whereas in others it is
not. In a nice series of studies in cultured nucleus basalis and locus
coeruleus neurons, Yamaguchi et al. (1990), Farkas et al. (1994), and
Velimirovic et al. (1995) found that Kir currents are
inhibited by substance P and neurotensin. In locus coeruleus neurons,
in which a Kir current is activated by somatostatin and
Met-enkephalin, activation is prevented by substance P. Whereas the
molecular identity of these currents has not been identified (Takano et
al., 1996), the actions of substance P (and probably neurotensin) in
nucleus basalis neurons are mediated by protein kinase C
(Takano et al., 1995). In contrast, activation of
Kir current in acutely isolated dorsal raphe neurons by the
5-HT1A receptor is not sensitive to stimulation of PKC
(Chen and Penington, 1996), demonstrating heterogeneity in the
interaction of PKC with Kir current activation. In AtT-20 cells we have also found receptor-specific effects; CB1-mediated activation of Kir current was sensitive to PKC stimulation,
whereas somatostatin activation was not. AtT-20 cells express mRNA for at least five subtypes of somatostatin receptor (Kaupman et al., 1993).
Presumably the G-protein-coupled signaling of at least one of the
somatostatin receptors expressed in AtT-20 cells is not interrupted by
PKC. By RT-PCR, AtT-20 cells express GIRK1 and GIRK2 but not GIRK3 or
GIRK4 (CIR) (J. Redell, B. L. Tempel, and K. Mackie, unpublished
results). GIRK1 and GIRK2 have multiple consensus sequences for PKC
phosphorylation. Nevertheless, activation of Kir currents
by cannabinoids in cells expressing the S317A-CB1 receptor and
activation by somatostatin were unaffected by stimulation of PKC. Thus
any phosphorylation by PKC of these GIRK proteins does not perturb
their activation by the G- subunits liberated after CB1 receptor
stimulation.
In summary, cannabinoid modulation of P/Q- and N-type calcium and
Kir channels is exquisitely sensitive to disruption by
protein kinase C stimulation. Based on our results, one might even
imagine that activation of phospholipase C-linked receptors could be
viewed as a potential "antidote" to psychoactive effects of
marijuana. Disruption of CB1-mediated P/Q-type calcium channel
inhibition and Kir current activation by PKC is chiefly a
consequence of phosphorylation of the third intracellular loop of the
CB1 receptor. This is the first demonstration that the phosphorylation
of a G-protein-coupled receptor by protein kinase C can disrupt the modulation of ion channels by the receptor. The high sensitivity of the
response provides a point of potential crosstalk and integration of
diverse signals, such as that between endogenous cannabinoids and
neuromodulators that activate phospholipase C-linked receptors. It
seems quite likely that modulation of channels by certain other G-protein-coupled receptors will be attenuated by PKC via a similar mechanism.
| |
FOOTNOTES |
Received Dec. 8, 1997; revised Jan. 22, 1998; accepted Jan. 22, 1998.
This work was supported by the W. M. Keck Foundation, an Alexander
von Humboldt Stiftung Fellowship, Universidad Nacional Autonoma de
Mexico DGAPA and Consejo Nacional de Ciencia y Tecnologia, and National
Institutes of Health Grants NS01588, DA08934, DA00286, and NS08174. We
thank A. Perdichezzi and B. Murphy for help with phosphorylation
assays, D. Anderson and L. Miller for technical help, W. Catterall, H. Cruzblanca, J. Isaacson, E. Kaftan, K.T. Kim, D.-S. Koh, J. Roche, and
M. Shapiro for reading this manuscript, Y. Lai and A. Nairn for
providing protein kinase C, and Sterling Winthrop for providing WIN
55,212-2.
Correspondence should be addressed to Dr. Ken Mackie, University of
Washington, Department of Anesthesiology, Box 356540, Seattle, WA
98195-6540.
| |
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Top
Abstract
Introduction
