From the Department of Pharmacology and Therapeutics,
Neuroscience Research Group, University of Calgary, Calgary, Alberta
T2N 4N1, Canada and the § Biotechnology Laboratory,
University of British Columbia,
Vancouver, British Columbia, V6T 1Z3 Canada
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ABSTRACT |
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The modulation of presynaptic calcium channel
activity by second messengers provides a fine tuning mechanism for
neurotransmitter release. In neurons, the activation of certain G
protein-coupled receptors reduces N-type channel activity by ~60%.
In contrast, activation of protein kinase C (PKC) results in an
approximately 50% increase in N-type channel activity, and subsequent
G protein inhibition is antagonized. Here, we describe the molecular
determinants that control the dual effects of PKC-dependent
phosphorylation. The double substitution of two adjacent PKC consensus
sites in the calcium channel domain I-II linker
(Thr422, Ser425) to alanines abolished
both PKC-dependent up-regulation and the PKC-G protein
cross-talk. The single substitution of Ser425 to glutamic
acid abolished PKC up-regulation but had no effect on G protein
modulation. Replacement of Thr422 with glutamic acid
eliminated PKC-dependent up-regulation and mimicked the
effects of PKC phosphorylation on G protein inhibition. Our data
suggest that Thr422 mediates the antagonistic effect of PKC
on G protein modulation, while phosphorylation of either
Thr422 or Ser425 are sufficient to increase
N-type channel activity. Thus, Thr422 serves as a molecular
switch by which PKC is able to simultaneously trigger the up-regulation
of channel activity and antagonize G protein inhibition.
Calcium influx through neuronal voltage-dependent
calcium channels mediates a range of cytoplasmic responses, such as
neurotransmitter release, proliferation, and the activation of
calcium-dependent enzymes. Most neurons express multiple
calcium channel types with distinct functional properties, and
molecular cloning has identified genes encoding at least eight
different neuronal calcium channel The physiological properties of presynaptic calcium channels are
extensively modulated by second messenger molecules, including protein
kinase C and G protein We have previously shown that cross-talk between G protein and PKC
pathways mainly occurs at the level of the calcium channel Molecular Biology--
DNA encoding wild type
A carboxyl-terminal deletion mutant lacking amino acid residues
1955-2336 was constructed by eliminating an XbaI fragment contained between an XbaI site in the Transient Transfection--
Human embryonic kidney TSA 201 cells
were grown in standard Dulbecco's modified Eagle's medium,
supplemented with 10% fetal bovine serum and 0.4 mg/ml neomycin. The
cells were grown to 85% confluency, split with trypsin EDTA, and
plated on glass coverslips at 10% confluency 12 h prior to
transfection. Immediately prior to transfection, the medium was
replaced, and the cells were transiently transfected with cDNAs
encoding for calcium channel Electrophysiology--
Immediately prior to recording,
individual coverslips were transferred to a 3-cm culture dish
containing recording solution composed of 20 mM
BaCl2, 1 mM MgCl2, 10 mM HEPES, 40 mM triethanolamine chloride, 10 mM glucose, 65 mM CsCl (pH 7.2). Whole cell
patch clamp recordings were performed using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA) linked to a personal computer equipped with pCLAMP version 6.0. Patch pipettes (Sutter borosilicate glass; BF150-86-15) were pulled using a Sutter P-87 microelectrode puller, fire-polished using a Narashige microforge, and showed typical
resistances of 2-3 megaohms. The internal pipette solution contained
105 mM CsCl, 25 mM triethanolamine chloride, 1 mM CaCl2, 11 mM EGTA, 10 mM HEPES (pH 7.2), supplemented with nystatin. Nystatin was
dissolved in Me2SO at 100 mg/ml and diluted directly into
the pipette solution. After seal formation, nystatin was allowed to
equilibrate into the patch for 5-10 min to permit electrical access.
Currents were typically elicited from a holding potential of The Domain I-II Linker Mediates both PKC and G Protein Modulatory
Effects on N-type Channels--
We have previously shown that N-type
(
Two putative protein kinase C consensus sites, Thr422 and
Ser425, are contained with this region of the channel (Fig.
1, inset). To test whether one or both of these residues
might mediate PKC/G
To elucidate which of the two residues mediate the effects of PKC, we
"permanently phosphorylated" either Thr422 or
Ser425 by replacing them individually with glutamic acid
residues. As shown in Fig. 2A,
the S425E mutant exhibits a somatostatin response comparable with that
of the wild type channel (p = 0.41), suggesting that
phosphorylation of Ser425 does not antagonize G protein
action. PMA treatment had no significant effect on current amplitude
(p = 0.83, paired t test; see also Fig.
3B), suggesting that replacing
Ser425 with glutamic acid mimics a permanently up-regulated
state of the channel. Nonetheless, PMA treatment significantly
attenuated the degree of somatostatin inhibition from 46 ± 7 to
15 ± 4% (p = 0.004) (Figs. 2B and
3A), suggesting that replacement of Ser425 with
glutamic acid does not preclude phosphorylation of the adjacent Thr422 residue and that cross-talk between PKC and
G
To confirm this hypothesis, we created two additional mutants in which
Thr422 and Ser425 were substituted individually
by alanines in order to further define the relative contributions of
the individual PKC consensus sites to the overall action of PKC. As
shown in Fig. 4A, T422A exhibits a somatostatin sensitivity that closely parallels that seen
with the T422A/S425A double mutant shown in Fig. 1. After treatment
with 100 nM PMA, the degree of somatostatin inhibition of
T422A did not decrease significantly (37 ± 5% (without PMA) versus 32 ± 3% (with PMA), p = 0.42)
and remained significantly (p < 0.003) larger than
that of the PMA-treated wild type channel (20 ± 5%). These data
indicate that cross-talk between PKC and G protein pathways is blocked
upon selective removal of the Thr442 PKC substrate.
Consistent with this notion, removal of the Ser425 PKC
substrate (i.e. S425A) did not significantly change G
protein sensitivity (p = 0.49), nor did it affect
cross-talk between the G protein and PKC pathways (48 ± 7%
(without PMA) versus 18 ± 3% (with PMA),
p < 0.002, Fig. 4A). Both T422A and S425A
exhibited a similar degree of PKC-dependent up-regulation
(Fig. 4B), which did not differ significantly from that
observed with the wild type channel (p > 0.79). This
further supports the notion that N-type channel activity is fully
up-regulated upon phosphorylation of either Thr422 or
Ser425, whereas only Thr422 is capable of
mediating the cross-talk between PKC and G protein pathways.
Voltage Dependence of G Protein Modulation--
To examine the
voltage dependence of the T422E mutant, we utilized a ramp protocol to
allow the acquisition of complete current-voltage relations without
contamination from receptor desensitization. Fig.
5 compares the somatostatin response of
wild type channels to that of the T422E mutant at a number of test
potentials. In both cases, the effect of somatostatin is dependent on
membrane potential and is consistent with the direct inhibition of
native N-type calcium channels by G proteins (18, 23, 24, 31, 32). As
evident from Fig. 5, somatostatin produced a significantly greater
inhibition of the wild type channels at all potentials. Although the
T422E mutant is capable of undergoing G protein modulation, the voltage
dependence of G Contribution of the
Overall, the data suggest that the carboxyl region mediates an
important role in stabilizing the G Protein Kinase C-dependent Up-regulation Is Mediated by
the Calcium Channel I-II Linker--
Whole cell currents of
exogenously expressed
Here, we show that the double substitution of Thr422 and
Ser425 for alanines abolishes the PKC-dependent
up-regulation of
Recently, Shistik et al. (35) showed that deletion of
N-terminal residues 2-46 abolished PKC-dependent
up-regulation of rabbit heart Model for the G Protein Inhibition of N-type Calcium
Channels--
Over the past several years, a number of studies have
examined the molecular determinants of G protein modulation of
presynaptic calcium channels (18, 28, 29, 31-34, 36-40). It is now
widely accepted that the G
In addition to the domain I-II linker, the carboxyl terminus (33, 34)
as well as domain I (38) and the amino terminus (39) of the calcium
channel
Kinetic modeling of G protein inhibition of single N-type calcium
channels has shown that G protein binding results in a reluctance of
channels to undergo transitions from the closed states to channel opening (27). Upon membrane depolarization, G proteins dissociate from
the channels prior to opening, and the associated increase in first
latency to opening results in a decrease in peak current amplitude.
Within the framework of this model, the binding of G
Overall, a model emerges in which G Implications of Dual PKC Sites for Calcium Channel
Modulation--
Our data indicate that up-regulation of N-type channel
activity occurs via phosphorylation of either Thr422 or
Ser425, whereas G protein modulation is antagonized by
phosphorylation of only Thr422. The selective
phosphorylation of Ser425 would result in up-regulation by
~50%, and the subsequent stimulation of the G protein pathway would
produce a ~50% inhibition of the PKC-enhanced current, resulting in
an overall inhibition of ~25%. Phosphorylation of Thr422
(or of both residues simultaneously) would also result in the 50%
up-regulation, but subsequent G protein inhibition would be attenuated
to ~20% and result in a net up-regulation by ~20%. Since
activation of PKC and G protein pathways individually would respectively produce a 50% increase and a 50% decrease in control current levels, Thr422 and Ser425 may function
as an integration center for inputs from PKC and G protein pathways to
produce multiple levels of calcium channel activity. Together with
recent reports that protein kinase C-dependent phosphorylation disrupts the interactions between syntaxin and the
calcium channel II-III linker (16), this convergence of second
messenger pathways directly at the level of the calcium channel
INTRODUCTION
Top
Abstract
Introduction
References
1 subunits (termed
1A through
1H). Functional expression in heterologous expression systems has revealed that
1A
encodes for P/Q-type calcium channels (1-3);
1B defines
an
-conotoxin GVIA-sensitive N-type channel (4-6);
1C,
1D and
1F are L-type calcium channels (7-9); and
1E is a unique calcium
channel with properties common to both high threshold and low threshold
calcium channels (10, 11). More recently,
1G and
1H have been shown to encode members of the family of
T-type calcium channels (12). Among the eight types of
1
subunits,
1A and
1B are predominantly located at more distal dendritic and presynaptic nerve terminals (13,
14) and are directly coupled to the presynaptic vesicle release
machinery (15, 16).
subunits (17-21). The activation of
certain G protein-coupled seven-helix transmembrane receptors mediates
a pronounced voltage-dependent inhibition of both N-type and P/Q-type calcium currents (Refs. 22-25; for a review, see Ref. 26). This inhibition is probably caused by direct 1:1 binding of G
protein
subunits to the calcium channel
1
subunit, resulting in a reluctance of the channels to undergo opening
in response to membrane depolarization (27, 28). In contrast,
stimulation of protein kinase C-dependent phosphorylation
results in a substantial up-regulation of N-type channel activity (19).
PKC1 and G
modulation are functionally coupled (termed cross-talk), such that
PKC-dependent phosphorylation of the channel antagonizes subsequent G protein inhibition (20, 21, 29).
1 subunit (29). In particular, the cytoplasmic linker
connecting domains I and II of the
1B subunit is a
crucial determinant of both G protein inhibition and PKC-G protein
cross-talk. This region has also been implicated in
PKC-dependent up-regulation of N-type calcium channels
(19), suggesting the possibility of a common mechanism underlying the
dual effects of protein kinase C-dependent phosphorylation.
Here, we identify individual amino acid residues within the
1B domain I-II linker that mediate up-regulation by PKC-dependent phosphorylation as well as the cross-talk
between PKC and G protein pathways. Using site-directed mutagenesis in combination with functional expression in human embryonic kidney cells,
we show that phosphorylation of either threonine 422 or serine 425 is
sufficient to mediate PKC-dependent up-regulation of the
channel. Whereas the nature of serine 425 does not affect G protein
inhibition, substitution of the threonine residue to glutamic acid to
create a permanent phosphoform drastically reduces the degree of G
protein inhibition. We propose a model whereby threonine 422 acts as a
molecular switch by which protein kinase C up-regulates the activity of
N-type calcium channels and concomitantly antagonizes their inhibition
by G protein
subunits. The remaining (~20%) degree of G
protein inhibition is further reduced upon deletion of the 3' third of
the
1B carboxyl terminus, suggesting that the calcium
channel domain I-II linker and the carboxyl terminus might
cooperatively interact with G protein
subunits.
MATERIALS AND METHODS
1B
channels was subcloned into the cytomegalovirus expression vector. The
1B-cytomegalovirus construct was cut with
SpeI and SplI, and the
SpeI-SplI fragment was then subcloned into a
modified pSL1180 Bluescript vector (which had been cut with
StuI and SmaI and recircularized to eliminate a Kpn2I site in the polylinker). A Kpn2I fragment
was excised from this construct and subcloned into a modified pSL1180
vector (in which a NarI-NarI fragment was
deleted). Site-directed mutagenesis of PKC consensus sites was carried
out on this construct using the Quick Change site-directed mutagenesis
kit (Stratagene). The mutations were confirmed via DNA sequencing. The
Kpn2I fragment was subsequently subcloned into the
SpeI-SplI construct in pSL1180, and the
SpeI-SplI fragment was ligated into the
full-length
1B construct in cytomegalovirus. After
completion of subcloning, the 900-base pair Kpn2I fragment
contained in the full-length sequence was completely sequenced to
confirm the presence of the mutations and to eliminate the possibility
of cloning and polymerase chain reaction artifacts.
1B
carboxyl terminus and a second XbaI site in the 3' portion
of the polylinker of the cytomegalovirus expression vector. The
construct was cut with XbaI and recircularized, and the
successful elimination of the XbaI fragment was confirmed
via enzyme analysis and DNA sequencing.
1B,
1b, and
2 subunits (at a 1:1:1 molar ratio) using a standard
calcium phosphate protocol. After 12 h, the medium was replaced
with fresh Dulbecco's modified Eagle's medium, and the cells were
allowed to recover for 12 h. Subsequently, the cells were
incubated at 28 °C in 5% CO2 for 1-2 days prior to recording.
100 mV
to various test potentials using Clampex software (Axon Instruments).
Current-voltage relations were generated by utilizing a ramp protocol
(dV/dt = 1 mV/ms) as reported previously (30). Somatostatin (RBI
Chemicals) was dissolved in water to give a stock solution of 1 mM, and PMA (RBI) was dissolved in Me2SO at a
stock concentration of 2 mM. These compounds were diluted
into the external recording solution at the appropriate final
concentrations and perfused directly onto the cell using a
gravity-driven microperfusion system. At the applicable concentrations,
Me2SO by itself had no effect on calcium channel activity.
In every case, peak current inhibition was assessed 15 s after
somatostatin application. Data were filtered at 1 kHz and recorded
directly onto the hard drive of the computer. Data were analyzed using
Clampfit (Axon Instruments). All curve fitting was carried out in
Sigmaplot 4.0 (Jandel Scientific). Unless stated otherwise, all error
bars represent S.E. values, numbers in parentheses displayed in the
figures reflect numbers of experiments, and p
values given reflect Student's t tests.
RESULTS
1B +
2
+
1b) calcium
channels transiently expressed in human embryonic kidney cells are
reversibly inhibited by 50-70% via activation of endogenous somatostatin receptors (Ref. 29; see Fig.
1C). In contrast, activation
of protein kinase C with 100-400 nM of the phorbol ester
PMA results in a pronounced (50-60%) up-regulation of
1B N-type currents (Ref. 29; see Fig. 1D).
Subsequent to PMA treatment, inhibition of N-type channel activity by
somatostatin is dramatically reduced (see Fig. 1D),
consistent with previous observations in intact neurons (20, 21).
Synthetic peptides directed against two subregions of the
1B calcium channel domain I-II linker block the
modulatory effects of exogenously applied G
subunits (29). One of the peptides is a substrate for in vitro
phosphorylation by protein kinase C, and when phosphorylated in
vitro, it loses the ability to interfere with G protein modulation
(29). These results suggested that cross-talk between protein kinase C
and G protein pathways might occur in this subregion of the
1B domain I-II linker.
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Fig. 1.
Effect of a double substitution of I-II
linker protein kinase C consensus sites for alanines on PKC and G
protein action. A, current records illustrating G
protein inhibition of the double alanine mutant. Somatostatin
reversibly reduces the peak current amplitude of the mutant channel to
65% of its control value. The currents were leak-subtracted using a
p/5 protocol. The holding potential was 100 mV, and the
test potential was +20 mV. B, application of PMA does not
affect current amplitude and has no adverse effects on G protein
inhibition mediated by somatostatin (holding potential
100 mV, test
potential = +20 mV). Inset, proposed transmembrane
topology of voltage-dependent calcium channels and amino
acid sequence of part of the
1B channel I-II linker. The
bar above the amino acid sequence indicates a
previously identified putative target region for G protein/PKC
cross-talk. The Thr422 and Ser425 residues were
substituted to alanine residues. C, degree of somatostatin
(100 nM) inhibition of wild type
1B channel
and the alanine (422 and 425) mutant channels (each coexpressed with
1b and
2) with or without prior
application of 100 nM PMA. Note that the alanine
substitution slightly but significantly reduces the degree of
somatostatin inhibition. While PMA reduces the G protein sensitivity of
the wild type channel, the G protein inhibition of the double mutant is
not affected. D, up-regulation of channel activity by 100 nM PMA. Note that the up-regulation seen with the wild type
channel is blocked by the double alanine substitution. Error
bars represent S.E. values; the test potential in
C and D was +20 mV.
cross-talk, we replaced both
sites with alanine residues. Fig. 1A depicts current records
obtained from the double alanine mutant in the absence and the presence
of 100 nM somatostatin. Similar to that observed with the
wild type channel (Fig. 1), activation of somatostatin receptors
mediates a reversible and pronounced inhibition of channel activity,
paired with a slowing of activation kinetics and an apparent slowing of
inactivation. Fig. 1B illustrates the effect of PMA on
channel activity and on somatostatin modulation of the alanine double
mutant. While application of 100 nM PMA had no detectable
effect on channel activity, the subsequent application of 100 nM somatostatin produced the same degree of G protein
inhibition as that observed in the absence of PMA. Fig. 1, C
and D, illustrates the effect of alanine substitution for a
number of experiments. There are two effects evident. First, the
alanine mutation per se significantly reduces the degree of
somatostatin inhibition seen with the wild type channel from 53 ± 5 to 36 ± 5% (p = 0.02) (Fig. 1C).
Second, while PMA treatment reduced the somatostatin effect for the
wild type channel, the somatostatin sensitivity of the double mutant
was not altered by PMA (36 ± 5 versus 37 ± 5%,
p = 0.86; Fig. 1C), indicating that
cross-talk between PKC and G protein pathways is blocked by the double
alanine substitution. In addition, the PMA-induced up-regulation
observed with the wild type channels was reduced from 49 ± 14 to
6 ± 5% when both PKC consensus sites were simultaneously
replaced with alanines (Fig. 1D). Thus, the critical
structures mediating up-regulation of channel activity and inhibition
of direct G protein action appear to reside within overlapping regions
of the calcium channel domain I-II linker and contain one or both of
Thr422 and Ser425.
modulation is probably mediated by
Thr422 rather than Ser425. If this is correct,
then mimicking phosphorylation of Thr422 (i.e.
T422E) should reduce the degree of G protein inhibition to those levels
observed after PKC phosphorylation of the wild-type channel. This is
supported by the current records shown in Fig. 2, C and
D, and the data presented in Fig. 3A. The T422E
mutant showed a significantly reduced somatostatin sensitivity, which was comparable in magnitude with that observed with the wild type channel after PMA treatment (WT (with PMA) = 20 ± 5%; T422E
(without PMA) = 20 ± 3%, p = 0.91). These data
indicate that replacing Thr422 with a negatively charged
side group mimics the antagonistic effect of PKC on G protein
inhibition. PMA application did not further affect somatostatin
sensitivity of T422E (T422E (with PMA) = 14 ± 3%,
p = 0.29). Also, PMA failed to increase the peak current amplitude of T422E (p = 0.71) (Figs.
2D and 3B), suggesting that similar to S425E, the
T422E construct is likely be tonically up-regulated. Overall, the data
suggest that whereas only Thr422 is capable of mediating
the cross-talk effect, phosphorylation of either Ser425 or
Thr422 is sufficient to fully up-regulate channel
activity.
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Fig. 2.
Effect of individual replacement of I-II
linker PKC consensus sites with glutamic acid residues. The
holding potential was 100 mV, the test potential was +20 mV, and
currents were leak-subtracted using a p/5 protocol.
A and C, inhibition of mutant channels by 100 nM somatostatin. Note that the threonine substitution
dramatically reduces G protein sensitivity. B and
D, effect of PMA on channel activity and on the degree of
somatostatin inhibition. Either mutation blocks up-regulation of the
channel by PMA. S425E shows a reduced sensitivity to somatostatin
inhibition following application of PMA (i.e. cross-talk
remains intact), while no additional effect of PMA on G protein
inhibition of T422E is evident.
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Fig. 3.
A, effect of somatostatin on calcium
channel N-type channel activity with or without prior application of
100 nM PMA. The S425E mutant exhibits a somatostatin
sensitivity that is not significantly different from that of the wild
type channel. With prior application of PMA, the degree of somatostatin
inhibition of both the wild type and the S425E mutant is reduced to
similar levels. In contrast, even in the absence of PMA, the T422E
mutant exhibits a somatostatin sensitivity that parallels that of a
PMA-treated wild type channel, and PMA does not further effect
somatostatin sensitivity. The holding potential was 100 mV, and the
test potential was typically +20 mV. Error bars
represent S.E. values. B, up-regulation of wild type and
mutant channels by 100 nM PMA. Note that the glutamic acid
substitution of either Thr422 or Ser425 blocks
up-regulation by PMA. Experimental conditions were as described for
A.
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Fig. 4.
Effect of individual alanine substitutions in
the I-II linker PKC consensus sites on PKC and G protein inhibition of
N-type calcium channels. Error bars
represent S.E. values, and numbers in
parentheses indicate the numbers of experiments. The
dotted lines indicate the levels of G protein
inhibition (A) or PKC-dependent up-regulation
(B) depicted in Fig. 1. A, inhibition of T422A
and S425A by 100 nM somatostatin with and without prior
application of 100 nM PMA. Note that T422A does not permit
PKC activation to antagonize somatostatin inhibition, whereas the S425A
mutant exhibits the behavior seen with the wild type channel. The level
of somatostatin-induced inhibition of T422A closely parallels that
observed with the T422A/S425A double mutant examined in Fig.
1B. The PKC-dependent up-regulation observed
with T422A and S425A is similar to that observed with the wild type
channel. The dotted line indicates the level of
up-regulation of the wild type channel.
dissociation appears be shifted
toward more hyperpolarized potentials in the mutant channel.
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Fig. 5.
Voltage dependence of G protein inhibition
for wild type (n = 7) and mutant T422E (n = 12) 1B channels elicited
by application of 100 nM somatostatin. The data were
obtained via ramp protocols as described under "Materials and
Methods" 15 s after somatostatin application. Note that the
voltage dependence of somatostatin inhibition is shifted toward more
hyperpolarizing potentials for the mutant channels, resulting in a
significantly greater inhibition of the wild type channels at each
potential. Error bars represent S.E.
values.
1B Carboxyl Region in PKC and G
Protein Modulation--
Several studies have implicated the carboxyl
terminus in the direct G protein modulation of
voltage-dependent calcium channels (33, 34). Furthermore,
the carboxyl terminus contains several putative protein kinase C
consensus sites. To examine the possibility that the carboxyl terminus
might contribute to the modulation of N-type calcium channels by PKC
and G proteins, we examined a deletion mutant in which the last third
of the carboxyl terminus of
1B (residues 1955-2336) was
deleted (
1B
COOH). Fig.
6B depicts a current record
obtained with the deletion mutant in the presence and absence of
somatostatin. Although somatostatin reduces the peak current amplitude
and mediates the slowing of activation kinetics typical of direct
G
modulation, the degree of inhibition is reduced
compared with the wild type channel (from 53 ± 5% to 32 ± 2%, p = 0.02). In contrast, up-regulation by PMA remains intact (Fig. 6C and inset), suggesting
that the deleted portion of the carboxyl-terminal does not directly
mediate PKC-dependent changes in channel activity.
Following pretreatment with PMA, somatostatin application resulted in
only a small effect on peak current amplitude (4 ± 2%
inhibition), at a test potential of +20 mV (Fig. 6D). Hence,
PKC-dependent phosphorylation in combination with deletion
of the carboxyl terminus further reduces the degree of
G
modulation. To further examine this observation, we
deleted the carboxyl-terminal region of the T422E mutant. As seen from
Fig. 6D, this mutant showed only a ~10% inhibition in response to somatostatin at a test potential of +20 mV. The degree of
inhibition did not differ significantly from that seen with mutant
T422E after PMA treatment (p = 0.1) but was
significantly lower than the sensitivity of the
1B
COOH construct (p = 0.025). These
data indicate that deletion of the carboxyl terminus and replacement of
Thr422 with glutamic acid produce additive effects on G
protein sensitivity. This particular construct did not express well in
HEK cells, and we were unable to systematically examine the voltage
dependence of somatostatin action.
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Fig. 6.
Effect of deletion of residues 1955-2336 of
the 1B carboxyl terminus on G
protein and PKC sensitivity. A, schematic
representation of the proposed calcium channel transmembrane topology
indicating the deletion of part of the carboxyl terminus region.
B, current record elicited by a step depolarization from
100 to +20 mV and leak-subtracted via a p/5 protocol.
Deletion of the 3' third of the carboxyl terminus region results in a
slowing of inactivation kinetics as well as in a reduced degree of G
protein inhibition as evident by application of 100 nM
somatostatin. C, time course of up-regulation of current
activity by 100 nM PMA. The bar
graphs depicted in the inset illustrate the
degree of PMA up-regulation as defined by the peak current ratio
I (with PMA)/I(without PMA) for the wild type
channel and the deletion mutant. D, degree of
somatostatin-mediated G protein inhibition of the wild type channel and
the deletion mutant in the presence and absence of 100 nM
PMA. Note that the degree of G protein inhibition is significantly
attenuated upon deletion of the 3' portion of the
1B
carboxyl terminus. Pretreatment with 100 nM PMA almost
completely abolished G protein inhibition at test potentials of +20 mV.
A double mutant (
COOH,T422E) also exhibited a reduced G
protein sensitivity that did not differ significantly from that seen
with T422E after PMA treatment. Error bars
represent S.E. values.
interaction with the calcium channel
1 subunit, especially when PKC sites
in the
1B domain I-II linker are phosphorylated.
DISCUSSION
1B N-type channels are
up-regulated by activation of protein kinase C by either phorbol esters
(such as PMA) or activation of coexpressed metabotropic glutamate
receptors (19). Here, we have used application of 100 nM
PMA to stimulate protein kinase C in human embryonic kidney cells
expressing
1B channels. Consistent with the results of Stea and co-workers (19), PMA application resulted in a pronounced increase in channel activity for wild type
1B channels
that was blocked by pretreatment with staurosporine. In their study,
Stea and co-workers (19) were able to confer aspects of PKC sensitivity of
1B onto the less sensitive
1A channels
by inserting the domain I-II linker of
1B into
1A. We have previously shown that a fusion protein
directed against the
1B I-II linker region is a
substrate for PKC-dependent phosphorylation (29). Two
considerations have led us to focus on a pair of PKC consensus sites
(Thr422 and Ser425) located within a 20-amino
acid stretch (residues 410-428) of the
1B I-II linker.
First, this stretch of residues is both a substrate for in
vitro phosphorylation by PKC and has also been implicated in the
PKC-mediated antagonism of G protein inhibition of wild type
1B channels (29). Second, certain amino acid
substitutions in the vicinity of the corresponding region in
1A increases PKC sensitivity of
1A.2
1B channels, suggesting that
phosphorylation of one or both of these residues is sufficient to
mediate up-regulation. Individual substitutions of these residues for
glutamic acid also precluded the effect of PKC stimulation. In
contrast, individual substitution of these two residues for alanines
had no adverse effect on PKC-dependent up-regulation. These
data imply that the effects of phosphorylation of the two PKC consensus
sites are nonadditive and that phosphorylation of either
Thr422 or Ser425 is sufficient to mediate
complete up-regulation of channel activity in an all or none manner. At
present, the molecular mechanisms by which the phosphorylation event
affects channel activity remain to be determined. It is possible that
phosphorylation induces a conformational change in the domain I-II
linker that directly affects activation. Alternatively, phosphorylation
of these residues might alter the interaction with the calcium channel
subunit, which in turn may affect channel activation. Such a
mechanism would be consistent with data of Stea and co-workers (19),
who reported that the calcium channel
subunit is required for
PKC-dependent up-regulation.
1C channels expressed in
Xenopus oocytes. The N-terminal region of rat brain
1B N-type calcium channel is 51 residues shorter than
that of the rabbit heart
1C channel and thus lacks the
motif identified by Shistik et al. (35). Furthermore, there is no counterpart to Thr422 present in the rabbit heart
1C sequence, and the analog to Ser425
(Ser499 in the rabbit heart
1C sequence) is
not part of a PKC consensus motif. This suggests that the molecular
mechanism underlying the PKC-dependent modulation of N-type
calcium channel activity is fundamentally different from that for the
rabbit cardiac L-type isoform.
subunits are the active G
protein species mediating the antagonistic effect on presynaptic
calcium channel activity (28, 29, 31, 32). The G protein
subunits are able to interact with two separate regions within the
calcium channel domain I-II linker (29, 36). We have previously
suggested that the PKC dependent phosphorylation of one of these two
I-II linker G
binding motifs might mediate the
previously identified antagonistic effect of PKC stimulation on G
protein sensitivity (29). Here, we present further confirmation for the
involvement of the domain I-II linker region in direct G protein modulation of N-type calcium channels. Each, the double alanine mutant
and the T422E and T422A constructs, exhibited a significantly reduced
sensitivity to somatostatin-induced G
modulation. Neither of these substitutions resulted in significant changes in
current kinetics or half-activation potential, minimizing the possibility of an indirect effect due to changes in channel gating. We
suggest the possibility that these substitutions more likely reduce the
affinity of the channel for binding G
.
1 subunit have all been implicated in direct G
protein modulation. Here, we present corroborating evidence that the
carboxyl terminus contributes to a portion of the overall G protein
inhibition of N-type channels. A deletion of one-third of the
carboxyl-terminal region significantly reduced but did not eliminate
the somatostatin-induced inhibition of
1B channels. The
deleted portion contains a highly conserved motif that was recently
implicated in G
binding to
1E channels (33). Together with our results, it appears that while the carboxyl terminus probably contributes to G
binding to N-type calcium channels, the major determinant of G protein action is the
domain I-II linker. That the carboxyl terminus contributes to
G
binding could account for the observation that
1B or
1E channel constructs containing
the
1C I-II linker remain sensitive to G protein
inhibition (33, 34)3 despite
the fact that
1C I-II linker fusion proteins do not bind
G
. It is possible that the carboxyl region
cooperatively enhances binding to the
1C I-II linker in
these chimeric constructs.
to the domain I-II linker and perhaps the carboxyl terminus mediates
the stabilization of the closed state by inducing a conformational
change in the channel protein. The translation of G protein binding
into a change in channel function may be mediated by residues located
in domain I, consistent with previous studies (34, 38, 39).
interacts with
two high affinity regions within the calcium channel domain I-II linker
(residues 353-389 and 410-428 (29, 36) and a lower affinity site in
the carboxyl terminus (33). PKC-dependent phosphorylation probably destabilizes G
binding to the second site
within the I-II linker G
binding domain
(i.e. residues 410-428), thereby shifting the voltage
dependence of G
dissociation to more hyperpolarized
potentials and perhaps reducing the increase in first latency to
opening associated with G
binding (27). Single
channel experiments will ultimately be required to confirm any effects
of phosphorylation (and of the T422E substitution) on first latency.
1 subunit would provide a mechanism to precisely control neurotransmitter release at presynaptic nerve terminals.
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FOOTNOTES |
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* This work was supported by operating grants from the Medical Research Council of Canada (MRC) (to G. W. Z. and T. P. S.) and by an operating grant from the Heart and Stroke Foundation of Canada (to G. W. Z.).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.
¶ Recipient of a studentship award from the Alberta Heritage Foundation for Medical Research (AHFMR).
Recipient of an MRC Scientist Award.
** Recipient of scholarship awards from the MRC and the AHFMR. To whom correspondence should be addressed: Dept. of Pharmacology and Therapeutics, University of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta T2N 4N1, Canada. Tel.: 403-220-8687; Fax: 403-283-8731; E-mail: Zamponi{at}acs.ucalgary.ca.
2 G. W. Zamponi and T. P. Snutch, unpublished observations.
3 E. Bourinet, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate.
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REFERENCES |
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