1Department of Neurobiology, Harvard Medical School; and 2Department of Cardiology, Brigham and Women's Hospital, Boston, Massachusetts 02115
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ABSTRACT |
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Greif, Gabriela J.,
Deborah L. Sodickson,
Bruce
P. Bean,
Eva J. Neer, and
Ulrike Mende.
Altered Regulation of Potassium and Calcium Channels by
GABAB and Adenosine Receptors in Hippocampal Neurons From
Mice Lacking Go.
J. Neurophysiol. 83: 1010-1018, 2000.
To examine the role of
Go in modulation of ion channels by neurotransmitter
receptors, we characterized modulation of ionic currents in hippocampal
CA3 neurons from mice lacking both isoforms of G
o. In
CA3 neurons from G
o
/
mice,
2-chloro-adenosine and the GABAB-receptor agonist baclofen activated inwardly rectifying K+ currents and inhibited
voltage-dependent Ca2+ currents just as effectively as in
G
o+/+ littermates. However, the
kinetics of transmitter action were dramatically altered in
G
o
/
mice in that recovery on washout
of agonist was much slower. For example, recovery from
2-chloro-adenosine inhibition of calcium current was more than fourfold
slower in neurons from G
o
/
mice
[time constant of 12.0 ± 0.8 (SE) s] than in neurons from G
o+/+ mice (time constant of
2.6 ± 0.2 s). Recovery from baclofen effects was affected
similarly. In neurons from control mice, effects of both baclofen and
2-chloro-adenosine on Ca2+ currents and K+
currents were abolished by brief exposure to external
N-ethyl-maleimide (NEM). In neurons lacking
G
o, some inhibition of Ca2+ currents by
baclofen remained after NEM treatment, whereas baclofen activation of
K+ currents and both effects of 2-chloro-adenosine were
abolished. These results show that modulation of Ca2+ and
K+ currents by G protein-coupled receptors in hippocampal
neurons does not have an absolute requirement for G
o.
However, modulation is changed in the absence of G
o in
having much slower recovery kinetics. A likely possibility is that the
very abundant G
o is normally used but, when absent, can
readily be replaced by G proteins with different properties.
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INTRODUCTION |
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Many effects of neurotransmitters are mediated by
heterotrimeric G proteins, composed of G and G
subunits.
Go is the most abundant type of G protein in the brain
(Huff et al. 1985
; Neer et al. 1984
;
Sternweis and Robishaw 1984
). However, the range of
effects mediated by Go is still unclear. Considering the
abundance of the protein, the effects of inactivating the
G
o gene in mice are surprisingly modest. Mice lacking
G
o are viable and have no gross abnormalities of the
brain, although they develop tremors and occasional seizures and have
impaired motor control (Jiang et al. 1998
;
Valenzuela et al. 1997
).
One role of G proteins is to mediate neurotransmitter control of
excitability by modulating the activity of ion channels (Hille 1994). G protein activation of inwardly rectifying
potassium-selective (GIRK) channels is one such pathway, mediated by
direct binding of G
subunits to the channels (see Clapham
and Neer 1997
). A second well-studied case of G
protein-mediated modulation is transmitter inhibition of
voltage-dependent Ca2+ channels, which can
proceed by multiple pathways (Beech et al. 1992
;
Diverse-Pierluissi and Dunlap 1993
;
Diverse-Pierluissi et al. 1995
; Dolphin
1995
; Shapiro and Hille 1993
). One pathway
prominent in many neurons is rapid, membrane-delimited inhibition
mediated by direct binding of G
subunits to
Ca2+ channels (De Waard et al.
1997
; Herlitze et al. 1996
; Ikeda
1996
; Zamponi et al. 1997
). Although G
subunits directly control GIRK channels and Ca2+
channels, the type of the G
subunit from which the G
is
released can determine specificity and kinetics of transmitter action. In addition, there is evidence for more direct involvement of G
subunits in at least some pathways (Furukawa et al.
1998a
,b
).
Based on sensitivity to pertussis toxin (PTX), all transmitters known
to activate GIRK channels appear to act through G released from
members of the Gi/Go family, as do most, but
not all, transmitters that inhibit Ca2+ channels
by rapid, membrane-delimited action (Dolphin 1995
;
Hille 1994
; Wickman and Clapham 1995
;
Zhu and Ikeda 1994
). For Ca2+
channel inhibition, Go has been specifically implicated by
studies in a variety of neurons and neuroendocrine cells using
antibodies or antisense oligonucleotides against specific G protein
subunits (Campbell et al. 1993
; Caulfield et al.
1994
; Ewald et al. 1988
; Hescheler et al.
1987
; Kleuss et al. 1991
; Lledo et al.
1992; McFadzean et al. 1989
, 1993;
Menon-Johansson et al. 1993
; Moises et al.
1994
; Taussig et al. 1992
)
Mice lacking both isoforms of Go present a useful model
to study the role of Go in G protein-mediated ion channel
modulation. Several alterations of channel modulation in
G
o
/
mice are already known. In
cardiac myocytes, acetylcholine activation of GIRK channels appears
normal but there is disruption of a pathway by which muscarinic
acetylcholine receptors regulate L-type Ca2+
channels (Valenzuela et al. 1997
). In neurons, a recent
study found that inhibition by opioid receptors of
Ca2+ channel in sensory neurons was diminished in
G
o
/
mice (Jiang et al.
1998
).
To further study the role of Go in control of neuronal
GIRK channels and Ca2+ channels, we examined
transmitter modulation in hippocampal CA3 neurons where a number of
transmitters can both inhibit Ca2+ channels and
activate GIRK channels. We find that modulation of
Ca2+ channels and GIRK channels by
GABAB and adenosine receptor agonists is
unchanged in magnitude in neurons from
G
o
/
mice but that kinetics of
transmitter modulation are dramatically different compared with neurons
from wild-type mice.
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METHODS |
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Cell preparation
Go
/
,
G
o+/
, and
G
o+/+ mice were obtained from +/
matings as described by Valenzuela et al. (1997)
.
Hippocampal CA3 pyramidal neurons were isolated enzymatically from the
brains of mice (aged 9-15 d). Mice were anesthetized with
methoxyflurane before decapitation and hippocampi were dissected out in
oxygenated ice-cold dissociation solution containing (in mM) 82 Na2SO4, 30 K2SO4, 5 MgCl2, 10 HEPES, 10 glucose, and 0.01% phenol
red indicator (pH 7.4, adjusted with NaOH) and cut into 400 µm thick
slices. After incubation with 3 mg/ml protease XXIII in dissociation
solution (37°C for 9 min), the enzyme solution was replaced with
dissociation solution containing 1 mg/ml trypsin inhibitor and 1 mg/ml
bovine serum albumin and the slices were stored at room temperature
with oxygen blown over the surface of the fluid. As cells were needed,
the CA3 region was dissected out of individual slices and triturated
mechanically with fire-polished glass pipettes to liberate individual
cells. Neurons were allowed to settle in the recording chamber for a few minutes and were superfused with Tyrode's solution containing (in
mM) 150 NaCl, 4 KCl, 2 CaCl2, 2 MgCl2, 10 glucose, and 10 HEPES (pH 7.4 with
NaOH). CA3 pyramidal neurons were identified morphologically
(Sodickson and Bean 1996
).
Electrophysiological methods
All recordings were done at room temperature using whole cell
voltage clamp techniques (Hamill et al. 1981). Patch
pipettes were pulled from 100 µl Boralex micropipettes (Dynalab,
Rochester, NY). Pipette resistance ranged 2-6 M
when pipettes were
filled with internal solution. Pipette capacitance was reduced by
wrapping the tip with thin strips of Parafilm. After the whole cell
configuration was obtained in Tyrode's solution, the cell was lifted
from the bottom of the chamber and control and agonist-containing
external solutions were applied from different reservoirs through
gravity-driven microcapillary perfusion pipes (internal diameter of 250 µm), positioned directly in front of the cell. Solutions were
exchanged (<1 s) by moving the pipes. Baclofen, 2-chloro-adenosine,
and somatostatin were from RBI (Natick, MA) and
N-ethyl-maleimide (NEM) was from Sigma. Agonists were stored
as concentrated aliquots at
70°C and a fresh aliquot was diluted
into external recording solution on the day of the experiment.
Data acquisition and analysis
Currents were recorded with an Axopatch 200 amplifier, filtered at 2 kHz, digitized at 20-50 kHz, and stored on a computer using a Digidata interface and pClamp6 software (Axon Instruments, Foster City, CA). Series resistance (~2-2.5 times higher than the pipette resistance) was compensated by 70-90%. Membrane potentials were corrected for liquid junction potential.
Solutions and voltage protocol
We used ionic solutions and a voltage protocol that allowed
simultaneous recording of Ca2+ and inwardly
rectifying K+ currents. The internal solution
contained (in mM) 189 Cs2HPO4, 9 CsCl,
9 HEPES, 9 EGTA, 14 Tris-creatinePO4, 4 Mg-ATP,
and 0.3 Tris-GTP (pH 7.4 with CsOH).
Tris-creatinePO4, Mg-ATP, and Tris-GTP were added
freshly each day to pipette solutions from aliquots stored at 70°C.
External recording solution consisted of modified Tyrode's solution
with 16 mM KCl, with KCl substituted for an equimolar amount of NaCl. 1 µM Tetrodotoxin (TTX) was included in the external solutions to block
sodium currents. With these solutions, transmitter-activated inwardly
rectifying K+ current (carried by 16 mM
[K+]o) could be recorded
as a transmitter-sensitive inward current at voltages negative to
60
mV, whereas voltage-activated calcium current could be recorded in
response to a depolarizing voltage steps to
12 mV (where outward
K+ current is blocked by the internal
Cs+).
To determine the time course of transmitter action, the voltage
protocol was delivered every 2 s. Time-course plots show current measured at the end of the steps to 142 and
12 mV. Kinetics of
K+ currents were analyzed only for cells in which
there was a stable baseline before and after application of
transmitters and where transmitter-activated current was
70 pA.
In some experiments, Ba2+ currents through Ca2+ channels were measured. These experiments used an external solution containing (in mM) 2 BaCl2, 160 NaCl, and 10 HEPES (pH 7.4 with NaOH), along with the standard Cs-based intracellular solution.
Data are expressed as mean ± SE.
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RESULTS |
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Simultaneous recording of baclofen effects on Ca2+ and K+ currents
We wished to examine modulation of both voltage-dependent Ca2+ currents and GIRK currents, both of which are modulated by a variety of transmitters in hippocampal CA3 neurons. Typically, these currents are examined in isolation using appropriate ionic substitutions (for example, blocking both inward and outward K+ currents when recording Ca2+ channel currents). To facilitate efficient collection of data from the minimal necessary number of animals, we used ionic solutions and a voltage protocol that allowed simultaneous recording of Ca2+ and K+ currents. The internal solution used Cs+ as the main cation, and the external solution was Tyrode's solution with increased (16 mM) KCl and containing 1 µM tetrodotoxin (TTX) to block sodium currents.
With these solutions, transmitter-activated inwardly rectifying
K+ current (carried by 16 mM
[K+]o) can be recorded
as a transmitter-sensitive inward current at voltages negative to 60
mV, because the internal Cs+ eliminates outward
K+ currents but not inward
K+ currents. Voltage-activated calcium current
can be recorded in response to depolarizing voltage steps positive to
50 mV or so, and the calcium currents are well-isolated because
outward K+ current that would otherwise overlap
with them is eliminated by the internal Cs+.
Currents were thus recorded using a voltage protocol consisting of a
strongly hyperpolarizing step (to
142 mV for 5 ms) and a depolarizing
step to
12 mV (10 ms), which gave maximal Ca2+
current. The currents elicited by this protocol, and the effects of
baclofen on them, are shown in Fig.
1A in a recording from a
neuron from a wild-type animal. The voltage steps were delivered from a
steady holding potential of
92 mV. The hyperpolarizing step was given
first because there was little time dependence of the current before or
after this step, whereas the calcium current during the depolarizing
step was followed by a slowly-decaying tail current (mainly from inward
K+ current through voltage-activated
K+ channels opened during the depolarization).
The experiments shown in Fig. 1, B and C, tested
the ability of this protocol to separate the effects of baclofen on
GIRK current and Ca2+ current in a single
application of transmitter. Resting current is inward at the holding
potential of
92 mV, and the step to
142 mV elicits increased inward
current with little time-dependence (the step is too short to activate
Ih, if present). Baclofen increases the inward current at both
92 mV and
142 mV, as expected for activation of GIRK channels. In the absence of baclofen, the step to
12 mV activates a time-dependent inward current that appears fully
activated at the end of the 10-ms step, consistent with a
voltage-activated Ca2+ current. The inward
current elicited by the depolarizing step is reduced by baclofen, as
expected for calcium channel inhibition. In the experiment shown in
Fig. 1B, the effect of baclofen was tested in the presence
of 300 uM external barium, which blocks both inward and outward current
through GABAB-activated potassium channels in rat
neurons (Sodickson and Bean 1996
). With GIRK current blocked, baclofen had no effect on the currents at
92 mV and
142 mV
but baclofen still effectively inhibited the depolarization-activated inward current. To test whether the current activated by depolarization to
12 mV was purely Ca2+ current, we repeated
the baclofen application in an external solution in which 2 mm
Co2+ replaced the 2 mM Ca2+
present in normal external solution. In
Co2+-containing solution, the current at
12 mV
was near zero both in the absence and presence of baclofen. Thus in
control conditions the current at
12 mV appears to be almost purely
voltage-activated Ca2+ current, and baclofen does
not activate or influence any other current at this voltage.
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The separation of baclofen effects on GIRK current and
voltage-activated Ca2+ current by the different
voltage-sensitivity of the currents is illustrated in Fig.
2, showing the current-voltage
relationship (determined with 5-ms steps from 92 mV) in the absence
and presence of baclofen. Baclofen induces inward current negative to
60 mV, and this baclofen-activated current is completely blocked by
300 uM external barium (Fig. 2A). Baclofen reduces
inward current activated by steps positive to
50 mV, and both this
current and the effect of baclofen are blocked by substitution of
Co2+ for Ca2+ (Fig.
2B). Voltage-activated Ca2+ current is
maximal at
12 mV, at which voltage the leak current remaining in 2 mM
Co2+ is near zero. Thus inward current at
12 mV
in Ca2+-containing solution provides an accurate
measurement of Ca2+current without requiring
significant correction for leak current. In each cell studied, we first
performed the voltage protocol with the 2 mM Co2+
solution before testing transmitters in the 2 mM
Ca2+ solution, and the Ca2+
current at
12 mV was corrected for any current (always small) remaining in Co2+-containing solution.
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The ability of 300 uM external barium to block the current activated by
baclofen negative to 50 mV is consistent with this being an inwardly
rectifying potassium current. To test further the identity of this
current, we characterized its reversal potential and current-voltage
relationship in experiments using intracellular solution containing
K+ in place of Cs+ ions
(Fig. 3). The current reversed at
62
mV, near the calculated equilibrium potential for potassium (
58 mV)
and had an inwardly rectifying current-voltage relationship. It
appeared identical in all respects to the baclofen-activated GIRK
current previously characterized in rat hippocampal neurons using the
same solutions (Sodickson and Bean 1996
).
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Modulation of Ca2+ and K+ currents in hippocampal CA3 neurons
We compared Ca2+ and
K+ current modulation by 2-chloro-adenosine and
the GABAB receptor agonist baclofen in freshly
dissociated pyramidal CA3 neurons of hippocampi from
Go
/
mice and from
G
o+/+ littermates. The genotypes of
the animals were confirmed after the experiment by PCR.
In CA3 neurons from Go+/+ mice,
baclofen reliably activated inwardly rectifying
K+ currents and inhibited
Ca2+ currents (Fig.
4A). Both baseline currents
and effects of baclofen were similar in cells from
G
o
/
mice (Fig. 4B).
Baclofen inhibition of Ca2+ current was identical
in magnitude (25 ± 1%; n = 34) in cells from
G
o
/
mice as in cells from
G
o+/+ mice (25 ± 1%;
n = 30) (Fig. 4E). Baclofen activation of
inwardly rectifying K+ currents was also little
different: baclofen-activated current (measured at
142 mV) was
6.6 ± 0.5 pA/pF (n = 34) in cells from G
o
/
mice compared with 7.9 ± 0.7 pA/pF (n = 30) in cells from
G
o+/+ mice (Fig. 4F).
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The adenosine receptor agonist 2-chloro-adenosine also reliably
activated inwardly rectifying K+ currents and
inhibited Ca2+ currents in CA3 neurons (Fig.
4C). 2-Chloro-adenosine was just as effective in cells from
Go
/
mice as in those from
G
o+/+ mice (Fig. 4,
D-F). We also tested somatostatin (5 cells from G
o
/
mice and 8 cells from
G
o+/+ mice) and the metabotropic
glutamate receptor agonist 1R,2S-ACDP (3 cells from
G
o
/
mice and 5 cells from
G
o+/+ mice). Although these series
of experiments were less systematic, there was no obvious difference in
the size of effects on cells from
G
o+/+ and
G
o
/
mice (data not shown). The
voltage-dependence of control Ca2+ currents and
the voltage-dependence of inhibition by baclofen or 2-chloro-adenosine
(which was most prominent for moderate depolarizations) were also not
distinguishable between cells from
G
o
/
and
G
o+/+ mice (data not shown).
Kinetics of K+ and Ca2+ current modulation by G protein-coupled receptors
Although the magnitude of modulation was not different
between neurons from Go+/+ and
G
o
/
mice, the kinetics of the
action of agonists were different, especially the time course of
recovery. Figure 5 shows the time course
of action of baclofen on K+ and
Ca2+ currents in
G
o
/
and
G
o+/+ mice, from recordings like
those in Fig. 2. In cells from
G
o+/+ mice,
K+ current was activated rapidly on exposure to
agonist and it also deactivated rapidly on return to agonist-free
solution. Both activation and deactivation of K+
current by baclofen were too fast to be clearly resolved with the 2-s
sampling period of the experiments (Fig. 5A). Interestingly, the kinetics of agonist action on Ca2+ current
were somewhat different from agonist activation of
K+ current in cells from
G
o+/+ mice, even when determined
simultaneously during the same application of agonist. Both onset of
inhibition of Ca2+ current and recovery from
inhibition were biphasic, with a predominant, rapid (
2 s) phase
followed by a smaller, slower phase.
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In comparing cells from Go+/+ mice
and G
o
/
mice, we focused on
recovery kinetics, which were dramatically different. In
G
o+/+ mice, recovery from
Ca2+ current inhibition by baclofen consisted of
an initial fast phase with a time constant
2 s, where 70-90% of
effect recovered, and a remaining small component with a time constant
of ~5-20 s. The fast phase was poorly resolved with the 2 s
sampling interval, but the magnitude (22 ± 4%) and time constant
(7.0 ± 0.7 s, n = 15) of the slower phase
could be resolved reasonably well.
In cells from Go
/
mice,
recovery from agonist was much slower than in
G
o+/+ mice (Fig. 5B). In
G
o
/
mice, recovery from
activation of K+ current by baclofen was slow
enough to be resolved and could be fit well by a single exponential,
with an average time constant of 7.1 ± 1.0 s
(n = 12). This is at least four- to fivefold slower than the recovery from activation of K+ current
in cells from normal mice (complete in <2 s). Recovery from baclofen
effects on Ca2+ current was also slower in cells
from G
o
/
mice. In
G
o
/
mice, the time course of
recovery from Ca2+ current inhibition was
sigmoidal, with a delay in recovery when the cell was removed from
baclofen. This was followed by a main phase of recovery that was fit
well with a single exponential. This had an average time constant of
14.5 ± 1.1 s (n = 23). It was notable that
recovery from the effects of baclofen on Ca2+
current was slower than decay of K+ current, even
when monitored simultaneously in the same cell (Fig. 5B).
The length of agonist exposure had no obvious effect on recovery
kinetics. If cells were continuously exposed for 1 min to 50 µM
baclofen, Ca2+ currents recovered from inhibition
with a similar time constant as for short exposure in the same cell
(for cells from both Go+/+ and
G
o
/
mice; data not shown).
Recovery from exposure to 2-chloro-adenosine was also slower in
cells from Go
/
mice. Figure
6 shows typical examples of the kinetics
of action of 2-chloro-adenosine in cells from
G
o+/+ (Fig. 6A) and
G
o
/
(Fig. 6B) mice.
In cells from G
o+/+ mice, recovery
from 2-chloro-adenosine was rapid. Effects of 2-chloro-adenosine on
K+ current recovered too fast to be resolved with
the 2-s sampling interval, as for baclofen effects in wild-type cells.
2-Chloro-adenosine effects on Ca2+ currents in
wild-type cells recovered more slowly than the effects on
K+ currents but (unlike those of baclofen) could
generally be fit well by a single exponential (Fig. 6A). The
average time constant was 2.6 ± 0.2 s (n = 12). In cells from G
o
/
mice,
recovery from 2-chloro-adenosine effects was much slower than in
G
o+/+ mice (Fig. 6B).
Recovery from activation of K+ current by
2-chloro-adenosine in G
o
/
mice
could be fit well by a single exponential, with an average time
constant of 7.9 ± 0.9 s (n = 12). Recovery
from 2-chloro-adenosine effects on Ca2+ current
was sigmoidal, with an initial delay followed by a main phase of
recovery that was fit well with a single exponential (Fig.
6B). The main phase had an average time constant of
12.0 ± 0.8 s (n = 16). As for the effects of
baclofen on cells from G
o
/
mice, recovery from effects on Ca2+ current was
slower than decay of agonist-activated K+ current
when monitored in the same cell (Fig. 6B).
|
The much slower recovery rates from agonist effects in cells from
Go
/
animals compared with
G
o+/+ littermates were also seen in
less systematic experiments with somatostatin and 1R-2S-ACPD
(not shown). Slowed recovery was similarly seen in experiments in which
2 mM Ba2+ rather than 2 mM
Ca2+ was the current carrier for calcium channel
current (not shown).
NEM-sensitivity of K+ and Ca2+ current
modulation in Go
/
and
G
o+/+
mice
The sulfhydryl alkylating agent N-ethyl-maleimide (NEM)
disrupts some but not other G protein-mediated transmitter pathways when applied briefly to the outside of the cell. Among the different pathways of Ca2+ current inhibition in
sympathetic neurons, NEM effects appear to be selective for pathways
that are also sensitive to pertussis toxin (Jeong and Ikeda
1998; Shapiro et al. 1994
; Wollmuth et al. 1995
). We attempted to study the pertussis toxin
sensitivity of transmitter effects in CA3 neurons, but the cells
deteriorated during overnight exposure to pertussis toxin. Thus
short-term NEM exposure, which can be performed quickly on freshly
dissociated cells, was a useful tool for distinguishing various
pathways. In rat CA3 neurons, brief (1 min) exposure to 50 uM NEM
completely blocks activation of inwardly rectifying
K+ current by baclofen (Sodickson and Bean
1996
), suggesting that this response is mediated by
Gi or Go.
Figure 7A shows the effect of
NEM exposure on baclofen effects in a CA3 neuron from a
Go+/+ mouse. Both baclofen
activation of K+ current and inhibition of
Ca2+ current were completely disrupted by NEM,
which had no effect on basal currents. However, in neurons from
G
o
/
mice, NEM had a
differential effect on K+ and
Ca2+ current modulation by baclofen (Fig.
7B). Whereas baclofen activation of K+
current was abolished, as in cells from
G
o+/+ mice, NEM reduced but did not
abolish the baclofen effect on Ca2+ current.
|
We also examined the effect of NEM on modulation by 2-chloro-adenosine
in both Go
/
mice and
G
o+/+ mice. Figure
8 shows the responses to
2-chloro-adenosine of the same neurons tested with baclofen in Fig. 7.
In the G
o+/+ cell, NEM blocked both
effects of 2-chloro-adenosine (Fig. 8A). NEM also completely
blocked both effects of 2-chloro-adenosine in the
G
o
/
cell (Fig. 8B).
Thus in the same cell, NEM blocked the effect of baclofen on
Ca2+ currents only partially and completely
blocked the effect of 2-chloro-adenosine. The results of the cells
shown in Figs. 7 and 8 were typical of the collected results on many
cells, summarized in Fig. 9.
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DISCUSSION |
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Interpretation of slowed kinetics
The simplest interpretation for the dramatically different
kinetics of transmitter action in mice lacking Go is that
in wild-type mice, Go is the predominant G protein used for
coupling both GABAB and adenosine receptors to
both K+ and Ca2+ channels
and that in Go
/
mice, different
G proteins take over this role, acting with equal efficacy but
different kinetics. Isoforms of G
i are the most likely candidates for mediating modulation by
GABAB and adenosine receptors in hippocampal
neurons from G
o
/
mice. The
complete elimination by brief exposure to NEM of the effects of
adenosine on both Ca2+ and
K+ current and the effects of baclofen on
K+ current are consistent with these being
mediated by G
i in
G
o
/
mice. The effects of
baclofen on Ca2+ current were only partially
blocked by NEM treatment in G
o
/
mice, suggesting an additional contribution by a G protein other than
G
i.
GTPase activity and recovery
It appears that Go is necessary for normal rapid
kinetics seen in current modulation by G protein-coupled receptors in
hippocampal CA3 neurons. What determines the rate of recovery from
transmitter effects is not known. The rate-limiting step could be GTP
hydrolysis by the G
subunit (see Breitwieser and Szabo
1988
; Zhou et al. 1997
). If G
o
does play a predominant role in coupling GABAB
and adenosine receptors to Ca2+ and
K+ channels in wild-type mice and is substituted
mainly by G
i in G
o
/
mice, it is not obvious why
the kinetics are slower, because the intrinsic rates of GTPase activity
of G
i1, G
i2, and
G
i3 are all similar to that of
G
o (Linder et al. 1990
). However, the
intrinsic GTPase rate for purified G
o subunits is only
about 2 min
1 (Kurachi 1995
;
Linder et al. 1990
), an order of magnitude slower than
the recovery from effects of transmitters on either GIRK channels or
Ca2+ channels (0.5-2
s
1). Recently, a family of regulators of G
protein signaling (RGS) proteins have been discovered which influence G
protein signaling by speeding up GTPase activity of various G
subunits (Watson et al. 1996
; Zerangue and Jan
1998
). RGS proteins appear to be widely distributed in the
brain (Gold et al. 1997
), and have been shown to speed
kinetics of K+ and Ca2+
channel modulation in heterologous expression systems (Doupnik et al. 1997
; Jeong and Ikeda 1998
;
Melliti et al. 1999
; Saitoh et al. 1997
).
A candidate RGS protein for the responses we studied is RGS8, which is
expressed in neural tissue (Gold et al. 1997
) and binds
to both G
o and G
i3 subunits
(Saitoh et al. 1997
). One possibility is that the
relevant RGS proteins in CA3 neurons are less efficacious on
Gi isoforms that substitute for Go in the
G
o
/
mice.
Kinetics of recovery of IK versus ICa
In Go
/
mice, kinetics of
recovery from agonist effects on both GIRK current and
Ca2+ current were slow enough to be
well-resolved. It was striking that, when monitored simultaneously in
the same cell, recovery of effects on Ca2+
current were slower (by a factor of 2-4) than recovery of GIRK current. Both effects are believed to be mediated by direct binding of
subunits to the channels. Why then do the kinetics differ? One
possibility is that the difference in recovery kinetics reflects different stoichiometry of
binding to GIRK channels and calcium channels. Activation of GIRK channels appears to require binding of
multiple
subunits (Ito et al. 1992
;
Krapivinsky et al. 1995
), whereas binding of a single
subunit might be enough to inhibit a calcium channel
(Zamponi and Snutch 1998
). If so, activation of GIRK
channels would be expected to have a steeper dependence on the
concentration of free
subunits. As the concentration of free
subunits declines after removal of agonist, significant inhibition of calcium channels might remain after the concentration is
too low for activation of GIRK channels.
Specificity and plasticity of G proteins
Overall, our results in hippocampal CA3 neurons are consistent with previous evidence from other neurons and cell lines suggesting that effects of many neurotransmitters on Ca2+ channels are mediated by Go. However, the results also show that other G proteins are capable of mediating inhibition with equal efficacy. This implies that there is no special need for Go in the coupling pathways. It may be that Go predominates in normal mice not because of any intrinsic selectivity for Go in the pathways but simply as a result of a large excess of Go compared with other G proteins.
Perhaps the most surprising result from our experiments is the lack of
change in the magnitude of modulation of Ca2+
channels and GIRK channels, despite the likelihood that Go
normally mediates modulation. An obvious possibility is that with loss of Go there is compensatory up-regulation of expression of
other G proteins. However, at least at the level of the whole brain, there is little or no change in expression of subunits of other G
proteins in G
o
/
mice
(Mende et al. 1998
), and there is a dramatic reduction
of G
(to ~30% of wild-type). Another possibility is that even
though the total level of Gi/Go family proteins
decreases, the local concentration of G proteins near the effector
channels may not change. Perhaps the concentration of G proteins used
for signaling to ion channels is tightly regulated in small subcellular
areas comprising clusters of receptors, G proteins, and ion channels (and probably RGS proteins). Comparing the speed of modulation with
two-dimensional diffusion, it can be calculated that receptors, G
proteins, and channels are within at least 1 u of one another (Hille 1992
; Zhou et al. 1997
),
suggesting the existence of mechanisms by which location and
stoichiometry of these proteins are coordinated. In fact, the elements
of the G-protein-coupled cascade mediating phototransduction in
Drosophila are known to be colocalized by means of a protein with five
PDZ domains (Tsunoda et al. 1997
). If an analogous
mechanism exists for regulation of Ca2+ channels
and K+ channels by G protein-coupled receptors,
the level of expression of G proteins is probably not the limiting
factor in formation of such complexes, because most G proteins are
present in large excess over receptors or effectors. Thus it may be
reasonable that a large reduction in total G protein expression could
result in little change in local concentration near receptors and
channels. Interestingly, adenylate cyclase activity is also unchanged
by the dramatic reduction in G
in
G
o
/
mice, also suggesting
compartmentalization or the existence of local membrane pools
(Mende et al. 1998
).
Consequences for brain function
The slowed kinetics of recovery from agonist effects in
Go
/
mice could contribute to
changes in neurological function. It seems likely that
GABAB-mediated inhibitory synaptic currents might
also decay more slowly in G
o
/
mice, because synaptic currents measured in brain slices (Otis et al. 1993
) have similar kinetics as GIRK currents in isolated cells (Sodickson and Bean 1996
).
GABAB receptors can also act presynaptically to
inhibit transmission (e.g., Dittman and Regehr 1997
;
Thompson and Gähwiler 1992
), with kinetics very
similar to that of inhibition of Ca2+ channels
(Dittman and Regehr 1997
; Pfrieger et al.
1994
). Thus presynaptic effects of GABA are also likely
prolonged in G
o
/
mice.
Prolonging GABAB effects at both presynaptic and
postsynaptic sites may change the dynamic control of synaptic strength
at many synapses and contribute to the neurological changes seen in
G
o
/
mice. Other changes,
including subtle changes in development, are also possible.
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ACKNOWLEDGMENTS |
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This work was supported by National Institutes of Health Grants HL-35034 to B. P. Bean and HL-52320 and GM-36359 to E. J. Neer.
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FOOTNOTES |
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Address for reprint requests: B. P. Bean, Dept. of Neurobiology, Harvard Medical School, 220 Longwood Ave., Boston, MA 02115.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 16 August 1999; accepted in final form 19 October 1999.
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REFERENCES |
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