Altered Regulation of Potassium and Calcium Channels by GABAB and Adenosine Receptors in Hippocampal Neurons From Mice Lacking Galpha o

Gabriela J. Greif,1 Deborah L. Sodickson,1 Bruce P. Bean,1 Eva J. Neer,2 and Ulrike Mende2

 1Department of Neurobiology, Harvard Medical School; and  2Department of Cardiology, Brigham and Women's Hospital, Boston, Massachusetts 02115


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 Galpha o. 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 Galpha o. In CA3 neurons from Galpha 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 Galpha o+/+ littermates. However, the kinetics of transmitter action were dramatically altered in Galpha 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 Galpha o-/- mice [time constant of 12.0 ± 0.8 (SE) s] than in neurons from Galpha 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 Galpha 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 Galpha o. However, modulation is changed in the absence of Galpha o in having much slower recovery kinetics. A likely possibility is that the very abundant Galpha o is normally used but, when absent, can readily be replaced by G proteins with different properties.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Many effects of neurotransmitters are mediated by heterotrimeric G proteins, composed of Galpha and Gbeta gamma 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 Galpha o gene in mice are surprisingly modest. Mice lacking Galpha 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 Gbeta gamma 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 Gbeta gamma subunits to Ca2+ channels (De Waard et al. 1997; Herlitze et al. 1996; Ikeda 1996; Zamponi et al. 1997). Although Gbeta gamma subunits directly control GIRK channels and Ca2+ channels, the type of the Galpha subunit from which the Gbeta gamma is released can determine specificity and kinetics of transmitter action. In addition, there is evidence for more direct involvement of Galpha 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 Gbeta gamma 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 Galpha o present a useful model to study the role of Go in G protein-mediated ion channel modulation. Several alterations of channel modulation in Galpha 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 Galpha o-/- mice (Jiang et al. 1998).

To further study the role of Galpha o 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 Galpha o-/- mice but that kinetics of transmitter modulation are dramatically different compared with neurons from wild-type mice.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell preparation

Galpha o-/-, Galpha o+/- , and Galpha 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 MOmega 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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1. Simultaneous monitoring of baclofen modulation of inwardly rectifying K+ current and voltage-activated Ca2+ current. Illustrated voltage protocol was applied every 2 s. Dashed lines: zero current. A: currents before and 4 s after application of 100 uM baclofen. B: currents with and without baclofen (in the same cell as in A) but in external solution containing 300 µM BaCl2. C: currents with and without baclofen recorded in external solution in which 2 mM CoCl2 replaced 2 mM CaCl2. Same cell as in A and B.

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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Fig. 2. Current voltage relationships for baclofen (100 uM) application for various ionic conditions. Pulses (5 ms) were applied every 100 ms from a steady holding potential of -90 mV. Current at end of 5-ms pulse is plotted vs. membrane potential. (Different cell than in Fig. 1.) A: 300 µM Ba2+ blocks baclofen activation of inward current negative to -60 mV but has no effect on inhibition of inward current positive to -50 mV. B: substitution of Co2+ for Ca2+ blocks inward current elicited by depolarization positive to -50 mV. Net current is near zero at -12 mV and baclofen has no effect on current at this voltage.

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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Fig. 3. Inwardly rectifying characteristic of baclofen-activated current. Recording was done with standard external solution containing 16 mM K+ and with a K+-based internal solution containing (in mM) 189 K2HPO4, 9 KCl, 9 HEPES, 9 EGTA, 14 Tris-creatinePO4, 4 Mg-ATP, and 0.3 Tris-GTP (pH 7.4 with KOH). A: current-voltage relationship was determined with a ramp voltage command (-162 to +38 mV in 500 ms) and repeated every second. Traces were signal-averaged from 2 such ramps in control and 4 in the presence of 100 uM baclofen. B: baclofen-sensitive current obtained by subtracting the traces in A.

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 Galpha o-/- mice and from Galpha o+/+ littermates. The genotypes of the animals were confirmed after the experiment by PCR.

In CA3 neurons from Galpha o+/+ 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 Galpha o-/- mice (Fig. 4B). Baclofen inhibition of Ca2+ current was identical in magnitude (25 ± 1%; n = 34) in cells from Galpha o-/- mice as in cells from Galpha 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 Galpha o-/- mice compared with 7.9 ± 0.7 pA/pF (n = 30) in cells from Galpha o+/+ mice (Fig. 4F).



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Fig. 4. Effects of baclofen (A and B) and 2-chloroadenosine (C and D) on ionic currents in hippocampal pyramidal neurons from Galpha o+/+ (A and C) and Galpha o-/- (B and D) mice. A and B: baclofen inhibition of Ca2+ current (carried by 2 mM Ca2+ and elicited by a step to -12 mV) and activation of inwardly rectifying K+ current (carried by 16 mM K+ and measured at -142 mV). Solutions and protocols as in Fig. 1. Thin trace: control. Bold trace: 50 µM baclofen. C and D: response to 25 µM 2-chloro-adenosine (same cells as A and B). E: collected results for inhibition of Ca2+ current by baclofen and 2-chloro-adenosine from cells of Galpha o+/+ mice (black bar) and Galpha o-/- mice (gray bar). Baclofen: 30 cells for Galpha o+/+ and 34 cells for Galpha o-/-. 2-Chloro-adenosine: n = 23 cells for Galpha o+/+ and 23 cells for Galpha o-/-. F: mean agonist-activated K+ current (normalized relative to cell capacitance) elicited by baclofen and 2-chloro-adenosine in cells from Galpha o+/+ (black bar) and Galpha o-/- (gray bar) mice. Same cells as E.

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 Galpha o-/- mice as in those from Galpha o+/+ mice (Fig. 4, D-F). We also tested somatostatin (5 cells from Galpha o-/- mice and 8 cells from Galpha o+/+ mice) and the metabotropic glutamate receptor agonist 1R,2S-ACDP (3 cells from Galpha o-/- mice and 5 cells from Galpha o+/+ mice). Although these series of experiments were less systematic, there was no obvious difference in the size of effects on cells from Galpha o+/+ and Galpha 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 Galpha o-/- and Galpha 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 Galpha o+/+ and Galpha 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 Galpha o-/- and Galpha o+/+ mice, from recordings like those in Fig. 2. In cells from Galpha 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 Galpha 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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Fig. 5. Time course of action of baclofen in hippocampal CA3 neurons of (A) Galpha o+/+ and (B) Galpha o-/- mice. Voltage protocol used in Fig. 1 was applied every 2 s. Gray bars: exposure of cells to neurotransmitter. Solid lines: best fits to the sum of 2 exponentials for cell from Galpha o+/+ mice and to 1 exponential for cell from Galpha o-/- mice.

In comparing cells from Galpha o+/+ mice and Galpha o-/- mice, we focused on recovery kinetics, which were dramatically different. In Galpha 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 Galpha o-/- mice, recovery from agonist was much slower than in Galpha o+/+ mice (Fig. 5B). In Galpha 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 Galpha o-/- mice. In Galpha 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 Galpha o+/+ and Galpha o-/- mice; data not shown).

Recovery from exposure to 2-chloro-adenosine was also slower in cells from Galpha o-/- mice. Figure 6 shows typical examples of the kinetics of action of 2-chloro-adenosine in cells from Galpha o+/+ (Fig. 6A) and Galpha o-/- (Fig. 6B) mice. In cells from Galpha 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 Galpha o-/- mice, recovery from 2-chloro-adenosine effects was much slower than in Galpha o+/+ mice (Fig. 6B). Recovery from activation of K+ current by 2-chloro-adenosine in Galpha 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 Galpha 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).



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Fig. 6. Time-course of action of 2-chloro-adenosine in hippocampal CA3 neurons of (A) Galpha o+/+ and (B) Galpha o-/- mice. Voltage protocol used in Fig. 1 was applied every 2 s. Gray bars: exposure of cells to neurotransmitter. Solid lines: best fits to 1 exponential.

The much slower recovery rates from agonist effects in cells from Galpha o-/- animals compared with Galpha 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 Galpha o-/- and Galpha 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 Galpha o+/+ 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 Galpha 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 Galpha o+/+ mice, NEM reduced but did not abolish the baclofen effect on Ca2+ current.



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Fig. 7. Effects of N-ethyl-maleimide (NEM) on K+ and Ca2+ current modulation by baclofen in hippocampal neurons from Galpha o+/+ and Galpha o-/- mice. A: CA3 neuron from Galpha o+/+ mouse. Top: effect of baclofen on currents elicited before (left) and after (right) exposure to 50 µM NEM for 1 min. Currents were elicited by voltage protocol as in Fig. 1 except that step to -142 mV was for 10 ms and step to -12 mV was for 20 ms. Thin trace: control. Bold trace: 50 µM baclofen (traces overlap after NEM). Bottom: time course of baclofen effects. Gray bars: exposure to baclofen. Interrupted x-axis indicates exposure of neuron to NEM for 1 min between measurements of baclofen responses. NEM had no effect on the basal currents. B: CA3 neuron from Galpha o-/- mouse studied with the same experimental protocol.

We also examined the effect of NEM on modulation by 2-chloro-adenosine in both Galpha o-/- mice and Galpha o+/+ mice. Figure 8 shows the responses to 2-chloro-adenosine of the same neurons tested with baclofen in Fig. 7. In the Galpha o+/+ cell, NEM blocked both effects of 2-chloro-adenosine (Fig. 8A). NEM also completely blocked both effects of 2-chloro-adenosine in the Galpha 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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Fig. 8. Effects of NEM on K+ and Ca2+ current modulation by 2-chloro-adenosine in hippocampal neurons from Galpha o+/+ and Galpha o-/- mice. Protocols as in Fig. 7. Gray bars: exposure to 25 µM 2-chloro-adenosine.



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Fig. 9. Collected results for effects of NEM on Ca2+ current inhibition (top) and K+ current activation (bottom) by baclofen (left) and 2-chloro-adenosine (right) in Galpha o+/+ and Galpha o-/- mice. Black bars: before NEM. Gray bars: after NEM exposure for 1 min. Baclofen: n = 3 cells from Galpha o+/+ and n = 9 cells from Galpha o-/-. 2-Chloro-adenosine: n = 3 cells from Galpha o+/+ and n = 7 cells from Galpha o-/-.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 Galpha o-/- mice, different G proteins take over this role, acting with equal efficacy but different kinetics. Isoforms of Galpha i are the most likely candidates for mediating modulation by GABAB and adenosine receptors in hippocampal neurons from Galpha 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 Galpha i in Galpha o-/- mice. The effects of baclofen on Ca2+ current were only partially blocked by NEM treatment in Galpha o-/- mice, suggesting an additional contribution by a G protein other than Galpha i.

GTPase activity and recovery

It appears that Galpha o 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 Galpha subunit (see Breitwieser and Szabo 1988; Zhou et al. 1997). If Galpha 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 Galpha i in Galpha o-/- mice, it is not obvious why the kinetics are slower, because the intrinsic rates of GTPase activity of Galpha i1, Galpha i2, and Galpha i3 are all similar to that of Galpha o (Linder et al. 1990). However, the intrinsic GTPase rate for purified Galpha 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 Galpha 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 Galpha o and Galpha 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 Galpha o-/- mice.

Kinetics of recovery of IK versus ICa

In Galpha o-/- 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 beta gamma subunits to the channels. Why then do the kinetics differ? One possibility is that the difference in recovery kinetics reflects different stoichiometry of beta gamma binding to GIRK channels and calcium channels. Activation of GIRK channels appears to require binding of multiple beta gamma subunits (Ito et al. 1992; Krapivinsky et al. 1995), whereas binding of a single beta gamma 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 beta gamma subunits. As the concentration of free beta gamma 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 alpha  subunits of other G proteins in Galpha o-/- mice (Mende et al. 1998), and there is a dramatic reduction of Gbeta gamma (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 Gbeta gamma in Galpha 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 Galpha o-/- mice could contribute to changes in neurological function. It seems likely that GABAB-mediated inhibitory synaptic currents might also decay more slowly in Galpha 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 Galpha 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 Galpha o-/- mice. Other changes, including subtle changes in development, are also possible.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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