 |
INTRODUCTION |
A large number of neurotransmitters including
adenosine, GABA (via GABAB receptors), serotonin,
dopamine, norepinephrine, acetylcholine, opioids, and somatostatin
modulate neuronal excitability by activating an inwardly rectifying
K+ current via a membrane-delimited, G
protein-mediated mechanism (Dascal 1997
; Yamada
et al. 1998
). The notion that G protein-activated, inwardly
rectifying K+ (GIRK) channels represent a common
target of many neurotransmitter systems is mainly based on the
following observations. 1) On the whole cell level, GIRK
currents display almost identical electrophysiological and
pharmacological properties, irrespective of the transmitter used to
evoke them. 2) The combined application of transmitters at
saturating concentrations produces subadditive and often occlusive GIRK
current responses (Andrade et al. 1986
; Dascal
1997
; McCormick and Williamson 1989
;
Sodickson and Bean 1998
; Yamada et al.
1998
). However, despite the large body of evidence suggesting a
common mechanism of GIRK current generation, it is still unclear
whether this notion also holds on the single channel level. Data on
elementary properties of GIRK channels in native CNS neurons are
sparse, and the findings published so far do not yield a uniform
picture. To our knowledge, elementary properties of adenosine- and
baclofen-activated GIRK channels in native CNS neurons have not been
reported yet. Here, we employed variance analysis of adenosine- and
baclofen-induced current fluctuations to determine and compare
properties of the underlying K+ channels.
 |
METHODS |
As described in detail elsewhere (Alzheimer et al.
1993
), a combined enzymatic/mechanic dissociation procedure was
employed to prepare acutely isolated pyramidal cell somata from the
sensorimotor cortex of anesthetized rats that were 2-3 weeks old. The
recording chamber was mounted on the stage of an inverted microscope
equipped with Hoffman modulation optics. Current signals from visually identified pyramidal cell somata recorded in whole-cell voltage-clamp mode were sampled at 20 kHz and filtered at 5 kHz (
3 dB) using an
Axopatch 200 amplifier in conjunction with a TL-1 interface and pClamp
6.0 software (all from Axon Instruments). All recordings were made at
room temperature (21-24°C). Standard bath solution was composed of
(in mM) 150 NaCl, 3 KCl, 2 CaCl2, 2 MgCl2, 10 HEPES, and 10 D-glucose (pH
7.4). After whole cell access was established, GIRK currents were
investigated in an extracellular solution containing (in mM) 85 NaCl,
60 KCl, 2 MgCl2, 2 CaCl2, 5 NaHEPES, 5 HEPES, and 10 D-glucose (pH 7.4). Patch pipets
were coated with Sylgard in the tapered region and filled with (in mM)
135 K-gluconate, 5 HEPES, 3 MgCl2, 5 EGTA, 2 Na2ATP, and 2 NaGTP (pH 7.25, adjusted with KOH).
Electrode resistance in the whole cell configuration was 5-15 M
before series resistance compensation (70-75%). Voltage readings were
corrected for liquid junction potential. If not noted otherwise,
holding potential (VH) was
80 mV. Cell
capacitance determined by means of the built-in whole cell capacitance
compensation circuit of the amplifier was used to estimate cell surface
area assuming a specific membrane capacitance of 1 µFcm
2. Adenosine and baclofen were applied at
increasing concentrations by means of a remotely controlled,
solenoid-operated Y tube system. Both drugs were purchased from Sigma
(Deisenhofen, Germany). Statistics are presented as means ± SE.
Statistical analysis (t-test) and curve fitting was done
with the use of Graphpad prism 2.0.
 |
RESULTS |
To increase current flow through GIRK channels,
extracellular K+ was elevated to 60 mM and GIRK
channel activity was recorded as inward current. Figure
1, A and B shows
the rapid and dose-dependent induction of GIRK currents by two
different concentrations of the GABAB receptor
agonist, baclofen (10 and 100 µM), in an acutely isolated rat
pyramidal neuron. Consistent with previous findings from other
preparations (Dascal 1997
; Sodickson and Bean
1998
), GIRK current responses to baclofen as well as those to
adenosine were inwardly rectifying and completely suppressed by
Ba2+ (200 µM, not shown). Dose-response
relationships of the two agonists were obtained by relating normalized
GIRK current to log agonist concentration. From the curves fitted to
the data points, we obtained EC50 values for
baclofen and adenosine of 25.6 µM and 2.0 µM, respectively, (Fig.
1, C and D). For noise analysis of agonist-evoked current fluctuations, we used stretches of current recordings obtained
at increasing agonist concentrations (Fig.
2, A and B). We
then calculated the current variance (pA2) at
each agonist concentration and plotted it as a function of the mean
GIRK current observed at the same concentration (Fig. 2, C
and D). To isolate membrane fluctuations associated with GIRK channel activity from background noise, the current variance of
control recordings was subtracted from the current variance determined
during agonist applications. The data points were fit to an equation of
the form
|
(1)
|
where
2 is the variance of the
current, i the unitary current amplitude, I the
whole cell current amplitude, and N the number of available
channels (cf. Traynelis and Jaramillo 1998
). The continuous solid line through the filled circles is the fit with Eq. 1,
giving estimated values for i and N of 1.48 pA
and 572 channels, respectively, for baclofen, and of 1.26 pA and of 584 channels, respectively, for adenosine. To obtain an estimate of GIRK
channel density, N was related to cell surface area and
density was expressed as channels µm
2.
Single-channel conductance (
) was determined using the relationship
|
(2)
|
where V is the holding potential, and
EK the equilibrium potential of
K+ under our recording conditions (
24 mV).

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Fig. 1.
G protein-activated inwardly rectifying K+ (GIRK) current
responses evoked by baclofen and adenosine in acutely isolated
pyramidal neurons from rat neocortex. A: morphology of
acutely isolated cell soma with patch pipet attached. B:
reversible and dose-dependent induction of GIRK current by baclofen.
Because of the recording condition ([K+]0: 60 mM, VH: 70 mV), GIRK currents were inward. C,
D: dose-response relationship of GIRK currents evoked by
baclofen (n = 11) and adenosine
(n = 12). Currents were normalized to the maximum
response. Dose response curves were fitted to the data points using an
equation of the form I = Imax/[(1 + (EC50/[A])n], where
Imax is the maximum current evoked by the
agonist, [A] the agonist concentration, EC50 the agonist
concentration yielding a half-maximal current response, and n the Hill
coefficient. Estimated EC50 value for baclofen was 25.6 µM (95% confidence interval 21.5-30.4 µM, Hill slope 0.98) and
for adenosine 2.0 µM (0.26-14.5 µM, Hill slope 0.69).
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Fig. 2.
Variance analysis of baclofen- and adenosine-induced membrane current
fluctuations. A and B: depiction of
original current traces used for the noise analysis shown in
C and D. At each agonist concentration,
current variance and mean current were independently determined for two
different current stretches 200 ms long.
|
|
The histograms of Fig. 3 depicting the
mean single channel conductance and the mean density of GIRK channels
evoked by baclofen and adenosine demonstrate that the two agonists
appear to activate GIRK channels of identical unitary conductance and
density. Average normalized channel density for adenosine-activated
GIRK channels was 0.49 ± 0.074 channels
µm
2 (n = 8), and 0.46 ± 0.097 channels µm
2 for baclofen-activated
GIRK channels (n = 6). At the
[K+]o/[K+]i
gradient of 60/151 mM used in our study, adenosine-induced GIRK
channels had an average single-channel conductance of 25.0 ± 0.89 pS (n = 8), which was not significantly different from the single-channel conductance of baclofen-induced GIRK channels (25.5 ± 1.57 pS, n = 6). Assuming that the
unitary conductance of inwardly rectifying K+
channels increases approximately proportional with the square root of
[K+]o (Sakmann and
Trube 1984
), the GIRK channels studied here should display
conductances between 5 and 6 pS at a physiological
[K+]o of 3 mM.

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Fig. 3.
Unitary conductance and normalized density of GIRK channels evoked by
adenosine (n = 8) and baclofen
(n = 6).
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 |
DISCUSSION |
This study is the first to report elementary properties of GIRK
channels mediating the hyperpolarizing effect of adenosine and baclofen
in native CNS neurons. In our hands, both transmitters activated
K+ channels of virtually identical single-channel
conductance, which was estimated as 5-6 pS under physiological
conditions. The simplest explanation of this finding would be that both
transmitters engage the same type of channel. Since N of
Eq. 1 indicates the total number of channels that can
potentially contribute to the whole cell current response, it should
reflect the pool of channels available for maximum transmitter
response. Given the fact that GIRK channels recruited by the two
agonists are expressed at equal densities, it appears reasonable to
assume that both agonists access the same pool of GIRK channels (or the
same pool of G proteins the 
-subunits of which then activate the
GIRK channels). This notion is consistent with previous findings
showing subadditive or often occlusive effects when saturating
concentrations of adenosine, baclofen or other agonists known to target
GIRK channels were applied simultaneously (Andrade et al.
1986
; Sodickson and Bean 1998
).
Whereas our data indicate that adenosine and baclofen recruit
electrophysiologically uniform channels, this does not necessarily imply that the channels are identical in terms of molecular structure. GIRK channels are tetrameric proteins of the Kir3.0 (GIRK) subfamily which comprises four cloned subunits (Kir3.1-3.4) in mammals
(Isomoto et al. 1997
). In the pyramidal cell layers of
rat neocortex, Kir3.1-3.3 subunits are abundantly expressed,
suggesting that the GIRK channels of these neurons are assembled from
these subunits (Karschin et al. 1996
). Coexpression of
adenosine A1 receptors with different combinations of Kir3.0 subunits
in Xenopus oocytes demonstrated effective coupling only when
A1 receptors were expressed in the presence of Kir3.1 combined with one
additional subunit, whereas other Kir3.0 assemblies including
homotetrameric channels yielded negligible current responses to A1
receptor activation (Pfaff and Karschin 1997
).
Single-channel recordings showed that heterologously expressed
Kir3.1/3.2 channel multimers displayed a unitary conductance compatible
with the one estimated here for adenosine- and baclofen-activated GIRK
channels (Lesage et al. 1995
; Spauschus et
al. 1996
). Based on these observations and the localization of
Kir3.0 subunits noted above, we propose that the GIRK current responses
described here were mediated by heterotetramers of Kir3.1/3.2 and/or
Kir3.1/3.3 subunits. Although our data do not allow us to resolve the
stochiometry of adenosine- and baclofen-activated GIRK channels in
neocortical pyramidal cells, they strongly suggest that the two
transmitters share a common transduction mechanism on the
single-channel level.
How do our findings relate to the properties of GIRK channels reported
from other CNS regions? Whereas we are not aware of single-channel data
on adenosine-activated GIRK channels, Premukar and Gage (1994)
reported
that, in cultured hippocampal neurons, baclofen induced a much larger,
67 pS K+ channel (in symmetric
K+ solution). However, because that baclofen
effect was observed in cell-attached recordings with the agonist in the
bath (requiring a diffusible second messenger), the
K+ channel is unlikely to belong to the GIRK
channel subfamily, which is typically activated in a membrane-delimited
fashion (Yamada et al. 1998
). Noise analysis of slow
IPSCs showed that synaptically released GABA acting on
GABAB receptors activates a
K+ channel, the conductance of which is within
the range reported here for baclofen-induced GIRK channels (De
Koninck and Mody 1997
). Unlike these channels, however, the
K+ channels mediating slow IPSCs were
nonrectifying or slightly outwardly rectifying suggesting that they
might represent a distinct channel type (De Koninck and Mody
1997
). Penington et al. (1993)
reported inwardly
rectifying K+ channels with multiple
subconductance levels up to 120 pS that were activated by serotonin in
dorsal raphe neurons in a membrane-delimited fashion, whereas two other
groups showed that, in hippocampal pyramidal neurons, serotonin induces
small-conductance GIRK channels resembling those described here
(Oh et al. 1995
; Van Dongen et al. 1988
).
GIRK channels of similar small conductance were also observed in rat
locus coeruleus neurons during application of somatostatin,
metenkephalin, and noradrenalin (Arima et al. 1998
; Grigg et al. 1996
). These latter findings, now
strengthened by our data on adenosine- and baclofen-activated GIRK
channels, lend support to the view that a variety of neurotransmitters
converges onto an electrophysiologically uniform, small conductance
type of K+ channel. The above discrepancies might
be explained by GABA acting on more than one type of
K+ channel, or by the fact that gating properties
of GIRK channels are influenced by the patch-clamp configuration used
to study them.
This work was supported by the Deutsche Forschungsgemeinschaft (SFB
391, Al 294/7, and Heisenberg fellowship) and the
Friedrich-Baur-Stiftung.
Address for reprint requests: C. Alzheimer, Dept. of Physiology,
University of Munich, Pettenkoferstr. 12, D-80336, Germany.
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.