Evidence that Neuronal G-Protein-Gated Inwardly Rectifying K^+ Channels are Activated by Gbetagamma Subunits and Function as Heteromultimers
ABSTRACT Guanine nucleotide-binding proteins (G proteins) activate K^+ conductances in cardiac atrial cells to slow heart rate and in neurons to decrease excitability. cDNAs encoding three isoforms of a G-protein-coupled, inwardly rectifying K^+ channel (GIRK) have recently been cloned from cardiac (GIRK1/Kir 3.1) and brain cDNA libraries (GIRK2/Kir 3.2 and GIRK3/Kir 3.3). Here we report that GIRK2 but not GIRK3 can be activated by G protein subunits Gbeta_1 and Ggamma_2 in Xenopus oocytes. Furthermore, when either GIRK3 or GIRK2 was coexpressed with GIRK1 and activated either by muscarinic receptors or by Gbetagamma subunits, G-protein-mediated inward currents were increased by 5- to 40-fold. The single-channel conductance for GIRK1 plus GIRK2 coexpression was intermediate between those for GIRK1 alone and for GIRK2 alone, and voltage-jump kinetics for the coexpressed channels displayed new kinetic properties. On the other hand, coexpression of GIRK3 with GIRK2 suppressed the GIRK2 alone response. These studies suggest that formation of heteromultimers involving the several GIRKs is an important mechanism for generating diversity in expression level and function of neurotransmitter-coupled, inward rectifier K^+ channels.
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ABSTRACT: GIRK channels control spike frequency in atrial pacemaker cells and inhibitory potentials in neurons. By directly responding to G proteins, PIP2 and Na(+), GIRK is under the control of multiple signaling pathways. In this study, the mammalian GIRK2 channel has been purified and reconstituted in planar lipid membranes and effects of Gα, Gβγ, PIP2 and Na(+) analyzed. Gβγ and PIP2 must be present simultaneously to activate GIRK2. Na(+) is not essential but modulates the effect of Gβγ and PIP2 over physiological concentrations. Gαi1(GTPγS) has no effect, whereas Gαi1(GDP) closes the channel through removal of Gβγ. In the presence of Gβγ, GIRK2 opens as a function of PIP2 mole fraction with Hill coefficient 2.5 and an affinity that poises GIRK2 to respond to natural variations of PIP2 concentration. The dual requirement for Gβγ and PIP2 can help to explain why GIRK2 is activated by Gi/o, but not Gq coupled GPCRs.eLife Sciences 07/2014; · 8.52 Impact Factor
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ABSTRACT: Despite a high level of sequence homology, tandem pore domain halothane-inhibited K+ channel 1 (THIK1) produces background K+ currents whereas THIK2 is silent. This lack of activity is due to a unique combination of intracellular retention and weak basal activity in the plasma membrane. Here, we designed THIK subunits containing dominant-negative mutations (THIK1DN and THIK2DN). THIK2DN mutant inhibits THIK1 currents whereas THIK1DN inhibits an activated form of THIK2 (THIK2-A155P-I158D). In situ proximity ligation assays and FRET experiments support a physical association between THIK1 and THIK2. Next, we expressed covalent tandems of THIK proteins to obtain expression of pure heterodimers. Td-THIK1-THIK2 produces K+ currents of amplitude similar to Td-THIK1-THIK1 but with a noticeable difference in the current kinetics. Unlike Td-THIK2-THIK2 that is mainly detected in the endoplasmic reticulum (ER), Td-THIK1-THIK2 distributes at the plasma membrane indicating that THIK1 can mask the ER retention/retrieval motif of THIK2. Kinetics and unitary conductance of Td-THIK1-THIK2 are intermediate between THIK1 and THIK2. Altogether, these results show that THIK1 and THIK2 form active heteromeric channels, further expanding the known repertoire of K+ channels.Journal of Biological Chemistry 08/2014; · 4.60 Impact Factor
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ABSTRACT: Keppen-Lubinsky syndrome (KPLBS) is a rare disease mainly characterized by severe developmental delay and intellectual disability, microcephaly, large prominent eyes, a narrow nasal bridge, a tented upper lip, a high palate, an open mouth, tightly adherent skin, an aged appearance, and severe generalized lipodystrophy. We sequenced the exomes of three unrelated individuals affected by KPLBS and found de novo heterozygous mutations in KCNJ6 (GIRK2), which encodes an inwardly rectifying potassium channel and maps to the Down syndrome critical region between DIRK1A and DSCR4. In particular, two individuals shared an in-frame heterozygous deletion of three nucleotides (c.455_457del) leading to the loss of one amino acid (p.Thr152del). The third individual was heterozygous for a missense mutation (c.460G>A) which introduces an amino acid change from glycine to serine (p.Gly154Ser). In agreement with animal models, the present data suggest that these mutations severely impair the correct functioning of this potassium channel. Overall, these results establish KPLBS as a channelopathy and suggest that KCNJ6 (GIRK2) could also be a candidate gene for other lipodystrophies. We hope that these results will prompt investigations in this unexplored class of inwardly rectifying K(+) channels. Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.The American Journal of Human Genetics 01/2015; 96(2). · 11.20 Impact Factor
Proc. Natl. Acad. Sci. USA
Vol. 92, pp. 6542-6546, July 1995
Evidence that neuronal G-protein-gated inwardly rectifying K+
channels are activated by GI3y subunits and function
PAULO KOFUJI, NORMAN DAVIDSON, AND HENRY A. LESTER*
Division of Biology 156-29, California Institute of Technology, Pasadena, CA 91225
Contributed by Norman Davidson, April 10, 1995
teins) activate K+ conductances in cardiac atrial cells to slow
heart rate and in neurons to decrease excitability. cDNAs
encoding three isoforms of a G-protein-coupled, inwardly
rectifying K+ channel (GIRK) have recently been cloned from
cardiac (GIRK1/Kir 3.1) and brain cDNA libraries (GIRK2/
Kir 3.2 and GIRK3/Kir 3.3). Here we report that GIRK2 but
not GIRK3 can be activated by G protein subunits Gj3, and
G-y2 in Xenopus oocytes. Furthermore, when either GIRK3 or
GIRK2 was coexpressed with GIRKI and activated either by
muscarinic receptors or by Gf3'y subunits, G-protein-mediated
inward currents were increased by 5- to 40-fold. The single-
channel conductance for GIRK1 plus GIRK2 coexpression
was intermediate between those for GIRKI alone and for
GIRK2 alone, and voltage-jump kinetics for the coexpressed
channels displayed new kinetic properties. On the other hand,
coexpression of GIRK3 with GIRK2 suppressed the GIRK2
alone response. These studies suggest that formation of het-
eromultimers involving the several GIRKs is an important
mechanism for generating diversity in expression level and
function of neurotransmitter-coupled, inward rectifier K+
Guanine nucleotide-binding proteins (G pro-
Binding of neurotransmitters or hormones to seven-helix
receptors regulates the activity of many ion-selective channels
(1). In cardiac atrium, stimulation of muscarinic receptors
leads to the opening of inwardly rectifying K+ channels (2, 3)
with a much faster time course (100-500 ms) (4, 5) than for
most effects of seven-helix receptors on channels. The atrial
pathway has the features expected for the involvement of
guanine nucleotide-binding proteins (G proteins) of the Gior
Goclass (2, 3) and proceeds without the mandatory mediation
of a diffusible, cytoplasmic second messenger (3, 6). The atrial
channel is also activated by exogenously applied G protein
Gf3y subunits to the cytoplasmic face of excised patches (7-9),
suggesting that direct channel-G protein association causes
channel activation (10).
While channel activation by this membrane-delimited path-
way was first described and characterized in atrial cells, it is
now clear that similar mechanisms are widespread in neurons
as well, underlying inhibitory actions of several nonpeptide and
peptide neurotransmitters (11). A pertussis-toxin-sensitive,
neurotransmitter-activated, inwardly rectifying K+ conduc-
tance is found in hippocampus (12, 13), dorsal raphe (14, 15),
and locus coeruleus (16, 17).
Recently, the muscarinic channel from cardiac atrium has
been cloned and named GIRKI (for G-protein coupled,
inwardly rectifying K+ channel 1), KGA, or Kir3.1 (18-20).
GIRK1 expression in Xenopus oocytes mimics the main fea-
tures of the native atrial channel, including activation by the m2
type muscarinic receptor (m2R), strong inward rectification of
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement" in
accordance with 18 U.S.C. §1734 solely to indicate this fact.
currents, and maintained activation when the channel is co-
expressed with G protein subunitsGo,B and Gy2 (18, 21). Two
additional clones have been isolated by low-stringency screen-
ing of a mouse brain cDNA library with a rat GIRKI probe
(22). These clones were named mbGIRK2 and mbGIRK3
[Kir3.2 and Kir3.3, respectively (20)] because they show
slightly greater sequence similarity to GIRK1 than to other
cloned channels. 6-opioid receptors couple functionally to
GIRK2 but not GIRK3 channels (22).
Although present evidence suggests that the cardiac channel
GIRK1 is activated by G/3y subunits (7-10), it has been
claimed that Ga0, subunits activate inwardly rectifying K+
channels in excised patches from hippocampal cells (23),
raising the question whether the neuronal channels GIRK2
and GIRK3 are activated by G protein Ga subunits and/or by
,3y subunits. Furthermore, given that GIRKI, GIRK2, and
GIRK3 mRNAs are present in brain (18, 19,22,24), we wished
to know whether these different GIRKs are able to associate
with each other to form functional multisubunit, heteromul-
timeric channels. Here, we show that GIRK2 but not GIRK3
can be directly activated on coexpression ofG protein subunits
Go,f and Gy2 inXenopus oocytes. Most important, we find that
coinjection of GIRK1 with GIRK2 or GIRK3 cRNA gives a
much large G-protein-induced current than the responses for
each cRNA expressed individually but that, surprisingly, coin-
jection of GIRK3 with GIRK2 mRNA suppresses the G
protein response of the latter. These results demonstrate the
formation of functional heteromultimeric channels in Xenopus
MATERIALS AND METHODS
Plasmids and DNAs. To isolate GIRK2 and GIRK3 from
mouse brain RNA, we designed oligonucleotides that anneal to
the first assigned methionine codon and the assigned stop
codon of mouse GIRK2 and GIRK3 (22). GIRK-specific
sequences were coupled to a 5'-end untranslated sequence
from alfalfa mosaic virus (25, 26) and a T7 RNA polymerase
recognition site to confer an optimal translational initiation
site and allow RNA synthesis in vitro (cRNA). At the 3' end,
a poly(A) tail was added to confer RNA stability. Total RNA
from adult mouse brain (purchased from Clontech) was used
as template for cDNA synthesis (27). PCR was conducted in a
thermal cycler (Perkin-Elmer) by using Vent Polymerase
(New England Biolabs), which minimizes misincorporations
during the amplification step. GIRK2 and GIRK3 PCR-
derived sequences were then inserted into the pNoTA vector
(5 Prime -> 3 Primer, Inc.) for subsequent sequence analysis.
Abbreviations: G protein, guanine nucleotide-binding protein; GIRK,
G-protein-coupled, inwardly rectifying K+ channel; ACh, acetylcho-
line; IKACh and IK, inward currents induced by ACh and high [K+],
respectively; m2R, m2 type muscarinic receptor; EK, K+ equilibrium
*To whom reprint requests should be addressed.
Proc. Natl. Acad. Sci. USA 92 (1995)
GIRK1 cloned from cardiac atrium (19) and inserted orig-
inally in pBluescript (Stratagene) was transferred to the vector
pMXT (gift from J. Yang; University ofTexas, Dallas) inwhich
the cloning site is flanked by 5' and 3' untranslated sequences
fromXenopus globin. G protein subunitsGo31and Gy2 cDNAs
were in the pFrogy vector (gift from L. Jan, University of
California, San Francisco), as described (28). The m2R cDNA
(gift from E. Peralta, Harvard University) was in the pGEM3
RNA Synthesis and Oocyte Injections. GIRK2 and GIRK3
cRNAs were synthesized directly from gel-isolated PCR prod-
ucts, while the remaining cRNAs were synthesized from
linearized plasmid DNAs. cRNAs were dissolved in sterile
water and injected into stage V or VI Xenopus oocytes as
described (29). Oocytes were maintained in ND96 solution (96
mM NaCl/2 mM KCI/1 mM CaCl2/1 mM MgCl2/5 mM
Hepes, pH 7.5 with NaOH/2.5 mM sodium pyruvate/0.5 mM
theophylline/50 mg of gentamycin per liter. Oocytes were
assayed 2-5 days after injection.
Electrophysiology. Whole-cell currents from oocytes were
measured by using an Axoclamp 2A or Geneclamp 500 amplifier
(Axon Instruments, Foster City, CA) in the two-electrode, volt-
age-clamp configuration. Current and voltage electrodes were
filled with 3 M KCl to yield resistances ranging from 0.5 to 1.5
Mfl. Recordings were started in an extemal solution (0 K+)
containing 98mM NaCl, 1 mM MgCl2, and 5 mM Hepes, pH 7.3.
In high-K+-containing solutions, the NaCl was replaced either
completely, by 98 mM KCI (98 K+), or partially, by 20 mM KCI
Cell-attached recordings of single channels were recorded
from Xenopus oocytes as described (30). Pipette solutions
contained. 150 mM KCI, 1 mM CaCl2, and 5 mM Hepes, pH
7.3 with KOH. Bath solution contained 150 mM KCl, 1 mM
MgC92,1 mM EGTA, and 5 mM Hepes, pH 7.3 with KOH. For
single-channel analysis, the current traces were filtered at 2
kHz and sampled at 10 kHz. Current amplitude histograms and
open-time durations were obtained by using FETCHAN and
pSTAT from pCLAMP 6.0 (Axon Instruments). All recordings
were performed at room temperature (-22°C).
GIRK1 is abundantly expressed in cardiac atrium and brain
(18, 19, 24, 31). When heterologously expressed in Xenopus
oocytes, GIRK1 induces strong inwardly rectifying K+ currents
either with m2R activation (19) or with coexpression of G
protein subunits Gf31 or G-y2 (21, 28).
Evidence from Activation via Muscarinic Receptors. Be-
cause expression of GIRK2 and GIRK1 was consistently
smaller than for other channels expressed in oocytes (see
Discussion), we have examined the effects of GIRK1 and
GIRK2 coexpression under conditions of maximal m2R acti-
vation [1AMacetylcholine (ACh)]. With the coinjection of
GIRK1 and GIRK2 (GIRK1 + 2) cRNAs (0.5 ng each per
oocyte), exposure to ACh in these oocytes led to development
of large inward currents when the voltage was jumped from 0
mV to more negative values (Fig. 1). By contrast, in oocytes
injected with GIRK2 or GIRK1 cRNAs (1 ng), receptor-
activated currents were much smaller in amplitude, compara-
ble to levels reported previously (18, 19, 22). On average,
GIRK1 + 2 currents [inward currents induced byACh(IY,ACh)
= -6.4 ± 0.8 ,LA; mean ± SEM; n = 5] were about 9-fold
larger than GIRK2 currents (IK,ACh = -698 ± 98 nA; n = 7)
and 17-fold larger than GIRK1 Currents (IKACh = -365 ± 45
nA; n = 5).
GIRK1 expressed in oocytes shows distinctive gating kinet-
ics, including slow phases of activation (several hundred ms)
during ajump from 0mV to more negative potentials (32) (Fig.
1). For a similar voltage jump, GIRK2 expressed in oocytes
showed kinetics that more closely resemble other strong
inward rectifiers, with a prominent phase of inactivation. We
believe that these differences are governed at least partially by
differences in the sequence of the P region (33). The coex-
pressed subunits showed a slow phase of activation (Fig. 1), but
the time course of this activation cannot be explained as a
98 K" + ACh
GIRK1 + 2
GIRK2 and GIRK1 coexpression. Xenopus oocyteswere
injected with m2R (1 ng per oocyte) cRNA, as well as
GIRK1 (1 ng) cRNA, GIRK2 cRNA (1 ng), or GIRK1
plus GIRK2 cRNAs (0.5 ng each). Whole-cell currents
were recorded 2 days postinjection in 98 K+ bath
solution in the presence and absence of 1 ,uM ACh
(saturating concentration for the activation of m2R).
Membrane potential was held at 0 mV for 1 sec, then
stepped to test potentialsvarying from -140 to +20 mV.
Data are typical of six oocytes at each condition. (Inset)
Further analysis of the traces superimposed for thejump
to -140 mV. Current traces are ACh-sensitive currents
obtained by subtracting traces on the left from those on
the right. Traces for GIRK1 and GIRK2 are expanded
to roughly half the amplitude of the trace for coexpres-
sion, then added (thin line). The summed trace is a poor
fit to the actual data, suggesting that the waveforms for
the coexpressed channels cannot be expressed as a linear
sum of the traces for GIRK1 alone and GIRK2 alone.
Enhancement of m2R-evoked responses by
Neurobiology: Kofujiet aL
Proc. Natl. Acad. Sci. USA 92 (1995)
simple weighted sum of the waveforms for GIRK1 alone and
GIRK2 alone (Fig. lInset). Thus, the relaxations for jumps to
voltages between -60 and -140 mV were well described by
two exponential components with nearly voltage-independent
time constants; at -80 mV the time constant of the slower
component was 213 ± 12 ms (n = 4), or less than half that for
GIRK1 (32). These kinetic differences indicate molecular
interactions between the GIRK1 and GIRK2 channels.
Evidence from Activation via fry Subunits. The large en-
hancement of the agonist-evoked currents for coexpressed
channels might be explained by effects on any component in
the receptor-channel signaling pathway, including receptors,
endogenous G proteins, or the channels themselves. To dis-
criminate among these possibilities, we uncoupled the receptor
from the channel by overexpression ofG protein subunitsGo31
and G-y2. Importantly, cells expressing G131y2and GIRK2
showed persistent inwardly rectifying currents at amplitudes
comparable or larger to those observed for m2R activation of
GIRK2 (current-voltage relationships for representative oo-
cytes are shown in Fig. 2A). Therefore, GIRK2, like GIRK1,
is activated by Gf3y subunits.
Cells coexpressing GIRK2 and GIRK1 (GIRK1 + 2) re-
sponded to high-K+ solution with much larger currents than did
cells expressing only GIRK1 or GIRK2, consistent with the
results obtained with m2R activation. On average, GIRK1 + 2
currents (IK = -4.3 + 1 uA; n = 7) were 14 times larger than
GIRK2 currents (IK= -305 ± 56 nA; n =7) and 40 times larger
than GIRK1 currents (IK =-104 + 10 nA; n = 6) (Fig. 2B).
Thus, these experiments demonstrate that the large mutual
potentiating effect of GIRK1 + 2 is independent of the method
of G protein activation and argue that the effects are on the
Evidence from Single-Channel Conductance and Kinetics.
An increase in the number of channels or modification of the
intrinsic channel properties might account for this potentiation.
Single-channel recordings of GIRK1 + 2 in combination or of
GIRK2 alone were made to test these possibilities. In oocytes
coinjected with cRNAs for GIRK2,Go31,and Gy2, single-channel
currents in the cell-attached configuration displayed features
consistent with macroscopic measurements-i.e., with hyperpo-
larization the unit conductance increased. No outward currents
were detected at membrane potentials positive to the K+ equi-
librium potential (EK; -0mV) (Fig. 3A). These channel openings
had a mean slope conductance of30 ± 2pS (n=4) overthe range
from -40 to -100 mV, significantly smaller than the value of 39
pS for GIRK1 alone (19), and showed bursts of flickery activity.
Mean open-time distribution could be described by a fast com-
with GIRK1. (A) Current-voltage relationship for representative
oocytes injectedwith cRNA for G protein subunitsGo31and Gy2 (5 ng
each), as well as for GIRK2 (1 ng), GIRK1 (1 ng), or GIRK1 plus
GIRK2 (0.5 ng each). Currents were measured 2 days postinjection.
Bath solution contained 20 mM K+. Note the large increase of inward
currents upon coexpression of GIRK1 and GIRK2. (B) Average
inward K+ currents in the presence ofhigh [K+] (IK) were obtained by
subtracting traces in 0 K+ from those in 20 K+ at -80 mV. Data are
from four to six oocytes for each condition.
GIRK2 enhancesG,3i1y2evoked currentswhen coexpressed
ponent of 0.1 ms (35% ofthe total number ofevents) and a main
slower component of 0.5 ms (65% of the total number of events)
The combination of GIRK1 + 2 produced unitary currents
with strong inward rectification and a mean slope conductance
of 35 ± 3 pS (n = 4), intermediate between the values for
GIRK1 alone and GIRK2 alone (Fig. 3A). In addition, GIRK1
+ 2 channels displayed markedly longer openings than GIRK2
channels (Fig. 3B). Recordings from GIRK1 + 2 channels
showed a 7-fold increase in the major component ofopen-time
duration (3.5 ms; 71% of the total number of events; there was
also a smaller component of 0.5 ms; 29% of the total number
of events) compared to GIRK2. Qualitatively, the GIRK1 +
2 patch recordings are rather similar to those observed for
GIRK1 in terms ofmean open time (see ref. 18), although the
typically low expression levels for GIRK1 alone vitiate sys-
tematic comparisons. While we did not accumulate systematic
data, it is our strong impression that the probability of finding
patches with multiple channels was greatly increased for
coinjected oocytes. At present, it is not known whether this
increase, like the increase in macroscopic currents on GIRK1
+ 2 coexpression, is solely explained by the increase in
open-time duration or whether the enhanced currents also
arise from an increase in the number of openings, either
because each channel opens more often or because there are
more functional channels.
Evidence from GIRK3 Coexpression with GIRK1 or
GIRK2.We also tested the effects ofGIRK3 coexpressionwith
GIRK1 or GIRK2. Injection of GIRK3,Go31,and Gy2 cRNAs
did not activate inward currents in high-K+-containing solu-
tion (data not shown); nor were currents activated by opioid
receptors (22). Nevertheless, GIRK3 had profound effects
when coexpressed with GIRK1 (Fig. 4A). Coexpression of
GIRK1 and GIRK3 (GIRK1 + 3) resulted in 7-fold larger IK
currents (IK =-3.4 ± 0.5 ,A; n = 6) than for GIRK1 alone
(IK =-481 ± 43 nA; n = 6). Such augmentation was seen for
each of several batches of oocytes and cRNAs and on activa-
tion of the m2R pathway (data not shown). Thus, these results
demonstrate that GIRK3, despite not being directly activated
by G protein subunits Gf81 and Gy2, can interact with and
increase G-protein-mediated responses of GIRK1.
Surprisingly, GIRK3 had a suppressing effect when coex-
pressed with GIRK2 (Fig. 4B). Gf3y-induced, inwardly recti-
fying currents in oocytes coinjected with GIRK2 and GIRK3
cRNAs (0.5 ng each per oocyte) (IK =-129 + 22nA;n = 7)
were much smaller than in oocytes injected with GIRK2cRNA
alone (1 ng per oocyte) (IK = -5.1 + 0.6 ,A; n = 9). This
dominant-negative effect of GIRK3 on GIRK2 currents was
also observed when we coexpressed m2R instead ofG protein
subunitsGo31and G-y2 (data not shown).
Expression of GIRK1 isolated from atria in Xenopus oocytes
induced currents with many of expected properties, including
activation by Ga-linked receptors (18, 19) and by G protein
subunits Gf3y (21, 28). However, some anomalies were noted
during the course of our studies with GIRK1. First, current
amplitudes were not much larger than those observed with
expression from tissue poly(A)+ RNA (compare refs. 19 and 34).
Maneuvers designed to raise the expression levels, such as
increasing the amount of injected GIRK1 cRNA or modifying
untranslated regions, were generally ineffective. Second, heter-
ologous expression ofGIRK1 hasbeen successful onlyinXenopus
oocytes. We and others have attempted to express GIRK1 in
mammalian cells by plasmid-mediated transient transfection or
vacciniavirus infection. We have observed in all cases the absence
of GIRK1-like currents in mammalian cells, despite the presence
of large amounts of infection/transfection-induced GIRK1
mRNA in these cells (Jun Li, personal communication). Such
Neurobiology: Kofujiet aL
Proc. Natl. Acad. Sci. USA 92 (1995)
GIRK1 + 2
-mrr.--- -20 mV
Open time, ms
coinjected with G,B1 and Gy2 cRNAs, as well as GIRK2 cRNA or GIRKI and GIRK2 cRNAs. Membrane potential was varied from -100 to +60
mV. Arrows indicate the zero current level. Current amplitudes were measured from all-points histogram data at each membrane potential. (B)
Open-time distributions for GIRK2 + 1 and GIRK2 channels at -80 mV. Current recordings were low-pass filtered at 2 kHz.
Single-channel recordings from oocytes expressing GIRK1 + 2 or GIRK2 channels. (A) Cell-attached patch recordings from an oocyte
idiosyncrasies of expression levels and cellular environment sug-
gested the involvement of accessory subunits for efficient assem-
bly of the G-protein coupled, inwardly rectifying K+ channels or
coupling with G proteins.
Here we have shown that GIRK2 and GIRK3, when coex-
pressed with GIRK1, are able to interact and produce large (5-
to 40-fold) enhancement of the G-protein-evoked currents.
Further analysis of GIRK1 + 2 expression shows that the
voltage-jump kinetics cannot be explained
weighted sum of the traces for the individual channels, and the
single-channel conductances are intermediate between those
for the individual channels. These data show that GIRKI with
GIRK2 and probably GIRK1 with GIRK3 can form hetero-
multimeric channels leading to larger G-protein-activated
currents with distinct physiological properties.
Heteromultimerization among distinct but highly similar
subunits is described for other channels, such as the amiloride-
sensitive sodium channel (35) and the retinal and olfactory
cyclic nucleotide-gated cation channels (36, 37). We cannot be
certain at this moment how coexpression of GIRK1 and
GIRK2 or GIRK3 results in such large potentiation of the
G-protein-evoked responses. Single-channel recordings from
the GIRK2 and GIRK1 combination show that individual
channel proteins remain open several times longer; but there
may be additional factors, such as more frequent openings or
an increased number of functionally competent channels.
While this manuscript was in preparation, it was reported that
in atrial cells (38), the GIRK1 polypeptide associateswith another
member of the inwardly rectifying K+ channel superfamily, CIR,
as a simple
demonstrated by immunoprecipitation of the heteromultimeric
channels from atrial membranes by GIRK1-specific antibodies.
CIR is identical in sequence to the channel previously identified
as KATP (39). However, the CIR channel has the features
expected from a G-protein-coupled channel: it is activated by
nonhydrolyzable analogs ofGTP and byG protein subunits Gfry
(38). Furthermore, GIRKi and CIR coexpression results in large
potentiation of the G-protein-gated currents inXenopus oocytes
(38). Such findings are consistent with the hypothesis that in the
atrial cells, the G-protein-gated channel is a heteromultimer of at
least two distinct, inwardly rectifying K+ channels (GIRK1 and
CIR). We speculate, as did Krapivinski et al. (38), that when
GIRK1 alone is expressed in oocytes, functional channels are
heteromers ofGIRK1 with a GIRK-like protein endogeneous to
Until immunoprecipitation experiments are done with brain
membranes, we can only speculate that GIRK2 also coas-
sembles with GIRKi in neuronal cells, but this seems likely
given the strong similarities between CIR (38) and GIRK2. (i)
CIR is more closely related to GIRK2 than to any other known
protein (20, 38). (ii) Both CIR and GIRK2 can be directly
activated by Gfry subunits (38). (iii) Homomeric CIR and
GIRK2 unitary currents display similarly brief, single-channel
openings. (iv) Both CIR and GIRK2 increase the magnitude
of evoked currents many fold upon coexpression with GIRK1.
Receptor-activated, inwardly rectifying K+ conductances are
found extensively in the brain and may underlie the postsynaptic
inhibitory action of many neurotransmitters and neuropeptides
(11). Which GIRK subunits account for these conductances?
Neurobiology: Kofujiet aL
Proc. Nati. Acad. Sci. USA 92 (1995)
-0- GIRKI +
differentially, as reported for other G protein effectors, such
as for the several isoforms ofadenylate cyclase (40);this would
allow a larger degree of flexibility on the control of this
importantform of neuronal signaling.
We thank Brad Henkle for preparing the oocytes, Craig Doupnik
andYinong Zhangforadvice, and Dr. Michel Lazdunski for sharing
apreprintof ref. 22 while it was inpress.This work was supported by
ICAgen, Inc., andbyNational Institutes of Health Grants GM29836
-0- GIRK2 + 3
gk - 41
shipsfortypical oocytes injectedwithGo,and Gy2 cRNAs (5 ng each),
as well as GIRK3 cRNA(1 ng), GIRK1 cRNA (1 ng), or GIRKI and
GIRK3 cRNAs (0.5 ng each). Bath solution contained 98 mMK+. Note
the large increase of inward currents upon coexpression of GIRK1 and
GIRK3. (Inset) Average K+
relationshipsfortypical oocytes injectedwithGo31 and Gy2 cRNAs, as
well as either GIRK2 cRNA(1 ng),GIRK3 cRNA (1 ng), or GIRK3 and
GIRK2 cRNA(0.5 ng each). (Inset) Average K+
Data are averaged from four to nine oocytes for each condition.
GIRK3 hasoppositeeffects on G-protein-evokedcurrents
or GIRK2.(A)Current-voltage relation-
currents at -80 mV. (B) Current-voltage
currents at -80 mV.
CIR mRNA is present in several brain regions (39) but is notably
poorly expressed in cerebral cortex and in the cerebellum (39),
two brain regions in which GIRK1
These resultsimplythat these twoinwardly rectifyingK+ channels
are notalwaysassociated andsuggestheteromultimerization of
GIRK2 or GIRK3 with GIRK1 in some brain areas. Moreover,
it isinterestingto note that in dorsal raphe and locus coeruleus
neurons, despitethe low levels of GIRK1 mRNA (24), a robust
G-protein-activatedconductance is described (14-17). Expres-
sion of still undescribed GIRKs in these brain regions may explain
GIRK3expressiondid not result in expression of measur-
able functional channels evenby coexpression of G protein
subunitsGo1,andG-y2.The facts that GIRK3 does enhance the
G-protein-evoked responseswith GIRK1 coexpression and
suppressestheG-protein-evoked responses with GIRK2 co-
expressionindicate thatGIRK3 can coassemble with GIRK1
and GIRK2. The reasonwhyGIRK3 alone does not give
functional channels inoocytesis not known. It may not be
targetedto theplasmamembrane without a partner; alterna-
tively, it may not bind activated G proteins.
Avery importantissue raisedbythe occurrence of hetero-
multimeric channels is their differential distribution in excit-
able tissues and thephysiological significance.Distinct GIRK
heteromultimersmaybe modulated or activated by G proteins
is abundantly expressed (24).
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