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INTRODUCTION |
G protein-coupled inwardly rectifying K+
(GIRK1; Kir3.x)
channels are targets for up- and down-regulation by receptors that couple to different classes of heterotrimeric G proteins, providing a
mechanism for dynamic regulation of cellular excitability (1, 2).
Activation of GIRK channels involves receptors that couple to pertussis
toxin (PTx)-sensitive G
subunits of the G
i or
G
o family, whereas inhibition of GIRK channels is
mediated by receptors that couple via PTx-insensitive G
subunits.
This dual regulation of GIRK channels has been noted in a number of
cellular contexts, including atrial cells of the myocardium (3, 4),
aminergic neurons of the brainstem (5, 6), and enteric neurons of the
peripheral nervous system (7). Because demonstration of this dual
regulation requires simultaneous activation of different receptor
classes, the phenomenon may be even more widespread than is currently realized.
Of the dual components of receptor-mediated GIRK channel regulation
(activation and inhibition), most emphasis has been on understanding
cellular mechanisms underlying GIRK channel activation. It is now well
established that, following agonist stimulation, G
subunits
released from receptor-bound heterotrimers bind directly to GIRK
channels to increase channel activity (reviewed in Refs. 1, 2, and 8).
It was recently found that G
activation of GIRK channels requires
permissive levels of membrane phosphatidylinositol bisphosphate
(PIP2) and that interactive effects of these mediators on
channel activity result from G
-mediated stabilization of PIP2 binding to GIRK channels (9-11).
In contrast to GIRK channel activation, mechanisms that contribute to
receptor-mediated inhibition of GIRK channels are not well understood.
It is clear that GIRK channel inhibition involves receptors that
typically couple via PTx-insensitive G proteins of the
G
q family, and it therefore seems reasonable to suspect involvement of that class of G
subunit. However, GIRK channel inhibition by these and/or other classes of PTx-insensitive G
subunits has not been directly examined. Moreover, it remains to be
determined if the signal for GIRK channel inhibition derives from the
subunit or 
dimer of the heterotrimeric G protein. In this
respect, we recently found that G
5-containing dimers can
inhibit G
-activated GIRK channels (12), a finding that is
especially intriguing in the current context of receptor-mediated GIRK
channel inhibition since G
5
dimers associate
preferentially with G
q-coupled receptors (13, 14).
Signaling mechanisms that contribute to GIRK channel inhibition
downstream of G protein subunits are also not known with any certainty,
although recently it was suggested that phospholipase C (PLC)
activation could contribute to GIRK channel inhibition by
G
q-coupled receptors in atrial myocytes by decreasing
membrane levels of PIP2 rather than by production of
downstream mediators (15-17). It remains to be determined if a similar
mechanism contributes to GIRK channel inhibition by similar receptors
in other settings.
Here, we prepared mammalian cell lines expressing GIRK channel subunits
together with two different classes of G protein-coupled receptors in
order to recapitulate dual regulation of GIRK channels in a
heterologous expression system. Using this system, we found that
G
q-coupled thyrotropin-releasing hormone (TRH) type 1 (TRH-R1) receptors can inhibit GIRK channels pre-activated by
G
i/o-coupled 5-hydroxytryptamine (5-HT) type 1A
(5-HT1A) receptors. GIRK channel inhibition involved
G
q family subunits, PIP2, and activation of
PLC, but it was independent of downstream signaling mediators typically
associated with PLC activation such as protein kinase C (PKC), inositol
trisphosphate (IP3), and intracellular calcium. These data
are consistent with the possibility that PLC-mediated decreases in
membrane PIP2 levels provide the signal for GIRK channel
inhibition by G
q-coupled receptors (15-17); if this is the case, PIP2 may be readily diffusible since inhibition
was robust even in cell-attached patches that were not exposed to agonist. These results further indicate that G
and G
subunits, each derived from a different class of receptor, can converge with
opposite effects on the same channel to dynamically regulate cellular excitability.
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MATERIALS AND METHODS |
Stable Cell Line Expressing GIRK Channels and TRH-R1 and
5-HT1A Receptors--
The derivation and maintenance of
stable human embryonic kidney HEK293 cells expressing GIRK1 (Kir3.1)
and GIRK4 (Kir3.4) channels (referred to as G1/4 cells) were described
previously (12). This cell line was further stably transfected with the TRH-R1 and 5-HT1A receptors (referred to as G1/4R cells).
Briefly, TRH-R1 (pBS; M. C. Gershengorn, Cornell University) and
5-HT1A receptor (pGEM3z; D. K. Grandy, Vollum
Institute) cDNAs were subcloned into pcDNA3 and transfected
together with pBabe-Puro (K. R. Lynch, University of Virginia)
into G1/4 cells and maintained under G418 (400 µg/ml; Life
Technologies, Inc.) and puromycin (200 ng/ml; Sigma) selection. A G418-
and puromycin-resistant cell line was selected based on strong
expression of inwardly rectifying K+ currents and robust
regulation of those currents by TRH and 5-HT (G1/4R cells). Both the
G1/4 and G1/4R cell lines were cultured in Dulbecco's modified
Eagle's medium/nutrient mixture F-12 with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin in the continued
presence of G418 (G1/4 cells) or G418 and puromycin (G1/4R cells).
Transfection of Stable Cell Lines Expressing GIRK
Channels--
Stable cell lines (G1/4 and G1/4R) were transiently
transfected with plasmids that direct expression of G
, G
, or G
subunits or of minigene constructs that interfere with G protein
signaling under the control of a cytomegalovirus promoter. G
subunits that bear a point mutation (Gln
Leu) rendering them
GTPase-deficient (i.e. constitutively active, indicated as
G
*) were used as follows: G
q* (in pcDNA3; W. F. Simonds, National Institutes of Health) and FLAG epitope-tagged
G
q* (in pSKAC; R. Iyengar, Mount Sinai School of
Medicine, Mount Sinai, NY); G
11* (in pCI; M. I. Simon, California Institute of Technology, Pasadena, CA); wild-type
G
14 (in pCIS; M. I. Simon), mutated by overlap
extension polymerase chain reaction to yield Q205L, subcloned into
pcDNA3, and sequenced; G
15* (in pBS; M. I. Simon), subcloned into pcDNA3; G
i1*,
G
i3*, and G
z* (in pCMV5; H. Itoh,
National Children's Medical Research Center, Tokyo, Japan);
G
i2* (in pCMV7; G. L. Johnson, National Jewish
Medical and Research Center); G
o* (in pBS; B. M. Denker, Harvard University), subcloned into pcDNA3;
G
s* and G
12* (both in pCMV5; G. L. Johnson); G
13* (in pcDNA3; M. Negishi, Kyoto University, Kyoto, Japan); and G
5 and G
2
(in pcDNA3; W. F. Simonds). The green fluorescent protein
(GFP)-tagged PLC
1-ct (where ct is the C-terminal domain; pEGFP-C1)
and RGS2 (pCI) G
q effector antagonist constructs were
obtained from S. R. Ikeda (Guthrie Research Institute) (18). Two
RGS constructs (RGS6 and RGS11) that bind specifically to
G
5 (19, 20) and interfere with signaling mediated by
G
5-containing G
dimers (21) were obtained in
pcDNA3.1 (A. G. Gilman, University of
Texas-Southwestern). Two additional constructs that interfere generally
with G
signaling were also used: a
-adrenergic receptor kinase
(
ARK) C-terminal domain construct,
ARK-ct (in pRK5; W. J. Koch, Duke University), was subcloned in frame with a myristic acid
attachment signal into pGTM, a pcDNA3 derivative (E. A. Golemis, Fox Chase Cancer Center) (22); and G
t
(transducin) was obtained in pcDNA1 (F. L. Kolakowski,
University of Texas at San Antonio). GFP-tagged constructs designed to
decrease membrane PIP2 levels (PLC
-PH (where PH is the
pleckstrin homology domain), AKT-PH, and 5'-phosphatidylinositol phosphatase (5'-PI-PTase)), provided by T. Meyer (Stanford University, Stanford, CA), were as previously described (23). Expression plasmids
were transfected in 35-mm culture dishes by CaPO4
precipitation (24) together with a plasmid (pGreenLantern, Life
Technologies, Inc.) that directs expression of an enhanced GFP at a
test plasmid/GFP plasmid ratio of 6:1 µg.
Western Blots--
Crude cell lysates were prepared from
transfected cells in the presence of a mixture of protease inhibitors
(2 µg/ml each leupeptin, pepstatin, and aprotinin and 100 µM phenylmethylsulfonyl fluoride). Proteins were resolved
by SDS-polyacrylamide gel electrophoresis, transferred to
nitrocellulose, and immunoblotted. Expression of FLAG epitope-tagged
constitutively active G
q subunits
(FLAG-G
q*) was detected using M2 anti-FLAG monoclonal
antisera (10 µg/ml; Sigma), and GIRK channel subunit expression was
determined with a rabbit anti-GIRK1 antibody (1:500 dilution; Alomone
Labs). Primary antibodies were detected with horseradish
peroxidase-conjugated mouse or rabbit IgG using enhanced chemiluminescence.
Electrophysiological Recordings from Transfected
Cells--
Cells were plated onto glass coverslips at a confluency
appropriate to obtain single cells for electrical recording 48-72 h
after transfection (except where noted). Coverslips were submerged in a
recording chamber at room temperature on microscopes equipped with
epifluorescent optics (Zeiss Axioskop FS or Nikon TE300). Individual cells that expressed GFP were identified using standard fluorescein isothiocyanate filter sets and targeted for recording.
Patch electrodes were made from borosilicate glass capillaries (Clark
Electromedical) and connected to the head stage of an Axopatch 200A
patch-clamp amplifier (Axon Instruments, Inc.). For whole cell
recordings, pipettes (2-4 megaohms) were filled with an internal
solution containing 120 mM KCH3O3S,
4 mM NaCl, 1 mM MgCl2, 0.5 mM CaCl2, 10 mM HEPES, 10 mM EGTA, 3 mM MgATP, and 0.3 mM
Tris-GTP (pH 7.2). The external solution contained 137 mM
NaCl, 6 mM KCl, 10 mM HEPES, 2 mM
CaCl2, 2 mM MgCl2, and 10 mM glucose (pH 7.3). For cell-attached patch recordings,
the same external solution was used, but pipettes were pulled to a higher resistance (7-10 megaohms) and contained 130 mM
KCl, 5 mM EGTA, 2 mM MgCl2, and 10 mM HEPES (pH 7.3).
Electrophysiological data were acquired and analyzed using the pCLAMP
suite of programs (Axon Instruments, Inc.). Series resistance was
typically <10 megaohms and compensated by ~70-80%. Membrane voltages were corrected for a 10-mV liquid junction potential. To
record inwardly rectifying whole cell currents, cells were held at
50
mV, and a slow voltage ramp command (
90 mV, 0.1 V/s) was applied
at 0.1 Hz. Membrane current was filtered at 0.5-1 kHz and sampled at
1-2 kHz. Measured variables included holding current at
50 mV and
slope conductance, obtained from a linear fit to current-voltage data
over the range of
100 to
120 mV. The effect of TRH on GIRK channel
conductance was determined following subtraction of endogenous
currents, defined by their resistance to 0.2 mM
Ba2+. Single channel data were filtered at 2 kHz (four-pole
Bessel) and collected on line using gap-free recording with a 10-kHz
sampling frequency. The slope of current amplitudes obtained from
gaussian fits to all-points histograms at three different patch
potentials was used to determine single channel conductance. Analysis
of the effects of agonist stimulation on GIRK channel activity in cell-attached patches was performed using Fetchan (Axon Instruments, Inc.) and the nPo freeware program (provided by J. L. Sui).
All data are presented as means ± S.E., and each data point
includes results obtained from at least two transfections.
Drugs and Reagents--
Compounds were prepared as concentrated
stock solutions, stored at
20 °C, and diluted to the indicated
concentrations in recording or pipette solutions, as needed. 5-HT
(serotonin, Sigma) was made up as a 10 mM stock solution
with 100 mM ascorbate and applied to cells at 50 µM. TRH (100 µM stock solution; Peninsula Laboratories, Inc.) was applied at 100 nM. Toxins from
Bordetella pertussis (132 µg/ml) and Pasteurella
multocida (50 µg/ml) were generously provided, respectively, by
E. L. Hewlett (University of Virginia) and L. J. Eaton (List
Biological Laboratories, Inc.); cells were incubated in toxins
overnight at 2 and 1 µg/ml, respectively. The PLC inhibitor U73122
and its inactive analog, U73343, were obtained from Research
Biochemicals Inc. and prepared as 2 mM stock solutions in
dimethyl sulfoxide; preincubation with U73122 or U73443 (both at 1 µM) in the culture medium was begun at least 30 min prior
to recording to test effects on receptor-mediated GIRK channel
inhibition and ~36-48 h before recording to test effects in cells
transfected with G
q*. The bisindolylmaleimide PKC
inhibitor (GF 109203X, Calbiochem) was stored as a 5 mM
stock solution, and continuous exposure to 5 µM
bisindolylmaleimide was started
30 min before recording. The phorbol
ester phorbol 12,13-dibutyrate (PDBu; Research Biochemicals
Inc.) and its inactive analog, 4
-PDBu (Alexis), were prepared as 1 mM stock solutions in dimethyl sulfoxide and applied in the
bath at 1 µM. IP3 (10 mM; Alexis)
was diluted to 100 µM in pipette solution.
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RESULTS |
Dual Modulation of GIRK1/4 Channel Currents in a Stable HEK293 Cell
Line--
To study mechanisms contributing to receptor-mediated
inhibition of recombinant GIRK channels in the context of a mammalian cell system, we prepared a stable HEK293 cell line that expresses Kir3.1/3.4 (GIRK1/4) channels. As we described previously for G1/4
cells, hyperpolarizing ramp voltage commands evoked substantial inwardly rectifying currents under non-stimulated conditions that were
due, in large part, to channel activation by free G
subunits endogenous to those cells (12). In cells transfected with the G
i/o-coupled 5-HT1A receptor and
G
q-coupled TRH-R1, whether expressed transiently in G1/4
cells or stably in G1/4R cells, the dual regulation of GIRK channels
that is seen in various native cell systems (5-7) was fully
recapitulated, i.e. GIRK channel currents pre-activated by
G
i/o-coupled receptors were inhibited by
G
q-coupled receptors. Thus, as illustrated in the
representative cell of Fig.
1A, the basal inwardly
rectifying conductance was increased by 5-HT (50 µM), and
this enhanced conductance was inhibited by TRH (100 nM).
Although it was often more convenient to use the G1/4R cell line stably
expressing the 5-HT1A and TRH-R1 receptors, we obtained
essentially identical results with transient receptor expression;
therefore, where available, data from experiments using transient and
stable receptor expression were combined. Averaged data from cells
treated with both 5-HT and TRH (n = 9) revealed that
5-HT caused an increase in GIRK channel conductance (42.2 ± 8.7%
of the initial conductance), whereas TRH inhibited GIRK channel
conductance (80.4 ± 3.8% of the conductance in 5-HT); when
referenced to the initial GIRK channel conductance before 5-HT, the
inhibition by TRH was often >100% (average of 118.3 ± 11.5%,
n = 9), indicating that signaling from
5-HT1A and TRH-R1 receptors converges on the same GIRK
channels, but with opposite effects.

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Fig. 1.
Dual regulation of GIRK channels in G1/4R
cells. A, dual regulation of GIRK channel currents in
HEK293 cells expressing GIRK1/4 channel subunits, the
G i/o-coupled 5-HT1A receptor, and
G q-coupled TRH-R1. Left panel, conductance
was determined as the slope of current-voltage curves between
100 and 120 mV under control conditions, during application of 5-HT
(50 µM) and during application of TRH (0.1 µM) in the continued presence of 5-HT. Right
panel, sample traces reveal the inwardly rectifying profile
of current responses to hyperpolarizing ramp voltage commands. Note
that 5-HT activated an inwardly rectifying current that was nearly
completely blocked by TRH. B, inhibition of GIRK channel
currents by TRH is PMT-sensitive, but is unaffected by PTx. Left
panel, shown is the time course of the effect of TRH on basal
conductance in a control cell (closed circles) and in a cell
treated overnight with PMT (1 µg/ml; open circles).
Right panel, shown are summary data on the effect of TRH in
control cells (n = 41) and in cells treated with PMT
(n = 15) and PTx (2 µg/ml, overnight;
n = 6) on TRH-induced inhibition of conductance
(percent of control conductance; means ± S.E.). The
asterisk denotes a statistically significant difference from
control cells (p < 0.05 by ANOVA with post
hoc Bonferroni's test).
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It is well known that activation of GIRK channels by 5-HT1A
and other receptors in native systems is mediated by PTx-sensitive G
proteins of the G
i/o class. Likewise, we found that GIRK
channel activation by 5-HT1A receptors was completely
blocked by PTx (2 µg/ml, overnight; 56.2 ± 7.3% activation in
control cells (n = 15) versus
11.9 ± 3.8% inhibition after PTx (n = 16)). On
the other hand, receptor-mediated GIRK channel inhibition in native systems is PTx-insensitive; and accordingly, we found no
effect of PTx on TRH-induced GIRK channel inhibition (Fig.
1B, right panel). Because TRH-R1 is typically
associated with G proteins of the G
q family, we tested
the effects of P. multocida toxin (PMT) (Fig. 1B,
left panel), which appears to directly activate and
permanently uncouple G
q subunits from receptors (25,
26); compared with the typical TRH-induced inhibition of GIRK channel conductance in a control cell (closed circles), the
inhibition by TRH was completely blocked in a representative cell
treated with PMT (1 µg/ml, overnight; open circles). The
degree of inhibition of GIRK channel conductance by TRH was >55% in
nearly all control cells (38 of 41 cells tested; 93%), whereas it
reached that level in only three PMT-treated cells (of 15 cells tested;
20%); as shown in Fig. 1B (right panel), the
averaged percent inhibition of GIRK channel conductance by TRH
was significantly decreased by PMT (34.5 ± 5.7%,
n = 15) compared with control cells (72.6 ± 2.0%, n = 41; p < 0.05). Thus, these
data suggest that TRH-R1 couples to G
q family subunits
to mediate inhibition of GIRK channels.
G
q Family Subunits and G
5-containing
G
Dimers Are Capable of GIRK Channel
Inhibition--
Receptor-mediated modulation of native GIRK channel
currents is invariably associated with G
q-coupled
receptors (reviewed in Ref. 1). To determine if members of the
G
q family can cause inhibition of GIRK channels
independent of receptor activation, we transfected constitutively
active (i.e. GTPase-deficient, Gln
Leu mutant)
G
q* subunits into G1/4 cells. We chose to use
constitutively active G
subunits because effects of wild-type G
subunits could result from promiscuous coupling to receptors; from
activity due to intrinsic GDP/GTP cycling; or indirectly, from effects
due to sequestering free G
subunits.
As evident in the sample records from representative cells shown in
Fig. 2A, basal GIRK channel
currents were substantially smaller in a cell transfected with FLAG
epitope-tagged G
q* compared with a control G1/4 cell.
Currents were essentially identical in cells transfected with either
FLAG-tagged or untagged G
q*, and data from experiments
with those constructs were combined in subsequent analyses. As shown in
the averaged data of Fig. 2C, GIRK channel currents were
significantly decreased in G1/4 cells transfected with
G
q*; indeed, the conductance in cells transfected with
G
q* approximated that seen in parental HEK293 cells not
expressing GIRK channels (data not shown), suggesting that channel
activity was essentially completely eliminated by G
q*.
Likewise, GIRK channel currents were drastically reduced in cells
transfected with constitutively active mutants of all other
G
q family members tested, including G
11*,
G
14*, and G
15* (Fig. 2C).
Expression of GIRK channel subunits was apparently unaffected by
G
q* transfection inasmuch as we found no difference in
GIRK1 levels by Western blot analysis (Fig. 2A,
inset). In addition, this inhibitory effect was specific to
G
q family subunits since GIRK channel currents were
unaffected by representative constitutively active members of all other
major classes of G
proteins. These included all members of the
G
i/o family (G
i1*, G
i2*,
G
i3*, and G
o*) as well as the
PTx-insensitive G
s*, G
12*, G
13*, and G
z* subunits (Fig.
2C).

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Fig. 2.
GIRK1/4 channel currents are inhibited by
G q family subunits and
G 5-containing
G dimers. A,
sample current traces from a representative control G1/4 cell and from
a cell expressing FLAG-G q*. The basal GIRK channel
conductance was essentially completely eliminated in the cell
expressing FLAG-G q*. Inset, immunoblots of
G1/4 cell lysates from control cells and from cells transfected with
FLAG-G q*. There was no effect on GIRK1 channel
expression (upper panel) in cells expressing
FLAG-G q* (lower panel). B, sample
current traces from a control G1/4 cell and from a cell expressing
G 5 2 dimers. Conductance was reduced in
the cell expressing G 5 2. C,
averaged data (means ± S.E.) from cells expressing the indicated
constitutively active G subunits and
G 5 2. Asterisks denote
statistically significant differences from control cells
(p < 0.05 by ANOVA with post hoc
Bonferroni's test; the number of cells in each group is shown in
parentheses). Note that among constitutively active G
subunits, only members of the G q family inhibited GIRK
channel conductance; 5 2 also inhibited
GIRK channel conductance. NS, not significant.
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G
subunits also convey signaling information to effectors
following receptor activation (reviewed in Refs. 1, 2, and 8). Many
G
and G
subunit genes have been identified by molecular cloning,
and it is now well established that multiple different combinations of
G
are capable of activating GIRK channels, including dimers
composed of G
1, G
2, G
3,
and G
4, together with various G
subunits (12, 27,
28). By contrast, we recently found that G
5 forms
G
dimers that cause inhibition, rather than the customary
G
-mediated activation of GIRK channel currents (12). As shown in
the representative records of Fig. 2B and in averaged data
of Fig. 2C, GIRK channel currents were consistently smaller in cells transfected with G
5
2 than in
control cells not expressing exogenous G protein subunits. The
observation that G
5-containing dimers are unique among
G
dimers in causing GIRK channel inhibition is particularly
cogent in the current context of GIRK channel inhibition by
G
q-coupled receptors since G
5-containing
dimers associate preferentially with G
subunits of the
G
q family (13, 14). Together, these data indicate that
G
q family subunits and G
dimers that contain
G
5 are capable of inhibiting GIRK channel currents.
Receptor-mediated GIRK Channel Inhibition Involves
G
q, but Does Not Require G
Dimers--
Our
experiments using chronic exogenous expression of G protein subunits
appear to constrain the possible mediators of GIRK channel inhibition
either to G
subunits of the G
q family or to G
dimers containing G
5 (12). The next series of
experiments was designed to determine the role of these subunits in
acute receptor-mediated inhibition of GIRK channels.
First, we examined TRH-induced inhibition of GIRK channel currents in
cells transfected with either G
q* or
G
5
2, as shown in Fig.
3. Consistent with the results presented
above, expression of both G
5
2 and
G
q* inhibited basal GIRK channel currents. When
challenged with TRH, however, GIRK channel currents were further
diminished in G
5
2-transfected cells, but
not in G
q*-transfected cells. When inhibition was
expressed as percent of control (Fig. 3, inset), it was
clear that TRH inhibited GIRK channel currents equally effectively in
control and G
5
2-expressing cells. These data are consistent with the possibility that TRH receptor-mediated GIRK channel inhibition was occluded by G
q*, but not by
G
5
2.

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Fig. 3.
Effect of
G q* and
G 5 2
on TRH-induced inhibition of GIRK channel currents. The effect of
TRH was tested in control cells (n = 41) and in cells
transfected with either G 5 2
(n = 8) or G q* (n = 9).
Averaged data (means ± S.E.) show basal conductance (black
bars) and conductance in the presence of TRH (gray
bars). Inset, TRH-induced inhibition of conductance
(percent of control conductance; means ± S.E.).
Asterisks denote statistically significant differences from
control cells (p < 0.05 by ANOVA with post
hoc Bonferroni's test). Inhibition of GIRK channel currents was
preserved in cells expressing G 5 2, but
not in cells expressing G q*.
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It is important to note that, although TRH was without effect on GIRK
channel currents in G
q*-expressing cells, the basal GIRK
channel currents were essentially eliminated even before TRH
application. Thus, although the results are consistent with the
interpretation that G
q* occluded receptor inhibition, it is also possible that chronic overexpression of G
q*
inhibited GIRK channel currents through a separate mechanism and that
TRH was without effect simply because there was no residual basal GIRK
channel current to inhibit. Therefore, we performed additional experiments in which cells were transfected with minigene constructs designed to interfere with signaling via either G
q or
G
subunits (Fig. 4).

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Fig. 4.
Receptor-mediated GIRK channel inhibition
involves G q. A,
the effect of TRH on GIRK channel conductance in representative cells
transfected with the G q sinks PLC 1-ct (left
panel) and RGS2 (right panel). Note that TRH had little
effect on conductance in cells expressing either of these constructs,
which interfere with signaling via G q family subunits.
B, the effect of TRH on GIRK channel conductance in cells
expressing the G 5 sinks RGS6 and RGS11, which inhibit
signaling by G 5-containing G dimers (21). TRH
caused a strong inhibition of GIRK channel conductance in these cells.
C, summary data showing inhibition of GIRK channel
conductance by TRH (percent of control conductance; means ± S.E.)
in cells transfected with constructs that disrupt signaling by
G q family subunits (i.e. G q
sinks) or G dimers (i.e. G sinks).
Asterisks denote statistically significant differences from
control cells (p < 0.05 by ANOVA with post
hoc Bonferroni's test; the number of cells in each group is
indicated in parentheses). Note that inhibition of GIRK
channel currents by TRH was significantly diminished by the
G q sinks, but not by the G sinks, consistent with
a primary role for G q in mediating the effects of
TRH.
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We chose to use two different inhibitors of G
q
signaling: RGS2 and a GFP-tagged C-terminal construct of PLC
1 (18).
Both of these constructs selectively bind activated
G
q-like proteins, and their overexpression interferes
specifically with receptor signaling mediated by G
q (18,
29, 30). Although each of these constructs exhibits GTPase
activity, the inhibition of signaling appears to be independent of that
activity and due rather to their ability to compete with effectors for
G
q binding (i.e. as "sinks" or
"effector antagonists") (30). We tested the effect of TRH on GIRK
channel currents in cells transfected with these two different inhibitors of G
q signaling. As is evident in the sample
records provided in Fig. 4A, the inhibition of GIRK channel
currents by TRH was diminished by PLC
1-ct or RGS2. Indeed, unlike
the control condition, in which GIRK channel current inhibition was
>55% in the overwhelming majority of cells (38 of 41 cells tested;
93%), inhibition by TRH was >55% in only 3 of 25 of cells
transfected with PLC
1-ct (12%) and in just 3 of 16 cells
transfected with RGS2 (19%). This was also borne out in summary data
from these cells, where the averaged percent inhibition by TRH was
significantly reduced by both constructs (Fig. 4C).
These data indicate that inhibition of GIRK channels mediated by the
TRH receptor involves signaling by G
q.
In contrast to the abrogating effects of the minigene inhibitors of
G
q signaling, we found that receptor-mediated GIRK
channel inhibition was largely preserved in cells transfected with a
number of different G
buffers. First, we overexpressed two RGS
proteins, RGS6 and RGS11, which, by virtue of their G
-like domains
that bind specifically to G
5 (19, 20), can interfere
with signaling mediated by G
5-containing G
pairs
(21). As shown in Fig. 4B, TRH produced a strong inhibition
of GIRK channel currents in cells transfected with either of the
G
-like domain-containing RGS proteins; the averaged percent
inhibition in cells transfected with RGS6 and RGS11 was not different
from that in control cells (Fig. 4C). To rule out possible
involvement of other G
dimers in receptor-mediated GIRK channel
inhibition (e.g. by their ability to activate PLC) (8), we
tested two additional, relatively nonselective G
sinks, wild-type
G
t and
ARK-ct. Neither had any effect on TRH-induced
inhibition of GIRK channel currents (Fig. 4C). Attesting to
the efficacy of G
sequestration by G
t and
ARK-ct, the basal GIRK channel conductance was significantly reduced
in G
t- and
ARK-ct-expressing cells (7.1 ± 0.7 nanosiemens in control cells versus 3.7 ± 0.6 and
2.2 ± 0.4 nanosiemens in G
t- and
ARK-ct-expressing cells, respectively; both p < 0.05) (see also Ref. 12); and moreover, expression of each completely blocked GIRK channel activation by 5-HT1A receptors, a
known G
-mediated effect (data not shown). Thus, these data
indicate that GIRK channel inhibition by TRH-R1 is due, at least in
large part, to G
q rather than G
signaling.
Receptor-mediated GIRK Channel Inhibition Involves PLC--
In
light of the above results that suggest a primary role for
G
q signaling in receptor-mediated GIRK channel
inhibition, we tested if activation of PLC, a well known
G
q effector, might also play a role. As illustrated in
Fig. 5, TRH-induced inhibition of GIRK
channels was markedly diminished in cells pretreated with the PLC
inhibitor U73122, but not with the control compound U73343 (both at 1 µM for at least 30 min.). The inhibition by TRH was
>55% in only three cells and averaged just 35.7 ± 4.4% (n = 19) during U73122 exposure, significantly less
than the inhibition recorded in the presence of U73343 (75.2 ± 3.9%, n = 18) or in control cells that were not
pretreated with either compound (72.6 ± 2.0%, n = 41).

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Fig. 5.
Receptor- and
G q*-mediated GIRK channel
inhibition involves PLC. A, the effect of TRH on
GIRK channel currents in representative cells pretreated (1 µM, >30 min) with the PLC inhibitor U73122 (left
panel) or its inactive analog, U73343 (right panel).
Note that TRH had little effect in the continued presence of U73122,
whereas it strongly inhibited GIRK channel conductance during exposure
to the control U73343 compound. B, averaged TRH-induced
inhibition (percent of control conductance; means ± S.E.) in
control cells and in cells treated with U73122 and U73343.
C, averaged conductance (means ± S.E.) in control
cells treated with U73122 (1 µM, ~36-48 h) compared
with that in G q*-transfected cells that were untreated
or treated with U73122 or U73343. GIRK channel conductance was smaller
in all cells transfected with G q*, but this effect was
significantly diminished in cells treated with the PLC inhibitor U73122
(i.e. the current reduction was not as great). *,
statistically significant difference from control cells; ,
statistically significant difference different from
G q*-transfected cells (p < 0.05 by
ANOVA with post hoc Bonferroni's test; the number of cells
in each group is indicated in parentheses).
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We also tested if PLC activation contributes to the sustained
inhibition of GIRK channels obtained by expression of constitutively active G
q* (Fig. 5C). Throughout the period
following transfection with G
q* (or empty vector), cells
were incubated continuously with U73122 at 1 µM
(~36-48 h). Note that we observed an apparent nonspecific inhibition
of GIRK channels in control cells chronically treated with U73122 (see
also Ref. 17). Thus, the averaged conductance in those
U73122-treated cells was smaller than that seen in untreated control
cells (3.1 ± 0.5 versus 9.2 ± 0.6 nanosiemens,
respectively; p < 0.05) (compare Figs. 5C
and 2C, first bars). Nevertheless, as shown in
Fig. 5C, the averaged conductance was further reduced in all
cells transfected with G
q*; and importantly, the
reduction associated with expression of G
q* was
significantly attenuated in cells treated with the PLC inhibitor
U73122. As a control, inhibition of GIRK channel currents by
G
q* was unaffected by identical treatment with the
inactive analog, U73343. Thus, activation of PLC by TRH and
G
q* contributes, at least in part, to GIRK channel inhibition.
Lowering Plasma Membrane PIP2 Levels Diminishes
Receptor-mediated GIRK Channel Inhibition--
It has recently been
established that the membrane phospholipid PI(4,5)P2
is an important coactivator of GIRK channels (9, 10, 31), and it has
been suggested that PLC-mediated decreases in PI(4,5)P2
levels might account for GIRK channel inhibition following receptor
activation in atrial cells (15-17). Since we found that TRH-induced
inhibition of GIRK channel currents also appears to involve PLC, we
tested the possibility that GIRK channel inhibition might be sensitive
to membrane PI(4,5)P2 levels.
To this end, we used two minigenes that deplete membrane
PI(4,5)P2 levels via different mechanisms (23). The first
was a GFP-tagged fusion protein that includes the pleckstrin homology domain of PLC
, a construct that sequesters PI(4,5)P2; as
a control for PLC
-PH, we used a different GFP-tagged pleckstrin
homology domain derived from AKT (AKT-PH), which interacts with a
separate isoform of the membrane phospholipid PI(3,4)P2.
The second construct was a GFP fusion protein that included a
constitutively active yeast 5'-PI-PTase that was targeted to the plasma
membrane by incorporation of a myristoylation-palmitoylation sequence.
When tested within 24 h of transfection, both the PLC
-PH and
5'-PI-PTase constructs are localized to the plasma membrane, and both
lower membrane PI(4,5)P2 levels (23). As shown in the
example records of Fig. 6, TRH caused
very little inhibition of GIRK channel currents in cells expressing
either the membrane-targeted 5'-PI-PTase (Fig. 6A,
upper panel) or PLC
-PH (middle panel)
construct, but was highly effective in cells expressing the control
AKT-PH construct (lower panel). Indeed, whereas GIRK channel
inhibition by TRH was >55% in nearly all control cells (38 of 41 cells tested; 93%) and AKT-PH-transfected cells (9 of 11 cells tested;
82%), TRH inhibition was >55% in only one 5'-PI-PTase-transfected
cell (of 12 cells tested; 8%) and in just four PLC
-PH-transfected
cells (of 15 cells tested; 27%). These differences were also reflected in summary data presented in Fig. 6B, in which the averaged
TRH-induced inhibition of GIRK channel currents was significantly
diminished in cells expressing the two inhibitors of PIP2
compared with either control cells or cells expressing AKT-PH.

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Fig. 6.
Receptor-mediated GIRK channel inhibition
requires permissive levels of PIP2.
A, the effect of TRH on GIRK channel conductance in
representative cells transfected with 5'-PI-PTase (upper
panel) and PLC -PH (middle panel), two constructs
that deplete membrane PI(4,5)P2, as well as a control
AKT-PH construct that does not bind to PI(4,5)P2
(lower panel). Note that TRH had little effect on
conductance in cells expressing either of the constructs that lower
membrane PI(4,5)P2 levels, but was highly effective in the
cell expressing the control construct that does not bind
PI(4,5)P2. B, summary data quantifying the
effects of TRH on GIRK channel currents in cells transfected with the
indicated constructs. Note that relative to the control, inhibition of
GIRK channel currents by TRH was significantly diminished by the two
constructs that lower membrane PI(4,5)P2 levels (5'-PI-PTase and PLC -PH), but not by the control construct
(AKT-PH). Cells expressing AKT-PH, 5'-PI-PTase, and PLC -PH were
recorded within 24 h of transfection (23). Asterisks
denote statistically significant differences from control cells
(p < 0.05 by ANOVA with post hoc
Bonferroni's test; the number of cells in each group is indicated in
parentheses).
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It should be pointed out that the basal GIRK channel conductance tended
to be somewhat lower in cells transfected with 5'-PI-PTase or PLC
-PH
compared with control cells (~4 versus ~7 nanosiemens), as expected given the demonstrated facilitating effects of
PI(4,5)P2 on GIRK channels (9-11). In many cells, however,
a substantial basal GIRK channel conductance was retained
(e.g. see records in Fig. 6A,
upper and middle panels),
suggesting that differences in initial current amplitudes could not
account for the diminished effect of TRH. It is also noteworthy that
PLC
-PH can bind IP3 (32), a product of PLC-mediated
hydrolysis. Although it is therefore conceivable that buffering
IP3 could be responsible for inhibitory effects of the
PLC
-PH construct (but see below), this could not account for the
effects of 5'-PI-PTase. Thus, depletion of membrane PI(4,5)P2 levels appears to disrupt receptor-mediated
inhibition of GIRK channel currents.
Receptor-mediated Inhibition of GIRK Channel Currents Is
Independent of PKC or IP3--
The data presented up to
this point indicate a role for PLC and its substrate,
PI(4,5)P2, in GIRK channel inhibition by TRH receptors.
Therefore, we tested if signaling pathways typically activated as a
result of PLC-mediated PI(4,5)P2 hydrolysis are involved in
GIRK channel current inhibition. We were particularly interested in
investigating a role for PKC since it has earlier been implicated in
inhibition of recombinant GIRK channels (Ref. 33, but see Ref. 34). As
illustrated in Fig. 7A,
neither the PKC-activating phorbol ester PDBu nor its inactive analog,
4
-PDBu, had any effect on GIRK channel currents (both applied via
the perfusate at 1 µM). Because some PKC isozymes are
insensitive to phorbol esters, we also tested if a competitive
inhibitor of ATP binding to the catalytic site of PKC, the
bisindolylmaleimide compound GF 109203X, could interfere with
TRH-induced inhibition of GIRK channel currents. As depicted in the
records from the representative cell shown in Fig. 7B
(left panel), TRH was highly effective at inhibiting GIRK
channel currents in cells pretreated with bisindolylmaleimide (5 µM for
30 min). Indeed, TRH inhibited 81.1 ± 2.6% of the initial GIRK channel conductance in
bisindolylmaleimide-treated cells (n = 8), clearly not
less than the TRH-induced inhibition in control cells (~73%). In
control experiments using HEK293 cells expressing mouse TREK-1
channels, we found that PDBu strongly inhibited TREK-1 currents and
that bisindolylmaleimide blocked those effects of PDBu, attesting to
the efficacy of both PDBu and bisindolylmaleimide compounds (data not
shown). These data argue against a role for PKC in TRH
receptor-mediated inhibition of GIRK channel currents.

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Fig. 7.
Receptor-mediated GIRK channel
inhibition is independent of PKC activation and intracellular
IP3. A, the effect on GIRK channel
conductance of a PKC-activating phorbol ester (PDBu) and its inactive
analog (4 -PDBu) in a representative cell (left panel).
The compounds were applied in the bath (1 µM) as
indicated; neither had any effect on GIRK channel currents, as is also
apparent in averaged data from cells treated with the two phorbol ester
compounds (percent of control conductance; means ± S.E.)
(right panel). B, the effect of a
bisindolylmaleimide inhibitor of PKC (GF 109203X) on TRH-induced
inhibition of GIRK channel currents in a representative cell pretreated
with 5 µM bisindolylmaleimide for 30 min
(left panel). TRH caused a strong inhibition of GIRK channel
currents in this bisindolylmaleimide-treated cell; averaged values for
the magnitude of TRH-induced inhibition (percent of control
conductance; means ± S.E.) indicated that it was equal in
magnitude in control and bisindolylmaleimide-treated cells (right
panel). C, the effect of IP3 (100 µM in the whole cell recording pipette) on GIRK channel
conductance and on TRH-mediated inhibition of GIRK channel conductance
in a representative cell (left panel). The conductance at
all time points (Gt) was normalized (norm.) to
the initial conductance (Gi) to illustrate the close
correspondence of the current rundown and the inhibition by TRH in
control and IP3-treated cells. Averaged data show the
magnitude of TRH-induced inhibition (percent of control conductance;
means ± S.E.) in these experiments and indicate that there was no
difference between control cells and those recorded with
IP3 in the pipette (right panel).
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Activation of PLC also leads to production of IP3 and
increases in intracellular calcium, either of which could conceivably lead to GIRK channel inhibition. It is unlikely that changes in intracellular calcium contribute to the effects of TRH since our whole
cell experiments were performed with intracellular calcium buffered to
~10
8 M by including 10 mM EGTA in the internal solution. To determine if
IP3 could have effects independent of altering
intracellular calcium, we introduced IP3 into cells by
including it in the recording pipettes (100 µM). As
illustrated in the example cells of Fig. 7C, we found that
the time- and TRH-dependent decreases in GIRK channel
current in IP3-dialyzed cells were not different from those
in control cells recorded with pipettes that did not contain IP3. As is evident in the averaged data of Fig.
7C, the TRH-induced inhibition of GIRK channel currents in
IP3-containing cells (75.5 ± 4.1%, n = 7) was essentially identical to that in control cells (i.e. ~73%). Thus, intracellular perfusion with
IP3 neither mimicked nor occluded the TRH receptor-mediated
inhibition of GIRK channel currents.
Receptor-mediated GIRK Channel Inhibition Involves a Readily
Diffusible Messenger--
Our experiments provide no evidence to
implicate the major signaling pathways downstream of PLC (either PKC or
IP3/Ca2+) in receptor-mediated GIRK channel
inhibition. To test whether other diffusible messengers might be
involved, we recorded the effects of bath-applied TRH on GIRK channel
activity in cell-attached patches. In this recording configuration, the
agonist does not have direct access to channels recorded within the
patch. Therefore, any actions of the agonist must be transmitted by
some intermediary that can move between the channels inside the patch
and the receptors outside the patch, either via the cytosol or within
the plane of the membrane.
Cell-attached patch recordings of K+ channels were obtained
in G1/4 cells either from the cell line stably expressing the TRH-R1 and 5-HT1A receptors (Fig.
8A) or from the parental G1/4
cell line, which does not express these receptors (Fig. 8B).
Single channel currents were clearly inwardly rectifying (data not
shown), with a unitary conductance of ~40 picosiemens (39.0 ± 1.4 picosiemens, n = 5) and a mean open time of ~1 ms
(0.9 ± 0.1 ms, n = 9) (Fig. 8C), as
expected for GIRK1/4 channels (35). Moreover, when exposed to
extracellular 5-HT in an inside-out patch configuration, the channels
were vigorously activated by adding GTP to the inside face of the patch
(data not shown). These are defining properties of GIRK channels (1, 2,
8).

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Fig. 8.
Receptor-mediated GIRK channel inhibition
involves a readily diffusible messenger. A: the effect
of TRH on GIRK channels recorded in a cell-attached patch of G1/4R
cells stably expressing TRH-R1. Left panel, GIRK channel
activity was quantified as NPo (in 1-s bins) and
plotted as a function of time (lower trace); TRH was added
to the perfusate for the indicated period. The patch holding potential
is provided (upper trace). Note that bath-applied TRH caused
a strong inhibition of channel activity within the patch. This was true
even after the patch potential was adjusted (to
Em 60 mV) to correct for the anticipated
TRH-induced depolarization of cell membrane potential (30 mV). For
clarity, periods of transient instability in the patch recordings were
blanked (indicated by stars). Right panel, shown
are the continuous records (~1 s) of GIRK channel activity under
control conditions (at a patch potential of Em 30
mV) and in the presence of TRH (at a patch potential of
Em 60 mV). Note that the membrane potential
correction resulted in current amplitudes in TRH that approximated
those in the control (see the dashed line). B:
the effect of TRH on GIRK channels recorded in a cell-attached patch of
G1/4 cells not expressing TRH-R1. Left panel,
GIRK channel activity was quantified as NPo (in 1-s
bins) and plotted as a function of time (lower trace); TRH
was added to the perfusate for the indicated period. The patch holding
potential is provided (upper trace), and periods of brief
patch instabilities were blanked (stars). As expected, TRH
had no effect in control cells that do not have TRH receptors.
Right panel, shown are continuous records (~1
s) of GIRK channel activity under control conditions and in the
presence of TRH (at a patch potential of Em 30
mV). C: left panel, shown is a schematic of the
experimental configuration. To modulate channels contained within the
patch, activation of receptors outside the patch must either produce an
inhibitory mediator that can diffuse to the channels or induce the
withdrawal of an activating substance from the patch. Middle
panel, shown are the averaged data (percent inhibition of
NPo by TRH; means ± S.E.) in patches from
G1/4R cells (i.e. with TRH-R1; n = 10) and
from control G1/4 cells (i.e. without TRH-R1;
n = 6). TRH inhibited channel activity by ~68%,
similar to the TRH-induced inhibition of whole cell GIRK channel
currents. Right panel, mean channel open time (±S.E.) was
determined before and during TRH application in those patches of G1/4R
cells that showed a robust response to TRH (n = 8).
Note that TRH caused a significant decrease in mean open time
(determined by paired t test).
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As illustrated in Fig. 8A (left panel), GIRK
channel activity (i.e. NPo) in a
cell-attached patch from a TRH-R1-expressing G1/4 cell was strongly
diminished by bath-applied TRH. Based on our whole cell recordings, we
expect that the TRH-induced decrease in basal GIRK channel currents
would cause a membrane depolarization of ~30 mV. To correct for
effects of this membrane depolarization on the patch potential, we
adjusted the patch holding potential by
30 mV in the presence of TRH.
This adjustment was apparently appropriate since the unitary current
was approximately the same amplitude in the presence of TRH, after
correcting for the membrane depolarization (see dashed line
in sample records of Fig. 8A, right panel).
Furthermore, since channel activity remained strongly inhibited even
after this correction, the inhibition by TRH could not be attributed
solely to a TRH-induced change in membrane potential. TRH inhibited
GIRK channel activity by >55% in 8 of 10 cell-attached patches, with
an average inhibition in all patches of ~68% (NPo decreased from 0.09 ± 0.02 to 0.03 ± 0.02, n = 10; p < 0.05) (Fig. 8C,
middle panel), which is very similar to the magnitude of
TRH-induced inhibition of whole cell GIRK channel currents. In those
patches with a robust inhibition of channel activity by TRH
(n = 8), the inhibition involved, at least in part, a
decrease in mean channel open time (Fig. 8C, right
panel). As expected, there was no effect of TRH on GIRK channel
activity in cell-attached patches from control G1/4 cells that did not
express TRH-R1, as shown in Fig. 8 (B and C,
middle panel). These data therefore indicate that GIRK
channel inhibition involves a readily diffusible messenger.
 |
DISCUSSION |
In this study, we examined dual regulation of GIRK channels using
mammalian cell lines that express GIRK1/4 channels together with two G
protein-coupled receptors that couple preferentially to distinct
classes of G
subunit. Unlike GIRK channel activation by
G
i/o-coupled receptors, which is mediated by G
dimers (reviewed in Refs. 1, 2, and 8), we found that GIRK channel
inhibition by G
q-coupled receptors involves principally
the G
subunit. Exogenous expression of constitutively active G
subunits revealed little selectivity within the G
q
family for GIRK channel inhibition, but clear specificity for
G
q family subunits over all other classes of G
subunits. Moreover, receptor-mediated inhibition of GIRK channel
currents was diminished by minigene constructs that interfere with
G
q signaling, but not by those that target G
subunits. Signaling downstream of G
q appeared to involve
PLC since receptor- and G
q*-mediated GIRK channel
inhibition was reduced by the PLC inhibitor U73122 and because GIRK
channel inhibition by G
q-coupled receptors was
diminished by two different constructs that reduce membrane
PIP2 levels. We found no evidence for involvement of PKC,
IP3, or intracellular Ca2+ in GIRK channel
inhibition, although cell-attached patch recordings indicated
involvement of a readily diffusible messenger. These data support
burgeoning evidence that PIP2, itself a potent activator of
GIRK channels (9-11), represents a diffusible signal responsible for
GIRK channel inhibition by G
q-coupled receptors
(15-17). It remains possible, however, that receptor activation of
G
q subunits and PLC initiates a separate
signaling pathway, yet undefined, that leads to GIRK channel inhibition.
G
q Family Subunits Mediate GIRK Channel
Inhibition--
In native systems, agonist-evoked GIRK channel current
inhibition is typically associated with receptors that couple via
PTx-insensitive G
q subunits (e.g.
1-adrenoreceptors in atrial myocytes (3) and NK1
receptors in locus ceruleus neurons (6)). Accordingly, we found that
receptor-mediated GIRK channel inhibition was not affected by PTx, but
was diminished by PMT, a toxin that selectively uncouples
G
q-type subunits from receptors (25, 26). Moreover, we
found that GIRK channel inhibition was a property unique among G
subunits to members of the G
q family; constitutively
active mutants of G
q family subunits caused essentially
complete inhibition of basal GIRK channel currents in G1/4 cell lines
stably expressing GIRK1/4 channels, whereas the corresponding
constitutively active members of other G
subunit families were
without effect. These data are consistent with most previous studies in
which other (non-G
q family) G
subunits pre-activated
with GTP
S had little effect on native and/or recombinant GIRK
channels in excised patches (1, 8, 36). In one earlier study, however,
purified recombinant G
s-GTP
S and
G
i1-GTP
S, but not G
i2-GTP
S or
G
i3-GTP
S, inhibited G
-activated GIRK channels
in inside-out patches from Xenopus oocytes (37). We
found no effect of any of these subunits in our whole cell assay using
G
subunits activated by a Gln
Leu point mutation. Although the
reason for these discrepant findings is not entirely clear, it is
possible that the inhibition of GIRK channels observed in oocyte
patches with G
i1 and G
s was due to
contamination with non-activated G
-GDP subunits, which will sequester G
and indirectly inhibit G
-activated GIRK channel activity. Indeed, we have found that multiple different wild-type G
subunits (i.e. those not activated by the Gln
Leu
mutation), including G
i and G
s subunits,
strongly inhibit basal GIRK channel currents when expressed in G1/4
cells (12). Moreover, it is important to point out that those earlier
results are difficult to reconcile with current understanding of
receptor signaling to GIRK channels since receptors that couple to
G
s typically have no effect on GIRK channel currents,
and those that couple to G
i cause GIRK channel
activation, not inhibition (1, 36, 38). By contrast, our results with
G
q* and closely related subunits are entirely concordant
with observations that inhibition of GIRK channels in native settings
involves receptors that couple via PTx-insensitive G
proteins of the
G
q family (1).
Our results provide additional evidence indicating that it is indeed
the G
subunit, and not the associated G
dimer, that provides
the signal for receptor-mediated GIRK channel inhibition. Thus, we
showed that so-called "effector antagonists" of G
q
subunits (i.e. RGS2 and PLC
1-ct) substantially decreased
GIRK channel current inhibition evoked by TRH-R1 stimulation. Likewise,
in atrial myocytes, a peptide that interferes with
G
q-mediated PLC activation disrupted GIRK channel
current desensitization by muscarinic receptors (15). By contrast,
expression of constructs that sequester G
subunits relatively
non-selectively (i.e. G
t and
ARK-ct), as
well as those that bind G
5 specifically (i.e.
RGS6 and RGS11), did not disrupt receptor-mediated GIRK channel current
inhibition. This result is perhaps not surprising since most
combinations of G
and G
subunits are known to activate, rather
than inhibit, GIRK channels (12, 27, 28). However, we showed here that activation of PLC, a known G
effector (8), is necessary for GIRK
channel inhibition, and we recently found that G
dimers including
the G
5 subunit are unique in causing inhibition of GIRK
channels, perhaps by competing with activating G
pairs for
binding to the channel (12, 14). Nevertheless, we found no evidence to
indicate that receptor-mediated inhibition of GIRK channel currents
requires downstream effects of G
dimers, including G
5
pairs. If G
5-containing dimers do
associate preferentially with G
q in vivo (13)
and exclude GIRK channel-activating G
pairs from receptor-bound
heterotrimers, this function is apparently not required for
receptor-mediated GIRK channel inhibition. Moreover, these data
indicate that G
subunits do not contribute appreciably to the PLC
activation that appears to be necessary for receptor-mediated GIRK
channel inhibition.
GIRK Channel Inhibition May Occur by PLC-mediated Decreases in
PIP2--
Consistent with the demonstrated involvement of
G
q family subunits, our data also indicate a key role
for PLC, the major downstream effector of G
q, in
receptor-mediated GIRK channel current inhibition. Thus,
agonist-induced GIRK channel inhibition was diminished
pharmacologically by U73122, a PLC inhibitor, and also disrupted by
minigene constructs that lower membrane levels of a PLC substrate,
PIP2. Despite this, however, we found no evidence to
implicate further effectors in the classical signaling pathway
downstream of PLC since GIRK channel inhibition was apparently independent of effects on PKC, IP3, and intracellular
calcium. Similarly, PLC involvement has been demonstrated in other
studies of receptor-mediated GIRK channel inhibition (15, 16), but there has been no evidence to indicate that either IP3 or
Ca2+ plays a role (3, 4, 17, 33). Although it was suggested that an atypical PKC could mediate inhibition of GIRK channels expressed in Xenopus oocytes (33), in other native and
heterologous expression systems, receptor-mediated inhibition of GIRK
channel currents was independent of PKC activation (3, 4, 16, 17, 34),
as we found here.
Therefore, inasmuch as our data implicate PLC, but exclude its usual
downstream mediators, and given compelling evidence indicating that
PIP2 is a potent activator of GIRK channels (9-11), the
data support the following mechanism for receptor-mediated GIRK channel inhibition: decreased membrane concentrations of PIP2 that
ensue following hydrolysis by receptor-activated PLC result in GIRK channel inhibition by promoting release of PIP2 from its
channel binding site. This same mechanism was proposed to account for muscarinic inhibition of KATP channels in COS-7 cells (39)
and, more recently, for inhibition of native GIRK channels in atrial myocytes by a variety of G
q-coupled receptors
(i.e. m3 muscarinic,
1-adrenergic, and
endothelin-1 receptors) (15-17). As in the present work with
recombinant GIRK1/4 channels, the molecular correlate of those atrial
GIRK channels (35), involvement of PIP2 was usually
inferred by exclusion when PLC was implicated in receptor-mediated GIRK
channel current inhibition, but downstream mediators were not. In each
study, a different experimental approach was used to test
PIP2 involvement; for example, receptor-mediated inhibition of atrial GIRK channel currents was disrupted by using the
phosphatidylinositol kinase inhibitor wortmannin to lower membrane
PIP2 levels (16) or by using intracellular perfusion of
excess PIP2 to overwhelm the GIRK channel
PIP2-binding site (17). Using a particularly elegant and
non-pharmacological approach, it was found that M1 muscarinic
receptor-mediated inhibition of homomeric mutant GIRK4 channels
expressed in Xenopus oocytes was diminished by amino acid
substitutions that increased the affinity of the channel for
PIP2 (15). In accord with these earlier results, we found here that GIRK channel inhibition was disrupted by membrane-targeted 5'-PI-PTase and PLC
-PH, two constructs that lower PIP2
levels (23). It is also interesting to note that
PIP2-induced increases in GIRK channel activity are
associated with increased mean channel open times (9), and further
consistent with a role for diminished levels of PIP2, we
found that receptor-mediated GIRK channel inhibition involved decreased
mean channel open time (see Fig. 8C). In sum, it therefore
appears that data from both native and heterologous systems converge on
the conclusion that decreases in PIP2 could account for
receptor-mediated GIRK channel inhibition. However, it is important to
point out that this interpretation is based on indirect tests of
PIP2 involvement and by exclusion of other pathways
downstream of PLC; since all other potential signaling pathways have
not yet been excluded, the possibility remains that some other
mechanism for inhibition of GIRK channels by G
q-coupled receptors may yet be discovered.
PIP2 as a Diffusible Mediator of GIRK Channel
Inhibition--
We found that bath-applied TRH was able to inhibit
GIRK1/4 channels in cell-attached patches. Similar results were noted
for native GIRK channel inhibition in atrial myocytes by
1-adrenoreceptors and endothelin-1 receptors (3, 16, 17)
as well as for metabotropic glutamate receptor inhibition of
recombinant GIRK channels in Xenopus oocytes (33). These
type of results are usually interpreted to imply that cytosolic second
messengers are involved in the modulatory mechanism. However, if
PIP2 maintains its membrane association, the GIRK channel
inhibition would be membrane-delimited even though it was initiated by
G
q-coupled receptors outside the patch. By contrast,
membrane-delimited receptor activation of GIRK channels, mediated by
direct binding of G
subunits to the channels, requires agonist
stimulation of G
i/o-coupled receptors within the patch
(2, 40). Thus, if decreased membrane PIP2 levels are indeed
involved in GIRK channel inhibition, as we and others now suggest
(15-17), the implication is that PIP2 may be more readily
diffusible within the membrane than are G
dimers. In this
respect, it has been suggested that the non-activated G protein
heterotrimer may serve to tether G
i/o-coupled
receptors in close proximity to GIRK channels (41), and this could keep membrane signaling by G
dimers relatively more localized than that by PIP2.
Dual Regulation of GIRK Channels--
In cells of the heart and
brain, dual regulation of native GIRK channels by receptors that couple
via distinct classes of the G
subunit represents a mechanism by
which convergent effects on a single target can provide dynamic control
of cell excitability. Here, we used a heterologous expression system
that fully recapitulates dual regulation in order to demonstrate that
receptor-mediated inhibition of GIRK channels is mediated by
G
q subunits and to support the proposition that it may
be due to PLC-catalyzed decreases in membrane PIP2 levels.
This mechanism is unlike that causing GIRK channel activation, which is
mediated by G
i/o-coupled receptors and involves direct
interactions of G
dimers with GIRK channels. Interestingly, a
similar dual regulation of AKT by G proteins was recently discovered;
as with GIRK channel modulation, AKT was activated by G
dimers
and inhibited by G
q (42). Thus, opposing actions of G
and G
to provide up- and down-regulation of a single effector may
be a more prevalent phenomenon than is currently appreciated.