From the Department of Pharmacology and the
** Department of Anatomy and Cell Biology, University of Illinois,
Chicago, Illinois 60612, and the
Department of
Pharmacology, University of Texas Southwestern Medical Center,
Dallas, Texas 75390
Received for publication, December 13, 2000, and in revised form, January 11, 2001
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ABSTRACT |
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G protein-coupled inward rectifier
K+ channels (GIRK channels) are activated directly by
the G protein Heterotrimeric G proteins transduce a variety of regulatory
signals from a large number of heptahelical receptors to effectors such as adenylyl cyclases, phosphodiesterases, phospholipases, and ion
channels (1). Each G protein oligomer contains a guanine nucleotide
binding The crystal structure of the The present study was undertaken to investigate the interaction sites
of Sf9 Cell Culture and Construction of Recombinant
Baculoviruses--
Sf9 cells were cultured in suspension in
IPL-41 medium containing 1% Pluronic F68 and 10% heat-inactivated
fetal bovine serum at 27 °C with constant shaking (150 rpm). The
site-directed mutagenesis of Purification of G Protein Subunits from Sf9
Cells--
Sf9 cells (1.5 × 106/ml) were
coinfected with amplified recombinant baculoviruses encoding wild type
or mutant Cultures of Locus Coeruleus Neurons--
Cultured neurons from
the locus coeruleus were made from 2-4-day-old postnatal Long-Evans
rats (Charles River Breeding Laboratories). The culture methods were
described previously (9, 10). Rats were anesthetized with ether, their
brainstems were removed, and the rats were killed by decapitation.
Brain slices were made from isolated brainstems using a Vibratome
(Lancer 1000). The locus coeruleus was visually identified under a
dissecting microscope and excised out. The excised pieces were
incubated in a papain solution, dissociated by trituration, and
cultured. The culture medium contained a minimum essential medium with
Earle's salts (Life Technologies, Inc., catalog no. 11430-030),
modified by adding D-glucose (5 mg/ml), NaHCO3
(3.7 mg/ml), and L-glutamine (0.292 mg/ml). The medium was
supplemented by heat-inactivated rat serum (2 or 5%, prepared in our
laboratory), L-ascorbic acid (10 µg/ml), penicillin (50 units/ml), and streptomycin (50 µg/ml). Dissociated neurons were
cultured on a squared plastic piece placed in a small well made at the
center of a 35-mm culture dish. Before plating the neurons, the well,
containing the plastic piece, was coated with rat collagen and a feeder
layer of glia cells obtained from rat brains. The cultures were
incubated at 37 °C in 10% CO2 and 90% air with
saturated humidity for 13.4 ± 2.5 days (mean ± S. D.).
Experiments were performed on large neurons (soma diameter, 24.8 ± 2.9 µm; mean ± S.D.).
Electrophysiology--
Electrophysiological experiments were
performed with the inside-out patch clamp techniques (11, 12). The
patch pipettes were made from thoroughly washed glass capillaries. The
patch pipette solution (external solution) contained 156 mM
KCl (or potassium gluconate), 2.4 mM
CaCl2, 1.3 mM MgCl2, 0.5 µM tetrodotoxin, and 5 mM HEPES-NaOH (pH
7.4). The GDP-containing cytoplasmic side solution (the bathing
solution) contained 141 mM potassium gluconate, 8.7 mM NaCl, 5 mM EGTA-KOH, 1 mM
MgCl2, 2 mM Na2ATP, 0.1 mM GDP, 5 mM HEPES-KOH, and ~5.5
mM KOH (pH 7.2). Membrane potential was corrected for the
liquid junction potential between the bathing solution and the patch
electrode solution.
The procedures of data analysis were similar to those previously
described (12). The data were analyzed with pCLAMP programs (version 6)
(Axon Instruments). The overall frequency response was set at 1 kHz
(
The experimental protocol was as follows. After an inside-out patch was
formed, the basal channel activity (in the GDP-containing bathing
solution) was recorded for a few minutes. The solution in the bath
(~0.1-0.17 ml) was then exchanged with 0.5 ml of various kinds of
When the inside-out patch configuration was established, usually the
patch produced a very infrequent activity of GIRK channels (basal GIRK
activity) and an activity of channels of small amplitude (of unknown
origin) in the GDP-containing bathing solution. In some patches, the
occurrence of large flickering channels (about 100 picosiemens with
[K+]o of 156 mM), whose activity
was not dependent on GTP, was observed (12). Frequent occurrence of
these large background channels hindered the analysis of GIRK channel,
and thus such patches were not included in our sample. Occasionally,
instead of an inside-out patch, vesicle formation occurred. (The
vesicle formation was inferred by the appearance of a current drooping
because of the existence of membrane resistance and capacity on the
opposite side of the patch.) (11). These patches were not included.
Sometimes we observed rather vigorous basal activity of GIRK-like
channels in the GDP-containing bathing solution before the application of
Stock solutions of Statistical Treatment of Electrophysiological Data--
The
distribution pattern of NPo was almost always
non-Gaussian; this can be inferred from substantial discrepancies between the mean and the median values (see Figs. 4A and 6).
This non-Gaussian distribution suggests the possible existence of more than one type of channel (e.g. solitary channels and
aggregates of channels). Because the mean value in our samples is
influenced greatly by a small number of patches with a large
NPo, the median was a more appropriate parameter to
represent the channel activity of a group. Also, statistical
comparisons of NPo were done, unless otherwise
noted, by using the nonparametric statistics (the Kruskal-Wallis ANOVA
and a posttest using the Mann-Whitney with the Bonferroni adjustment).
We used two different types of wild type
In Vitro Assays for Phospholipase C and Adenylyl Cyclase
Activity--
Phospholipase C activity was measured using sonicated
micelles containing 50 µM phosphatidylinositol
4,5-bisphosphate, 500 µM phosphatidylethanolamine, and
[inositol-2-3H]phosphatidylinositol
4,5-bisphosphate (PerkinElmer Life Sciences) (2500 cpm/assay) in
a solution containing 50 mM NaHEPES (pH 7.5), 0.42 mM EDTA, 3 mM EGTA, 2 mM
MgCl2, 1.7 mM CaCl2, 42 mM NaCl, 47 mM KCl, 4 µM GDP,
0.125 mg/ml bovine serum albumin, 1 mM
dithiothreitol, and 0.375% octyl glucoside with 0.1 nM
PLC
To measure adenylyl cyclase activity, purified Activation of GIRK Channels by 10 nM Is an Almost Saturated Concentration for
To test the hypothesis that regions on the side surface of the
First, we surveyed all these outer blade mutants (eight mutants
altogether) for their ability to activate GIRK channels. We used
Effects of Detergent--
We also tested whether channel
activities were affected by the detergent in which the protein was
dissolved. We tested the buffer with 0.033% octyl glucoside, 0.66 mM HEPES, 0.033 mM EDTA, and 6-17
µM dithiothreitol, corresponding to those used for 100 nM GIRK channels are activated directly by the G protein It should be noted that in this study, we have concentrated on
analyzing mutations that produced a large functional deterioration in
the K+ channel activation. This study does not exclude the
possibility that other mutants may have a moderate defect in
their ability to interact with K+ channels.
It was previously shown that the activation of PLC Our results on It is known that subunit. The crystal structure of the G protein
subunits reveals that the
subunit consists of an N-terminal
helix followed by a symmetrical seven-bladed propeller structure.
Each blade is made up of four antiparallel
strands. The top surface
of the propeller structure interacts with the G
subunit. The outer
surface of the
torus is largely made from outer
strands of
the propeller. We analyzed the interaction between the
subunit and
brain GIRK channels by mutating the outer surface of the
torus.
Mutants of the outer surface of the
1 subunit were
generated by replacing the sequences at the outer
strands of each
blade with corresponding sequences of the yeast
subunit, STE4. The
mutant
1
2 subunits were expressed in and
purified from Sf9 cells. They were applied to inside-out patches
of cultured locus coeruleus neurons. The wild type
1
2 induced robust GIRK channel activity
with an EC50 of about 4 nM. Among the eight
outer surface mutants tested, blade 1 and blade 2 mutants (D1 and CD2)
were far less active than the wild type in stimulating GIRK channels.
However, the ability of D1 and CD2 to regulate type I and type II
adenylyl cyclases was not very different from that of the wild type
1
2. As to the activities to stimulate
phospholipase C
2, D1 was more potent and CD2 was less potent than the wild type
1
2.
Additionally we tested four
1 mutants in which mutated
residues are located in the top G
/
interacting surface. Among
them, mutant W332A showed far less ability than the wild type to
activate GIRK channels. These results suggest that the outer surface of
blade 1 and blade 2 of the
subunit might specifically interact with
GIRK and that the
subunit interacts with GIRK both over the outer
surface and over the top G
interacting surface.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit and a high affinity dimer of
and
subunits.
The agonist-bound receptor activates the G proteins and generates
GTP-bound
subunits and free
subunits. Both GTP
and
can regulate downstream effectors. The hydrolysis of GTP to GDP
on
subunits leads to the reassociation of
and
subunits
to form inactive heterotrimers.
subunit reveals that the
subunit consists of an N-terminal
helix followed by a symmetrical seven-bladed propeller structure based on WD repeat sequences, repeating motif of about 40 amino acids (2, 3). Each blade consists of
four antiparallel
sheets. The top surface of the propeller
structure interacts with the
subunit. The bottom surface of the
torus is the major site for interaction with the
subunit. The outer
surface of the torus is largely made up of the outer
strands of
seven blades.
subunits directly regulate various effectors,
including phospholipase C
s
(PLC
s),1 adenylyl
cyclases, and ion channels. Experiments using mutated
subunits have
demonstrated that some of the amino acid residues, which are located at
the interaction sites between G
and -
subunits, are crucial for
the interaction between
and the effectors, and each effector
demonstrates its specific domain of interaction on the
subunit (4,
5). Further mutational analysis showed that the activation of
PLC
2 also involves residues in the outer strands of
blades 2, 6, and 7. However, mutations of
subunits that
affected PLC
2 activity did not influence the interaction between
and type I or type II adenylyl cyclase (6). These results suggest that
interacts with effectors through both the
G
binding surface and the outer surface of the propeller structure
and that each effector is interacting with
using different
regions of its outer surface.
1 with brain GIRK channels. We focused our
investigation on the outer surface of the
1 subunit. We
made sets of
1 mutants in which the amino acid residues
located on or near the outermost
strand of each blade were replaced
with the corresponding residues of the yeast
subunit (STE4), a most
distantly related member of
from mammalian
subunits. In
this study we show that mutating residues located on or near the outer
strands of blade 1 and blade 2 disrupt GIRK activation by
1
2. In contrast, the same mutations did
not substantially affect the ability of
1
2 to regulate adenylyl cyclases,
indicating that no global disruption of
1 occurred because of the mutations. It is possible that the sites of the
1 subunit in blade 1 and blade 2 could specifically be
involved in brain GIRK activation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 cDNA was performed
using the Muta-Gene in vitro mutagenesis kit (Bio-Rad). The
amino acid sequence of each mutation is shown in Fig. 3. The mutations
were confirmed by sequencing the mutating region. Mutated
1 cDNAs were subcloned into the pVL1392 transfer vector, and the resulting plasmids were cotransfected into Sf9 cells with BacPac6 viral DNA linearized with Bsu36I
(CLONTECH) by LipofectAMINE (Life Technologies,
Inc.). Recombinant baculoviruses were plaque-purified and amplified as
described (7). Baculoviruses encoding L117A, D224S, D228R, and W332A
were generated and generously provided by the late Dr. Eva J. Neer
(Harvard University) (4). Recombinant baculoviruses encoding wild type
1,
2, and
His6-G
i1 have been described
previously (8).
1,
2, and His6-G
i1. Cells were harvested after
48 h, and recombinant
protein was purified from the
membrane extract using an nickel-nitrilotriacetic acid column as
described (8). The elute fractions from the nickel-nitrilotriacetic
acid column were concentrated, and the buffer was exchanged into 20 mM NaHEPES (pH 8.0), 1 mM EDTA, 1 mM dithiothreitol, 50 mM NaCl, and 0.5%
octyl glucoside with Centricon-30 (Amicon). Recombinant
G
i2 was purified from Sf9 cells as described previously (8).
3 db by an 8-pole Bessel filter) and digitized at 10 kHz.
Transitions between the closed and the open states were registered if a
level crossed a threshold and lasted for more than 100 µs. The
threshold was set at 2.0 pA from the baseline when the membrane was
held at
101 mV (1.6 pA when the membrane was held at
80.4 mV); this
procedure excluded most of the small background channels (12). We
expressed the channel activity by a variable NPo
(i.e. the open probability of an elementary channel
multiplied by the number of channels in the patch).
subunits dissolved in the same GDP-containing bathing solution.
The exchange was done manually by using a pipetter wrapped up with a
grounded aluminum foil. The degree of solution exchange was tested by
using osmolarity changes as an indicator; the test showed that this
manual method resulted in the replacement of about 86% of
the original solution (using the bath volume of 0.15 ml).
. This basal activity might have originated from the local presence of overexpressed
s or from the basal activity of the receptors. Because the present objective was to analyze the GIRK channel activation induced by the application of exogenous
proteins, we did not pursue the investigation of the patches with a
high frequency basal activity (more than about once per second).
proteins were diluted with the GDP-containing
bathing solution to a final concentration of 1-100 nM. At
10 nM G
, the solution contained the following
buffer/detergent: 0.0033% octyl glucoside, 0.066 mM HEPES,
0.0033 mM EDTA, and 0.24-2.1 µM
dithiothreitol. Experiments were done with a bath temperature of
~21 °C.
1
2: with and without hexahistidine
tagging at the N terminus of
1. The median value
of NPo for the channel activated by the
hexahistidine-tagged wild type
1
2 was
0.109 (n = 47), and the median value of
NPo by nontagged
1
2
was 0.099 (n = 35) (difference not significant; p > 0.5). We will refer to these two types simply as
1
2.
2 and the indicated amount of
subunit. The
mixture was incubated at 30 °C for 8 min, and the amount of
IP3 generated was quantitated as described (4).
subunits were
reconstituted with 10 µg of membranes from Sf9 cells
expressing type I or type II adenylyl cyclase for 3 min at 30 °C in
a final volume of 20 µl. Assays were then performed for 7 min at
30 °C in a total volume of 50 µl containing 4 mM
MgCl2 and 0.2% octyl glucoside as described (4).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
2 in
Locus Coeruleus Neurons--
When a gigaseal was formed in the on-cell
mode in locus coeruleus neurons, some channel activity was usually
observed. Upon making an inside-out patch, this activity started to
subside, reaching a low level within a minute (presumably because the
intracellular GTP was washed away in exchange of the GDP-containing
solution). We then applied wild type
1
2
(10 nM), which induced, after a latency, vigorous channel
activity (Fig. 1A) (13, 14).
The channels showed typical GIRK-like characteristics (12, 14) with a
chord conductance of ~30-35 picosiemens, exhibiting a mixture of short openings and long openings, the latter sometimes showing bursts (Fig. 1A, record b). The current-voltage
relationship of the channels showed an inward rectification (Fig.
2A). Thus, these channels
activated by
1
2 in locus coeruleus
neurons probably belong to the GIRK (Kir3) subfamily (15, 16).
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Fig. 1.
Effects of wild type
1
2
and mutant D1 on GIRK channels from locus coeruleus neurons; single
channel recordings using the inside-out mode. A, the
time course of GIRK channel activation (NPo) induced
by wild type
1
2. Each circle
represents the NPo every 30 s. The thick,
solid horizontal line indicates the period when the GDP-containing
control solution (Ctr) was exchanged with wild type
(Wt)
1
2 (10 nM)-containing solution. About 2 min later, the
NPo started to increase. The upper left
panel (a) shows a segment of the record during the
control period (Ctr). Openings of GIRK channels were
observed very infrequently; the record was taken 120 s after the
beginning of the record (see the main graph). The upper right
panel (b) shows that the application of wild type
1
2 (Wt) produced robust GIRK
channel activity. The record was taken 501 s after the start of
the record. The downward direction corresponds to inward currents.
B, lack of activation of GIRK channels by the application of
D1 (10 nM). The thick solid horizontal line
indicates the period when the control cytoplasmic side solution
(Ctr) was exchanged with a solution containing 10 nM D1. The upper left panel (a) shows
a record segment during the control period (Ctr); the record
was taken 159 s after the start of the record. The upper
right panel shows a record (b) after the application of
D1 (10 nM) (465 s after the start of the record). In both
A and B, the holding potential was
101 mV, and
the patch pipette contained the 156 mM KCl solution.
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Fig. 2.
A, current-voltage relation of GIRK
channels from locus coeruleus neurons; single channel recordings using
the inside-out mode. The channel activity was induced by 10 nM wild type 1
2. The
solid line is the fit of the data to a second order
polynomial. Five patches (represented by different symbols) were used.
The inset records show single channel currents at different
membrane potentials. The experiment was done in the 156 mM
potassium gluconate pipette solution. B, dose-response
relationship of wild type
1
2. For each
patch, two or three different concentrations (including the standard
concentration, 10 nM) of wild type
1
2 were applied in ascending order. The
channel activity was determined by averaging NPo
during the 5-9 min of introducing a new concentration of
1
2 (except that values at zero
concentration were determined by NPo during 1 min
before introducing
1
2). For each patch,
the activity was normalized to the NPo value at the
standard concentration (10 nM). The mean
NPo at the standard concentration (solid
square; 100%) was 0.265. The vertical lines are S.E.
The continuous line is drawn by fitting to a logistic
equation using EC50 = 3.78 nM and Hill's
coefficient = 2.03. The number of patches was 7 at 30 nM, 22 at 10 nM, 7 at 3 nM, 5 at 1 nM, and 5 at 0.3 nM.
1
2--
Fig. 2B shows a
concentration-response relation in wild type
1
2. Values of NPo over
5 to 9 min after the introduction of
1
2
were plotted. The wild type
1
2 activated
the brain GIRK with an EC50 of about 4 nM, and
the activation almost saturated with 10 nM. These
results are in approximate agreement with previous studies on cardiac
and cloned GIRK channels (17-20).
1 Mutations at Outer Blades: K+
Channel--
Within the
subunit family, the STE4 gene product of
Saccharomyces cerevisiae is most distantly related to
mammalian
subunits. So far, no evidence has been presented to
suggest the regulation of adenylyl cyclase, PLC, or K+
channel activity by yeast
subunits. Therefore, we suspected that
exchanges of the effector-interacting domains of the
subunit with
the corresponding sequences from STE4 would result in mutant proteins,
which are incapable of interacting with mammalian effectors. Indeed,
Peng et al. (21) have recently reported that STE4 does not
activate GIRK channels.
torus are important for the regulation of effectors, we mutated
residues of an outer strand of each blade into the corresponding sequence of STE4 (Fig. 3). Each
mutant was coexpressed with His6-G
i1
and
2 in Sf9 cells, and mutant
subunits
were purified as described under "Experimental Procedures."
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Fig. 3.
Mutation of outer blades of
1 subunit. The amino acid sequence
of bovine
1 (
1) is aligned with the
corresponding STE4. For each type of mutation (D1 through D7, except
D2R), the amino acid sequence of the outer blade of the bovine
1 subunit (underlined) was replaced with the
corresponding sequence of STE4 (aligned underneath,
underlined). WD represents WD repeat motif. Because blade 5 (WD 6) is interacting extensively with the
2
subunit, it was excluded from the mutational analysis. In mutant
D2R, valine was changed into glycine; this mutation has been
shown to inhibit the ability of STE4 to transmit mating signaling (25).
The names of the mutants are indicated in bold letters above
the sequence (such as D1 and CD2). The empty parentheses in
WD6 of STE4 represents the following sequence:
(LFRGYEERTPTPTYMAANMEYNTAQSPQTLKSTSSSYLDNQ).
1
2 mutants at a concentration of 10 nM, which is, for the wild type
1
2, an almost saturating dose for
activating the K+ channel (Fig. 2B). The
variability of NPo among different patches was quite
large (see "Experimental Procedures") (Fig. 4A). Nevertheless, the mutant
D1 clearly showed far less ability to activate the GIRK channel than
did the wild type (p < 0.001) (Figs.
1B and 4A). The mutant CD2 was also significantly
less effective in activating the channels compared with the wild type (p < 0.05). Mainly because of the large variation of
NPo, we could not obtain significant differences in
NPo between the wild type and each of the other
mutants (D2F, D2R, D3, D4, D6, and D7) (Fig. 4A). We,
therefore, focused on D1 and CD2 mutants and tested a higher
concentration for their ability to activate GIRK channels. Even
at 100 nM, both D1 and CD2 produced only a small amount of
channel activity. Comparison of the channel responses by D1 and CD2
with the wild type dose-response curve clearly indicates the
impaired ability of these two mutants (CD1 and CD2) to activate the
K+ channel (Fig. 4B).
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Fig. 4.
A, effect of replacing residues on the
outermost strands of 1 with the corresponding sequences
of yeast STE4 on GIRK activity in locus coeruleus neurons. The
thick horizontal lines represent median values, and the
heights of the columns represent mean values of
NPo. The vertical lines
represent S.E. The number in parentheses indicates the
number of patches; for each patch, the channel activity was determined
by averaging NPo during the 5-9 min after
introducing various types of
1
2. The mean
basal activity (before applying various types of
1
2) was 0.0051 in NPo
(n = 74) and mainly originated from the small
background channel activity (12). The 156 mM KCl pipette
solution was used. Comparison with the wild type: *,
p < 0.05 (p = 0.035); ***,
p < 0.001 (nonparametric ANOVA and the posttest).
B, comparison between wild type dose-response relation with
the responses to D1 and CD2 mutants at 10 and 100 nM.
Square symbols represent the dose-response relationship of
the wild type. For each patch, the data were normalized to the response
at the standard
1
2 concentration (10 nM). This figure was derived from the same experiment as in
Fig. 2B; here, NPo was averaged during
the 5-7 min (instead of 5-9 min) of the solution exchange (see legend
for Fig. 2B for details). Circles and
triangles, respectively, represent data for D1 and CD2
mutants (at 10 and 100 nM) obtained in separate experiments
from those in A. The response (NPo) was
averaged during the 5-7 min of the exchange of the solution. The data
were not normalized. Solid symbols represent the mean
values, whereas the open symbols represent the median
values. For D1 at 10 nM, S.E. = 0.010 and n = 9. For D1 at 100 nM, S.E. = 0.007 and n = 7. For CD2 at 10 nM, S.E. = 0.024 and n = 7. For CD2 at 100 nM, S.E. = 0.0167 and n = 5. The mean of NPo for the wild type obtained during
the D1 experiments was 0.105 (median 0.067; n = 18).
The mean obtained during the CD2 experiments was 0.145 (median 0.106;
n = 16). The difference between 10 nM wild
type and 10 nM D1 was significant (p < 0.01; nonparametric ANOVA and posttests). The difference between 10 nM wild type and 10 nM CD2 was marginally
significant (p = 0.13). Because we performed two
independent tests on the differences between wild type and CD2 (10 nM) (A and B), we calculated the
overall significance level by which wild type and CD2 (10 nM) are different; it gives p = 0.03. In
contrast, no significant differences were obtained (ANOVA, posttest)
between 10 nM D1 and 100 nM D1 or between 10 nM CD2 and 100 nM CD2.
1 Mutations at Outer Blades: Adenylyl Cyclases and
PLC
2--
Despite the impaired ability of D1 and CD2 to
activate GIRK channels, these two mutants were as active as the wild
type to stimulate type II adenylyl cyclase (Fig.
5A). They could also inhibit
type I adenylyl cyclase, similarly to wild type
1
2 (data not shown). Both D1 and CD2 were
capable of stimulating PLC
2. The mutant D1 was, however,
more active and the mutant CD2 was less active than the wild type
within the concentration range of the assays (Fig. 5B).
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Fig. 5.
Activation of type II adenylyl cyclase and
PLC 2 by purified mutant
subunits. A, the
indicated amount of each
mutant was reconstituted with 10 µg
of Sf9 cell membrane expressing type II adenylyl cyclase in the
presence of 100 nM GTP
S-Gs
. Adenylyl cyclase activity
was measured as described under "Experimental Procedures."
B, the indicated amount of each
mutant was
reconstituted with 0.1 nM PLC
2, and the
synthesis of IP3 was measured over 8 min at 30 °C as
described under "Experimental Procedures." The data of each panel
show the average of duplicate determinations from a single
experiment that is representative of three such experiments. The
vertical bars indicate S.E.
1
2. This buffer alone did
not induce channel activity during ~10 min of application (mean
NPo: 0.000189 before application; 0.000232 after
application; n = 5; p > 0.7; paired
t test). We also compared the effect of 10 nM
wild type
1
2 in the standard buffer with
that in the high detergent buffer. Again, no marked difference was
observed. The NPo during the 4-6 min after the
start of application of the wild type
1
2
in the standard buffer was 0.233 (mean, n = 7), and
that in the high buffer/detergent was 0.14 (n = 5).
(The medians were 0.065 and 0.17, respectively; p > 0.6.)
1 Mutations at G
/
Interaction
Sites--
1 mutants (L117A, D228R, D246S, and W332A),
which are mutated at a residue of the interface between
and
subunits, were previously tested for their ability to regulate PLC
s
and adenylyl cyclases (4). We also tested these four mutants at 10 nM for their ability to activate the GIRK channels. Fig.
6 summarizes the values of
NPo during the 5-9 min after the start of
1
2 application. Two of these mutants
(D246S and W332A) produced channel activity significantly lower than
the wild type (p < 0.01 and p < 0.001, respectively).
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Fig. 6.
Effect of mutating some of the
G /
interaction sites
on GIRK activity in locus coeruleus neurons. The thick
horizontal lines represent the median values, and the heights of
the columns represent the mean values of NPo. The
vertical lines represent S.E. The number in
parentheses indicates the number of patches. For each patch, the
channel activity was determined by averaging NPo
over 5-9 min after introducing various types of
1
2 (wild-type and mutants). The mean
basal activity (NPo) (before applying
various types of
1
2) of this sample was
0.0057 (n = 69). The 156 mM KCl pipette
solution was used. Comparison with the wild type: **,
p < 0.01; ***, p < 0.001 (nonparametric ANOVA and posttest).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit. The interaction sites of
with GIRK have been
investigated on the G
interacting surface of the
subunit (5).
In the present study, we have demonstrated that regions outside of the G
/
interaction surface of the
subunit also participate in the
interaction with GIRK channels. We characterized the interaction of
with brain GIRK by using
1 mutants on the outer strands of the seven-bladed
-propeller structure. Mutations of
certain residues on the outer strands of blade 1 (D1) and blade 2 (CD2) resulted in the severe disruption of their ability to activate GIRK
channels. However, these mutants could regulate adenylyl cyclases
similarly to the wild type, suggesting that the mutations of the D1 and
CD2 areas did not produce a global disruption of
1
structure. Thus, the results suggest that the mutated residues on the
side of blade 1 and blade 2 of the
torus might be specifically involved in the regulation of GIRK channels.
2
involves the outer strands of blades 2, 6, and 7 (6). Interestingly, CD2 also showed a defect in its ability to activate
PLC
2. The results further support the importance of
blade 2 for the interaction with PLC
2. As shown in Fig.
5, D1 was 2-3-fold more potent than the wild type in its
ability to stimulate PLC
2. The exact reason for this
difference is currently unclear.
mutations over the G
/
interaction surface
indicate that W332A showed far less ability to activate GIRK channels.
Although only one concentration (10 nM) of
1
2 was tested, we observed a very large
difference in their activity between W332A and the wild type.
Because 10 nM is an almost saturating dose for the wild
type, this result suggests that W332A is much less active than the wild
type in its ability to stimulate GIRK channels. Thus,
/GIRK
interaction sites partially overlap with the G
/
interaction
sites, but they are not identical. This is similar to the case of
subunit interaction with PLC
2 or adenylyl cyclase (4,
5).
can interact with effectors only if G
is
dissociated from
. X-ray crystallographic studies have shown that
the
subunit does not undergo conformational changes when it is
dissociated from G
(3). It has been demonstrated that GDP-bound G
could sever the
/GIRK association quickly (22, 23). Because the
interaction sites of GIRK overlap with the G
interaction surface on
G
, this effect of G
could be explained by a simple spatial
(three-dimensional) competition on the
subunit between G
and
GIRK (4). It is also possible that the association of GIRK to
induces a conformational change of the
subunit, and this change
would favor the binding of
and GIRK. Conformational changes of the
subunit are demonstrated in the complex of
with phosducin (24). The binding of phosducin to the
subunit produces a distinct conformational change in blade 6 and blade 7 of the
subunit. The phosphorylation of phosducin on Ser-73 reduces its
affinity for
and the released
subunit and then switches
back to the conformation of free
or that of the heterotrimer (24). In the case of
complexed with GIRK, when
G
i interacts with a certain region of G
/
interaction sites, the conformation of
could return to the
resting state (before GIRK was attached), and this could decrease the
affinity of GIRK with
. The determination of the structure of
complexed with GIRK will be necessary to answer this question.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Alfred. G. Gilman for
support and encouragement throughout this study. We thank the
late Dr. Eva J. Neer (Harvard University) for baculoviruses
encoding mutant 1 subunits (L117A, D228R, D246S, and
W332A), Dr. Paul C. Sternweis for PLC
2 protein and
valuable comments on the manuscript, and Dr. Marlos A. G. Viana
(University of Illinois at Chicago) for advice on statistics.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants AG06093 (to Y. N.) and GM61454 (to T. K.) and American Heart Association Texas Affiliate Grant 96G-105 (to T. K.).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.
§ These authors contributed equally to this work.
¶ Present address: Dept. of Clinical Pharmacy and Pharmaceutical Technology, College of Pharmacy, University of Science and Technology, Irbid, Jordan.
To whom correspondence should be addressed. 835 S. Wolcott
Ave., Chicago, IL 60612. Tel.: 312-413-0111; Fax: 312-996-1225; E-mail:
tkozas@uic.edu.
Published, JBC Papers in Press, January 17, 2001, DOI 10.1074/jbc.M011231200
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ABBREVIATIONS |
---|
The abbreviations used are:
PLC, phospholipase C
;
GIRK, G protein-coupled inward rectifier
K+;
ANOVA, analysis of variance;
GTP
S, guanosine
5'-3-O-(thio)triphosphate.
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