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INTRODUCTION |
The G protein-gated inwardly rectifying K+ channels
(GIRKs)1 were the first
channels to be shown to be gated by a direct interaction with the

subunits of GTP-binding proteins (1, 2, 3). This mechanism
mediates the coupling of m2-muscarinic receptors with GIRK channels in
the atria, and the generation of inhibitory postsynaptic currents by
neurotransmitters acting on GABAB, 5HT1A and
A1 receptors, in hippocampal neurons (4). More recently, other regulators of GIRK channels have been identified, which can
activate these channels in the absence of G proteins. Recombinant GIRK1/GIRK4 channels and the native atrial channels were shown by Sui
et al. (5) to be activated by a rise in cytosolic
[Na+], with a threshold for activation of 3-10
mM and an EC50 of ~40 mM. In
isolated membrane patches, activation by Na+ required the
hydrolysis of ATP. Subsequently, Sui et al. (6) showed that
phosphatidylinositol 4,5-bisphosphate (PIP2) mimics the
ATP effects and that depletion or block of PIP2 inhibits
the activation of GIRK channels by both G
and Na+.
Huang et al. (7) reported similar findings and showed also that activation of GIRK channels, by applying PIP2 in the
presence of Na+, precluded further activation by
G
.
Activation by internal Na+ is not unique to atrial
GIRK1/GIRK4 channels. GIRK1/GIRK2 heteromeric channels have also been
shown to be activated by 20 mM Na+ in the
presence of ATP (8), and a mutation in the pore region of GIRK2, which
renders these channels permeable to both Na+ and
K+, causes a large increase in their G
-independent
activity (9, 10, 11).
Some of the molecular aspects of G protein gating of GIRKs have already
been elucidated. G proteins have been shown to interact with both the
cytoplasmic N- and C-terminal regions of GIRK subunits. Huang et
al. (12) made a series of GIRK1 C-terminal fusion proteins and
reported that the region between amino acids 318 and 462 was involved
in binding purified G
. A recent study by Krapivinsky et
al. (13) identified a single region within the C-terminal tail of
GIRK4 that lies in close proximity to the second transmembrane segment
(TM2) (amino acids 209-245) as being critical for G
binding and
channel activation. A point mutation within the proximal C terminus of
GIRK4, substituting threonine for cysteine 216, drastically reduced the
potency of G
in activating not only GIRK4 homomeric channels but
also GIRK1/GIRK4 heteromeric channels (13).
The aim of our study was to identify the region of GIRK1/GIRK2 channels
that mediates their activation by internal Na+. Using a
chimeric approach, combined with site directed mutagenesis of the GIRK
subunits, we have identified the proximal C-terminal region of GIRK2 as
playing a crucial role in the Na+-dependent
activation of both GIRK2 homomeric and GIRK1/GIRK2 heteromeric
channels. Within this region, GIRK2 has an aspartate at position 226, which is conserved in GIRK4, but is an asparagine in GIRK1.
Substituting asparagine for aspartate 226 in GIRK2, abolished
Na+-dependent activation of both the homomeric
and GIRK1/GIRK2 heteromeric channels without affecting their ability to
be activated via the m2-muscarinic receptors. The reverse mutation in
the GIRK1 subunit (N217D) was sufficient to recover
Na+-dependent activation of the heteromeric channel.
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EXPERIMENTAL PROCEDURES |
cDNA Clones--
Standard PCR procedures were used to
construct a strong Kozak consensus sequence GCCGCCACC immediately
upstream of the ATG initiation codon in cDNA clones for GIRK1 and
GIRK2. We used the longer of the GIRK2 splice variants, which contains
423 amino acids (14). These constructs were then subcloned into the
EcoRI site of the pBG7.2 vector, which provides the 5'- and
3'-untranslated regions of the Xenopus
-globin gene.
GIRK4 was also subcloned into the pBG7.2 vector, and the human
muscarinic m2 receptor was inserted into pBluescript KS(II)+.
Construction of Chimeras and Point Mutants--
The construction
of chimeras 1211 and 1222 was as described in Stevens et al.
(15). Briefly, silent restriction sites were introduced for
AflII and BssHII into the GIRK2 cDNA at
positions corresponding to amino acids 87 and 199, respectively. These
sites lie at the beginning of the first proposed membrane-spanning
segment (TM1) and just downstream of the second membrane-spanning
segment (TM2). GIRK1 has a BssHII site at the equivalent
position (amino acid 190), downstream of TM2, and in addition, an
AflII site was introduced at the DNA sequence corresponding
to amino acid position 78. Standard subcloning, in which the N- and
C-terminal hydrophilic regions of GIRK1 and GIRK2 were substituted
individually and together, generated 1211 and 1222 chimeric constructs.
For construction of 1221 and 1212 chimeras, an NheI
restriction site was introduced into chimera 1211 chimera at the
position corresponding to amino acid 361 using standard PCR methods.
GIRK2 cDNA has an NheI site at the equivalent position
(amino acid 370). 1221 and 1212 chimeric constructs were generated by
exchanging the transmembrane and proximal C-terminal regions of 1211 and 1222 using AflII and NheI sites. Point
mutants, GIRK2D226N and GIRK2E234S, were generated by
oligonucleotide-mediated mutagenesis using standard PCR methods. 1211N217D mutant was generated using GeneEditorTM in vitro
site-directed mutagenesis system (Promega). GIRK1N217D mutant was made
by exchanging the transmembrane regions of GIRK1 and 1211N217D using
AflII and BssHII sites. The sequence of all
PCR-amplified products and point mutations were verified by DNA
sequence analysis.
Preparation and Microinjection of Oocytes--
Oocytes were
surgically removed from female Xenopus laevis anesthetized
with 0.3% (w/v) 3-amino benzoic acid (Sigma) and dissociated from
connective tissue using 0.3% (w/v) collagenase (Sigma) in Ca2+-free buffer (mM): 82.5 NaCl, 2.5 KCl, 1 MgCl2, 5 HEPES, pH 7.6 with NaOH). Isolated oocytes were
microinjected with 50 nl cRNAs dissolved in water. The in
vitro transcription of cRNAs was as described previously in
Stevens et al. (15). A similar total amount of channel
subunit cRNA was injected into each oocyte. In some experiments, the
human m2-muscarinic receptor cRNA was coinjected. Oocytes were
incubated in ND96 at 18 °C.
Electrophysiology--
Two electrode voltage clamp recordings
were performed 3-6 days after microinjection using an OC-725B
amplifier (Warner Instruments), interfaced to a Macintosh Quadra 700 computer using an ITC16 AID board (Instrutech) with Pulse acquisition
software (version 7.89; HEKA Electronics, Lambrecht, Germany).
Microelectrodes filled with 3 M KCl had resistances ranging
between 0.5 and 2 megaohms. Oocytes were continually perfused with
standard recording solution (mM): 90 KCl, 1 MgCl2, 1 CaCl2, 1 HEPES (pH 7.4 with KOH).
Currents were recorded in the presence of 3 µM carbacol
(CCh) before and after application of 1 mM
Ba2+. Current records were filtered at 1 kHz and digitized
at 5 kHz. Experiments were carried out at room temperature
(22 °C).
Patch clamp experiments were performed using an Axopatch 200A patch
clamp amplifier, and currents were recorded at 10 kHz and filtered at 2 kHz. Patch pipettes had resistance of between 1 and 2 megaohms when
filled with pipette solution and were coated with wax. Excised
inside-out patch pipettes were transferred to a small chamber with a
volume of ~100 µl. Solutions were exchanged by perfusing through
this small chamber a volume of 500 µl. Continuous records of channel
activity were obtained at a holding potential of
80 mV. Ten-second
ramp tests from
140 mV to +80 mV were performed throughout the
recording to check that there were no noninwardly rectifying channels
present in the patch. Data were acquired using Pulse acquisition
software (version 8.11; HEKA Electronics).
The pipettes solution contained (mM): 96 KCl, 1 MgCl2, 1.8 CaCl2 and 10 HEPES, pH 7.2 with KOH;
while the bath solution contained (mM): 96 KCl, 2 MgCl2, 5 EGTA, 10 HEPES, pH 7.2 with KOH. Gadolinium at 100 µM was routinely added to the pipette solution to
suppress native stretch-activated channel activity in the oocyte
membrane. In some experiments, CCh at 3 µM was added to
the pipette solution. NaCl (1-200 mM) or NMDG (10-100
mM) was added to 96 mM KCl bath solution
without compensation for changes in osmolarity and ionic strength.
Solutions containing MgATP were prepared fresh each day and the pH was
readjusted to 7.2 after addition of MgATP. The concentration of free
Mg2+ was maintained at ~1.9 mM and calculated
using EQCAL software (BioTools). PIP2 (a gift from R. Irvine) was purified from pig brain tissue. The stock of purified
PIP2 was in chloroform (8.7 mM) and was kept at
20 °C in an air-tight glass container under nitrogen gas. Test
solutions were prepared freshly before each experiment by evaporating
the chloroform and redissolving in 100 µl of Mg2+-free 96 mM KCl bath solution under sonication for 1-2 min. The final concentration of PIP2 was adjusted by adding the
appropriate volume of Mg2+-free 96 mM KCl bath solution.
Data Analysis--
To measure channel activity, the mean current
of each continuous recording segment was calculated by first
subtracting the baseline current and then summing the amplitudes of all
of the sample points and dividing by the number of sample points within the continuous recording segment (Igor Pro, version 3; Wavemetrics, Lake Oswego, OR). We did not correct for the small reduction in the
unitary current amplitude at high [Na+]. For each
Na+-activated dose-response experiment, the mean currents
at different Na+ concentration were normalized to the
response obtained in the presence of 100 mM
Na+. For each Na+-inhibited dose-response
experiment, the mean currents were normalized to the response obtained
at 0 mM Na+. Dose-response curves were fitted
to the Hill equation: normalized mean current = A + (B
A)/(1 + ([Na+]/C)h), where
A is the maximum, B is the offset, C
is either the EC50 or IC50, and h is
the Hill coefficient. Single channel data were analyzed using TAC
software to determine unitary current amplitudes and open time
distributions (version 2.51; SKALAR Instruments, Seattle, WA). Data are
expressed as mean ± S.E.
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RESULTS |
Gating of GIRK1/GIRK2 Heteromeric Channels by Intracellular
Na+ and PIP2--
We measured the effects of
raising internal [Na+] on GIRK1/GIRK2 heteromeric
channels heterologously expressed in Xenopus oocytes. Fig.
1A shows a multichannel
recording firstly in the cell-attached mode and then following patch
excision in a solution lacking ATP, GTP, and Na+. Channel
activity ran down over a period of 1-2 min and was partly restored by
application of 5 mM MgATP and further increased by application of 20 mM Na+ in the continued
presence of ATP. In contrast, application of 20 mM
Na+ in the absence of MgATP had little effect on channel
activity (Fig. 1B), and 5 mM AMP-PNP was unable
to substitute for ATP in activating the channel. Even in the presence
of 100 µM GTP
S plus 5 mM MgATP,
application of 20 mM Na+ further increased
channel activity (Fig. 1C), and the effects of
Na+ were not blocked by 100 µM GDP
S (Fig.
1D). Thus, Na+-dependent activation
of GIRK1/GIRK2 channels appears to require ATP hydrolysis, but is
independent of G proteins. Sui et al. (6) suggested that the
production of PIP2 by hydrolysis of MgATP is responsible
for the ATP dependence of GIRK1/GIRK4 channel activity. They applied 1 µM PIP2 in the absence of Na+ or
G
and showed little change in GIRK1/GIRK4 channel opening frequency, but a large increase in channel activity upon subsequent application of 20 mM Na+. We applied 50 µM PIP2 for 5 min in the absence of
Na+ or G
and saw a dramatic increase in the opening
frequency of the channel (Fig. 1E), with no apparent change
in the single channel conductance. Subsequent application of 20 mM Na+ further increased the open probability
(Popen) by ~2-fold (1.93 ± 0.16, n = 3). Thus PIP2 appears to be an
activator of GIRK channels in the absence of either Na+ or
G
, but the actions of PIP2 do not preclude further
activation by Na+.

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Fig. 1.
Activation of GIRK1/GIRK2 heteromeric
channels by intracellular Na+ and PIP2.
Multichannel recordings from oocytes coexpressing GIRK1 and GIRK2.
A, channel activity recorded in the cell-attached mode ran
down upon formation of an inside-out, isolated patch (arrow). Application of 5 mM MgATP and 20 mM Na+, for the duration indicated by the
bars, re-activated GIRK1/GIRK2 channels. B,
application of 20 mM Na+ alone and together
with 5 mM AMP-PNP was unable to reactivate GIRK channels.
C, 100 µM GTP S in the presence of 5 mM MgATP maintained channel activity in an inside-out
patch; subsequent application of 20 mM Na+
further enhanced channel activity. D, channel activation by
20 mM Na+ was not blocked by 100 µM GDP S. E, recordings from an inside-out
patch, before and after application of 50 µM purified
PIP2. PIP2 dramatically increased the channel
opening frequency in the absence of Na+ or G .
Subsequent application of 20 mM Na+ further
increased the channel open probability. The holding potential was 80
mV, and the solution in the bath and in the pipette contained 96 mM K+.
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Fig. 2 shows the dose-response
relationship for Na+-activation of GIRK1/GIRK2, GIRK2, and
GIRK1/GIRK4 channels in the presence of 5 mM MgATP. The
data were fitted with the Hill equation with EC50 values
ranging between 27 and 44 mM. The Hill coefficients were
close to 2 for GIRK1/GIRK2 and GIRK1/GIRK4 and close to 4 for the GIRK2
homomeric channels (Table I). This
suggests that all four subunits within the homomeric GIRK2 channel
complex bind Na+, whereas in the heteromeric channels, only
two of the four subunits are Na+-sensitive. GIRK1 subunits
do not form functional homomeric channels and so to test the
possibility that GIRK1 subunits are Na+-insensitive, we
looked at the effects of Na+ on a chimeric channel (1211)
containing the N- and C-terminal tails of GIRK1 and the TM and H5
regions of GIRK2 (Fig. 2C). We have shown previously that
this chimeric subunit (previously called 121), expressed with the m2
receptor in Xenopus oocytes, gives rise to large ACh-induced
currents (15). In contrast to the GIRK1/GIRK2 or GIRK2 currents the
1211 currents were inhibited by increasing internal Na+,
with an IC50 of ~23 mM and a Hill coefficient
of ~1.0 (Fig. 2D). This inhibition appears to be partly
caused by a reduction in the single channel conductance at
Na+ levels greater than 10 mM (Fig.
2E) and partly by a decrease in the channel
Popen. Interestinly the 1211 channels were more sensitive to MgATP than were the wild type channels. Application of 2 mM MgATP, in the absence of Na+ or GTP, caused a
4.9 ± 0.5 (n = 3)-fold increase in the Popen of
1211 channels (Fig. 2F) and only a 2.1 ± 0.5 (n = 3)-fold
increase in the Popen of GIRK1/GIRK2 channels. Thus,
the loss of Na+-dependent activation does not
appear to be caused by a decrease in the ATP sensitivity of the
channel.

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Fig. 2.
Dose-response relationships for activation of
GIRK1/GIRK2 channels and inhibition of 1211 chimeric channels by
internal Na+. A, an inside-out patch
containing multiple GIRK1/GIRK2 channels in the presence of increasing
concentrations of internal Na+ and 5 mM MgATP.
B, dose-response relationships for
Na+-dependent activation of GIRK1/GIRK2, GIRK2,
and GIRK1/GIRK4 channels in the presence of 5 mM MgATP. The
data were fitted with a Hill function, and n values and
EC50 values are given in Table I. C, inhibition
of chimera 1211 by increasing concentrations of internal
Na+ in the presence of 5 mM MgATP.
D, dose-response relationship for
Na+-dependent inhibition of chimera 1211 fitted
with a Hill equation. E, increasing internal Na+
reduced the Popen and single channel conductance
of chimera 1211 (Vm = 80 mV). F,
application of increasing millimolar concentrations of MgATP-activated
1211 channels. G, inhibition of GIRK1/GIRK2 by internal
NMDG, in the presence of 5 mM MgATP. Bars
indicate duration of NMDG application in millimolar. H,
dose-response curve for NMDG inhibition of GIRK1/GIRK2 was fitted by a
Hill equation with an IC50 of 20 mM and a Hill
coefficient of 1.2.
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NMDG had a similar effect to Na+ on the 1211 currents, and
it also inhibited the GIRK1/GIKR2 currents. Fig. 2G shows
GIRK1/GIRK2 currents in the presence of increasing concentrations of
NMDG and then following wash out. The inhibition by NMDG was fully reversible and the dose response relation was fitted with a Hill equation with an IC50 of ~20 mM and a Hill
coefficient of 1.2 (Fig. 2H). These results suggest that
Na+ may have a dual action on the GIRK1/GIRK2 channels, an
inhibitory action that is masked by the ability of Na+ to
activate the channel by another mechanism.
Na+-dependent activation of GIRK1/GIRK2
channels appears to require the cytoplasmic tail regions of the GIRK2 subunit.
The Proximal C-terminal Region of GIRK2 Confers
Na+-dependent Activation--
To locate the
Na+-sensing region of GIRK2 we looked at the effects of
Na+ on the 1222 chimeric subunit. This differs from chimera
1211 in that it has the GIRK2 C-terminal tail and only the N-terminal tail of GIRK1. 1222 subunit (previously called 122) does not appear to
form functional homomeric channels but does form functional heteromeric
channels when coexpressed with GIRK1 in Xenopus oocytes (15). Increasing internal Na+ activated this channel with
an EC50 of ~43 mM and a Hill coefficient of
2.3 (Fig. 3, A and
B), suggesting that the C-terminal tail of GIRK2 is
sufficient to restore the Na+-dependent
activation of GIRK channels. To further narrow down the region crucial
for Na+ gating we generated two additional chimeric
subunits, 1221 and 1212, which divided up the GIRK2 C terminus into two
segments: amino acids 199 to 369, which are highly conserved within the GIRK family, and the poorly conserved region downstream of residue 369 (Fig. 3C). Two electrode voltage clamp measurements showed that both of these chimeric subunits produced much larger whole cell
currents when coexpressed with GIRK1 than when expressed alone,
indicating that they formed heteromeric complexes with GIRK1. We
compared the effects of Na+, in the presence of MgATP, on
the homomeric chimeric channels and the heteromeric channels. The 1221 and GIRK1/1221 channels were both reversibly activated by
Na+, whereas the 1212 and GIRK1/1212 channels were
inhibited (Fig. 3). The Na+ dose-response curve for 1221 had a Hill coefficient of 3.8, whereas the curve for GIRK1/1221 had a
Hill coefficient of 1.6 (Table I). Thus, the region of the GIRK2
C-terminal tail that lies proximal to TM2 appears to be responsible for
the Na+-dependent activation of GIRK1/GIRK2
channels.

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Fig. 3.
The proximal region of the GIRK2 C-terminal
tail is required for Na+-dependent
activation. A, activation of GIRK1/1222 heteromeric
channels by internal Na+ in the presence of 5 mM MgATP. B, the dose-response relationship
fitted with a Hill equation with EC50 of 43 mM and Hill coefficient of 2.3 (n = 4). C, diagram to illustrate the structure of the chimeric
subunits. D, the mean amplitudes of the
Ba2+-sensitive basal and CCh-induced currents recorded
using the two-electrode voltage clamp method from oocytes injected with
the subunit cRNAs indicated. All of the values shown were obtained from
the same batch of oocytes and represent the mean ± S.E. for at
least six oocytes. Coexpression of GIRK1 with the chimeric subunits
dramatically increases the size of the currents, indicating the
formation of functional GIRK1/1212 and GIRK1/1221 heteromers.
E and G, GIRK1/1221 currents (E) and
GIRK1/1212 currents (G), recorded from inside-out
macropatches in the presence of increasing internal Na+.
Recovery represents return to 0 mM Na+.
F and H, Na+ dose-response
relationships fitted with Hill equations.
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Aspartate 226 Is Important for
Na+-dependent Activation of GIRK2 and
GIRK1/GIRK2 Channels--
We compared the sequences of GIRK1, GIRK2,
and GIRK4 within the proximal C-terminal region and looked for
positions where there is a negative charge in GIRK2 and GIRK4 that is
not present at the equivalent position in GIRK1 (Fig.
4A). There are 7 acidic residues conserved between GIRK2 and GIRK4 but not GIRK1. Two of these
residues are located within the first 45 amino acid segment downstream
of the TM2 region (Fig. 4A). There is an aspartate at
position 226 in GIRK2, which is also present in GIRK4, but is an
asparagine in GIRK1. There is a glutamate at position 234, where GIRK1
has a serine. We generated two GIRK2 mutants, GIRK2D226N and
GIRK2E234S, and expressed these individually and together with GIRK1,
plus the m2-muscarinic receptor. The mutants displayed similar
characteristics to wild type GIRK2 in that they produced small whole
cell currents when expressed individually but much larger currents when
coexpressed with GIRK1 indicating the formation of functional
heteromeric channels (Fig. 4B). The time course of the
GIRK1/GIRK2E234S and GIRK1/GIRK2D226N heteromeric currents and their
degree of inward rectification were indistinguishable from wild type
GIRK1/GIRK2 currents (Fig. 4C and Ref. 15). The mutant GIRK2
homomeric currents also rectified strongly, suggesting that neither
glutamate 234 nor aspartate 226 plays a key role in determining the
rectification properties of these channels. Finally, all of the mutant
channels were activated by application of CCh, indicating that coupling
to the m2-muscarinic receptor was not disrupted by the mutations.

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Fig. 4.
Functional expression of GIRK2E234S and
GIRK2D226N mutants. A, amino acid alignment for
C-terminal region immediately proximal to TM2 of Kir subunits.
Positions of mutated residues are shown boxed. B,
Ba2+-sensitive basal and CCh-induced currents at 80 mV,
recorded by two-electrode voltage clamp from oocytes injected with the
cRNAs indicated. All of the values shown were obtained from the
same batch of oocytes and represent the mean ± S.E. for at least
six oocytes. C, CCh-induced currents and their
current-voltage relationship.
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GIRK2E234S and GIRK1/GIRK2E234S channels were activated by increasing
internal Na+, similar to the wild type channels; the
dose-response relationships were fitted with Hill functions with
EC50 values of ~42 and ~30 mM,
respectively, and Hill coefficients of 4.0 and 1.6, respectively (Fig.
5, A and B). In
contrast both GIRK2D226N channels and GIRK1/GIRK2D226N channels were
inhibited by increasing internal Na+ (Fig. 5, C
and D), similar to the 1211 channels. Thus the aspartate at
position 226 in GIRK2 appears to play a crucial role in
Na+-dependent activation of both GIRK2
homomeric and GIRK1/GIRK2 heteromeric channels. Two other mutations at
this position, D226K and D226E, failed to produce functional channels,
either when expressed alone or coexpressed with GIRK1. As a result, we
were not able to further investigate the relationship between the
structure of the side group of this residue and activation by
Na+.

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Fig. 5.
Effects of intracellular Na+ on
GIRK2E234S, GIRK2D226N, 1211N217D, and GIRK1N217D mutants.
A, C, E, and F, inside-out
patches from oocytes injected with the cRNAs indicated, in the presence
of increasing concentrations of Na+ and 5 mM Mg
ATP. B, D, F, and H,
Na+ dose-response relationships for the channels indicated.
Values for EC50 and Hill coefficients are given in Table
I.
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The reverse mutation in the 1211 chimera, N217D, was able to confer
Na+-dependent activation to this channel. The
dose-response relationship was best fitted with a Hill coefficient with
an EC50 of ~37 mM and a Hill coefficient of
3.6 (Fig. 5, E and F, and Table I). This is very
similar to the dose-response relationship for
Na+-dependent activation of the GIRK2 homomeric
channel. The reverse mutation in the GIRK1 subunit, N217D, was also
sufficient to recover Na+-dependent
activation to GIRK1N217D/GIRK2D226N heteromeric channels (Fig.
5, G and H, and Table I).
Single channel records of the wild type and mutant channels in
the cell-attached configuration are shown in Fig.
6. The E234S mutation, while not
affecting the Na+-dependent activation of the
channel, did alter the intrinsic gating of the GIRK2 homomeric
channels; all of the traces show a rapid flickering between the open
and closed states of the channel. However, the D226N mutation did not
appear to change either the kinetic behavior or the unitary conductance
of the homomeric and heteromeric channels. GIRK2 and GIRK2D226N
openings were very brief; the open time distributions were fitted with
single exponential with time constants of 0.51 and 0.50 ms,
respectively. The heteromeric channels displayed longer openings; there
was an additional slower component to the open time distribution with a
time constant of 2.8 ms (36%) for GIRK1/GIRK2D226N and 3.8 ms (23%)
for GIRK1/GIRK2. Thus the loss of Na+-dependent
activation of the mutant channels does not appear to be caused by a
change in their intrinsic gating behavior. Interestingly the reverse
mutation in the 1211 chimera, N217D, did alter the intrinsic gating of
the channel. In the cell-attached recording mode, this mutant displayed
very brief opening events (
= 0.21 ms), and correspondingly very
small whole cell currents (Fig. 4B), unlike the 1211 chimera, which produces large whole cell currents (15) and displayed
much longer openings;
1 = 0.85 (61%),
2 = 2.5 ms (39%).

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Fig. 6.
Single channel currents recorded in the
cell-attached mode from oocytes expressing either wild type or mutant
channels. Holding potential = 80 mV.
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DISCUSSION |
We have shown that the aspartate residue at position 226 within
the proximal C-terminal region of GIRK2 plays a crucial role in the
Na+-dependent activation of GIRK1/GIRK2
heteromeric channels and GIRK2 homomeric channels. When D226 in GIRK2
was substituted for an asparagine, activation of GIRK2D226N homomeric
channels and GIRK1/GIRK2D226N heteromeric channels by Na+
was lost, and instead Popen decreased with
increasing internal [Na+]. The wild type GIRK1 subunit
was not able to support Na+-dependent
activation of the heteromeric channel. The reverse mutation, N217D, in
the GIRK1 subunit was sufficient to recover Na+-dependent activation of the
GIRK1N217D/GIRK2D226N channel, and similarly this mutation in the 1211 chimera introduced Na+-dependent activation to
1211N217D channels. The D226N mutation in GIRK2 did not inhibit the
activation of the channel via the m2 receptor, suggesting that G
protein gating of GIRKs occurs via a different mechanism to
Na+-dependent activation.
Substituting asparagine for aspartate 226 in GIRK2 could be disrupting
Na+-dependent activation of the homomeric and
heteromeric channels in at least three different ways: 1) it could
reduce the binding affinity of Na+ to the channel; 2) it
could interfere with the mechanism by which Na+ binding, to
either GIRK2 or an accessory protein, is coupled to channel opening; 3)
it could disrupt the intrinsic gating of the channel in such a way as
to prevent the channel from opening in response to any stimulus. We can
rule out the third possibility, because the mutant and chimeric
channels that were not activated by Na+ were still
activated by both internal MgATP and external CCh. Also, the single
channel properties of GIRK2D226N channels and GIRK1/GIRK2D226N channels
in the cell-attached recording configuration did not differ
substantially from the wild type homomeric and heteromeric channels.
Thus, the intrinsic gating of these channels does not appear to have
been dramatically altered.
We have no direct evidence that Na+ acts by binding to
aspartate 226 or anywhere on the GIRK2 subunit. However,
Na+-dependent activation is observed in
isolated membrane patches, indicating that other cytosolic proteins are
not required for mediating its actions. Also, other negatively charged
residues within the proximal C-terminal region of Kir subunits are
involved in the binding of Mg2+ and polyamines (16, 17),
indicating that this region of Kir subunits is accessible to
internal cations. Aspartate residues have also been shown to
mediate the allosteric actions of internal Na+ on other
transmembrane proteins, for example the D2-dopamine receptor (18).
Aspartate 226 in GIRK2 and aspartate 223 in GIRK4 are only 7 residues
downstream of the cysteine that appears to be crucial for
G
-mediated activation of GIRK4 and GIRK1/GIRK4 channels (13). A
peptide from this region of GIRK4 has also been shown to compete with
binding of the G
to the native channel (13). The binding site for
PIP2 also appears to be located within this proximal
C-terminal region. PIP2 regulates the activity of several of the inward rectifiers, including ROMK1 (Kir1.1) and the
ATP-sensitive K+ channel (SUR/Kir6.2), as well as GIRK
channels. Neutralization of the arginines at positions 176 and 177 in
Kir6.2, and position 188 in Kir1.1, reduce PIP2 sensitivity
(19, 7, 20). Thus, the proximal C-terminal region immediately following
the second transmembrane segment appears to be an important domain for
mediating the effects of internal ligands.
Huang et al. (7) suggested that G
activates GIRK
channels by stabilizing interactions between PIP2 and the
channel. They showed that in the presence of G
, the rate of
channel activation by PIP2 was increased, and the rate of
dissociation of PIP2 was decreased. They also showed that
activation of GIRK channels by PIP2, in the presence of 20 mM Na+, precluded further activation by
G
. One possible mechanism for
Na+-dependent activation of GIRK channels is
that Na+ interacts with aspartate 226 (or aspartate 223 in
GIRK4) to promote the binding of the anionic PIP2 to a
nearby region of the C terminus. However, the results of Sui et
al. (6) suggest that PIP2 and Na+ activate
GIRK channels via different mechanisms. They reported that 1 µM PIP2 increased the mean open time of
GIRK1/GIRK4 channels, whereas subsequent application of Na+
specifically increased the opening frequency. In our experiments, a
5-min application of 50 µM PIP2 produced a
dramatic increase in the apparent open frequency of GIRK1/GIRK2
channels, but it did not preclude further activation of the channels by
Na+. Thus, we propose that Na+ increases the
Popen of PIP2-bound channels. It
remains to be tested whether or not it also increases the affinity of
PIP2 binding to GIRK channels.
For those mutant GIRK channels lacking aspartate 226, the decrease in
the open probability as a result of increasing internal [Na+] might be caused by a reduction in PIP2
binding. Shyng and Nichols (20) compared the ability of different
phospholipids to activate ATP-sensitive K+ channels
(SUR1/Kir6.2) and concluded that a negatively charged head and a lipid
tail are necessary to stimulate these channels. Screening of negative
charges by application of polycations, such as Ca2+ or
polylysine, inhibited PIP2-stimulated KATP
channel activity. In our experiments, increasing internal [NMDG]
appeared to be as effective as increasing [Na+] in
reducing the open probability of these mutant GIRK channels, and it
also inhibited GIRK1/GIRK2 currents. The Na+ and NMDG were
added to the bath solution without compensation for changes in ionic
strength. The resultant increase in the ionic strength of the internal
solution might have reduced long range electrostatic interactions
between PIP2 in the membrane and the GIRK channels.
Neither the D226N nor E234S mutation in GIRK2 appeared to change the
rectification of the whole cell currents. The E234S mutation did alter
the channels gating characteristics, causing it to flicker rapidly
between an open and shut state. Mutating the equivalent glutamate in
IRK1 channels has been shown to produce a similar flickering behavior,
but it also decreases the sensitivity of IRK1 to the internal blockers
Mg2+ and polyamines (16, 17), thus reducing rectification.
Our results, together with the fact that GIRK2 does not possess a negatively charged residue within its TM2 region, suggest that the
residues involved in determining the rectification of GIRK2 differ from
those shown to be important in the rectification of other inward
rectifier channels.
GIRK channels are located in postsynaptic neurons in several areas of
the central nervous system (21, 11), where the influx of
Na+ occurs through both ligand-gated receptors and
voltage-gated Na+ channels (22). During periods of rapid
firing, large increases in subplasmalemmal [Na+] are
likely to occur in the vicinity of these channels. Yu et al.
(23) recently reported that NMDA receptors are activated by
[Na+]i over a similar range of concentrations as
required for the activation of GIRK channels. They showed in
hippocampal neurons that Na+ entry through either NMDA
receptors or voltage-gated Na+ channels could activate
neighboring channels that were isolated within a cell-attached membrane
patch. Thus, it seems likely that GIRK channels will be activated by
Na+ influx into dendrites under physiological conditions,
and this may provide a negative feedback mechanism for suppressing
neuronal firing following periods of high activity.