(Received for publication, June 11, 1995; and in revised form, August 9, 1995)
From the
Four cDNA-encoding G-activated inwardly rectifying K channels have been cloned recently (Kubo, Y., Reuveny, E.,
Slesinger, P. A., Jan, Y. N., and Jan, L. Y.(1993) Nature 364,
802-806; Lesage, F., Duprat, F., Fink, M., Guillemare, E.,
Coppola, T., Lazdunski, M., and Hugnot, J. P. (1994) FEBS Lett. 353, 37-42; Krapivinsky, G., Gordon, E. A., Wickman, K.,
Velimirovic, B., Krapivinsky, L., and Clapham, D. E. (1995) Nature 374, 135-141). We report the cloning of a mouse GIRK2 splice
variant, noted mGIRK2A. Both channel proteins are functionally
expressed in Xenopus oocytes upon injection of their cRNA,
alone or in combination with the GIRK1 cRNA. Three GIRK channels,
mGIRK1-3, are shown to be present in the brain. Colocalization in
the same neurons of mGIRK1 and mGIRK2 supports the hypothesis that
native channels are made by an heteromeric subunit assembly. GIRK3
channels have not been expressed successfully, even in the presence of
the other types of subunits. However, GIRK3 chimeras with the amino-
and carboxyl-terminal of GIRK2 are functionally expressed in the
presence of GIRK1. The expressed mGIRK2 and mGIRK1, -2 currents are
blocked by Ba
and Cs
ions. They are
not regulated by protein kinase A and protein kinase C. Channel
activity runs down in inside-out excised patches, and ATP is required
to prevent this rundown. Since the nonhydrolyzable ATP analog AMP-PCP
is also active and since addition of kinases A and C as well as
alkaline phosphatase does not modify the ATP effect, it is concluded
that ATP hydrolysis is not required. An ATP binding process appears to
be essential for maintaining a functional state of the neuronal inward
rectifier K
channel. A Na
binding
site on the cytoplasmic face of the membrane acts in synergy with the
ATP binding site to stabilize channel activity.
Inward rectifier K channels were first
described in skeletal muscle and egg-cell
membranes(1, 2) . They are now found in many cell
types and are characterized by the following properties: (i) an
activation by hyperpolarization negative to the reversal potential for
K
( E
), (ii) an activation
potential shifting with E
, (iii) a blockade by
Cs
and Ba
(3) .
A class of
inward rectifier K channels is gated via G-proteins
(GIRK). In atrial cells, acetylcholine released by stimulation of the
vagal nerve causes the opening of a GIRK channel (I
) via
the activation of a m2-muscarinic receptor. The induced
hyperpolarization results in a slowing of cardiac
frequency(4, 5) . GIRK channels also exist in a
variety of neuronal cells and the modulation of such channels generates
slow synaptic potentials(6, 7) . They are coupled to
various neurotransmitter receptors such as the muscarinic cholinergic,
µ,
, and
opioid,
-adrenergic,
somatostatin, substance P, and GABA
receptors(8, 9, 10) .
A GIRK
channel, termed GIRK1 (11) or KGA(12) , was cloned from
rat heart, and two structural homologs were cloned from mouse brain,
mGIRK2 and mGIRK3(13) . Another close structural parent of the
GIRK family (rcK) was described initially as an
ATP-sensitive K
channel(14) , i.e. a
channel for which activity is controlled by intracellular
ATP(15) . However, it has been shown recently that the
functional I
channel stimulated by the G-protein
subunits is a heteromultimer composed of two GIRK subunits,
GIRK1 and CIR, a channel subunit which is nearly identical with
rcK
(16) .
A mouse analog of
rcK/CIR that we have cloned, and designed in this paper
as mGIRK4, presents the characteristic features of a
G-protein-activated inward rectifier K
channel. In
contrast to mGIRK1, which is present both in heart and brain,
mGIRK4/CIR is specifically localized in the heart, whereas mGIRK2 and
mGIRK3 are expressed mainly in the brain. By using different
strategies, co-localization of transcripts, immunoprecipitation, and
electrophysiology, we present evidences for a heterologous GIRK subunit
assembly in the brain. The paper also describes the main
electrophysiological properties and the modulation by ATP and
Na
of the currents expressed by mGIRK2 and the mGIRK1
+ mGIRK2 combination in Xenopus oocyte.
To construct the chimera mGIRK2/3/2, the mGIRK3 sequence was mutated at positions 151 (the A of the initiation codon taken as base 1) and 1012 to introduce MunI and NheI restriction sites, respectively, without modification of the amino-acid coded sequence. Site-directed mutagenesis was performed using oligonucleotide primers according to the manufacturer's protocol (Promega). The central mGIRK3 sequence between these two sites was exchanged with the corresponding mGIRK2 sequence in which a MunI site was created at position 255. The NheI site is found naturally in the mGIRK2 sequence.
TSA201 cells were transfected
with mGIRK2 and mGIRK4/CIR subcloned into the expression vector pcDNA
(Invitrogen) by the calcium phosphate method. After 48 h, cells were
harvested and microsomes were prepared. Briefly, cells were homogenized
in 150 mM NaCl, 3 mM EDTA, 0.1 mM
phenylmethylsulfonyl fluoride, 0.7 µg/ml pepstatin A, and 10 mM Tris-HCl (pH 8) buffer, centrifuged at 1000 g,
and the supernatant was pelleted at 100,000
g for 30
min. Pellets were dissolved in the homogenization buffer and stored at
-20 °C. Aliquots of 50 µg of microsomes were solubilized
in 100 µl of homogenization buffer containing 1% Triton X-100.
After 1 h at 4 °C, the volume was adjusted to 400 µl with
homogenization buffer (final Triton X-100 concentration of 0.25%).
Preimmune or immune sera were added for 3 h at a 200-fold dilution,
followed by addition of 10 µl of protein A immobilized on Sepharose
CL-4B (Sigma) for 1 h at 4 °C under slow rocking. Pellets were
washed five times with the homogenization buffer containing 0.25%
Triton. Immunoprecipitated proteins were resolved by SDS-polyacrylamide
gel electrophoresis (10% polyacrylamide) and transferred onto
nitrocellulose membrane (Hybond-C extra, Amersham). Blots were
saturated with phosphate-buffered saline containing 3% low-fat dry milk
and incubated with affinity-purified guinea pig polyclonal antibodies
diluted 400-fold. Blots were revealed with a
F(ab`)
-purified horseradish peroxidase-conjugated goat
anti-guinea pig antibody (Cappel) and then incubated with substrate for
ECL (chemiluminescence method, Boehringer).
Preparation of Xenopus laevis oocytes and cRNA injections have been described
previously(20) . 33 ng of GIRK cRNAs and 833 pg of
subunit cRNAs were injected per
oocyte.
Figure 1:
Sequences and PCR detection of mGIRK2
splice variants. a, nucleotide and deduced amino acid
sequences of the carboxyl termini of mGIRK2 splice variants.
Nucleotides are numbered from the first initiation ATG codon, and amino
acids are numbered beginning with the initiating Met. Nucleotides that
differ between mGIRK2 and mGIRK2A are printed in italics. The
carboxyl-terminal 11 amino acids specific to the mGIRK2A variant are
shown in bold. Sequences corresponding to the oligonucleotides
sense (P1 and P2) and antisense (P3 and P4) used in b are boxed. b, reverse transcription-PCR amplification of both
splice variants. Specific DNA fragments were amplified from mouse brain
cDNA by using P1 and P3 primers for mGIRK2 and P1 and P4 primers for
mGIRK2A. PCR products were blotted and probed with the P-labeled P2 oligonucleotide.
The entire coding region of the mGIRK4/CIR cDNA was cloned from
heart mouse cDNA (GenBank accession number U33631). It
shares 95% and 97.6% sequence identity with the rat CIR (16) /rcKATP (14) at the nucleotide and amino acid
levels, respectively. The percentages of amino acid identities between
mGIRK4/CIR and the other mGIRKs were 64.3% (mGIRK1), 71% (mGIRK2A), and
70% (mGIRK3). These values fall down to 44% and 46% in comparison
between mGIRK4/CIR and ROMK1 and IRK1, respectively. The highest degree
of sequence conservation between all these channels was found in a
central core, starting approximately 50 residues upstream of the first
transmembrane domain and ending 175 residues downstream of the second
transmembrane domain.
Figure 2:
Distribution of GIRK transcripts. a, the expression of GIRK transcripts was analyzed by Northern
blot in adult mouse brain (B) and heart (H).
Poly(A) RNAs (5 µg/lane) were isolated, blotted,
and hybridized as described under ``Experimental
Procedures.'' For GIRK2, the DNA probe corresponds to the
5`-coding sequence conserved in the two splice variants. b,
dark field photomicrographs of emulsion autoradiograms illustrating the
co-expression of mGIRK1 and mGIRK2 transcripts in CA3 pyramidal cells (P) of the hippocampus. Scale bar: 500
µm.
In situ hybridization studies have shown that the gene expression patterns of GIRK1, GIRK2, and GIRK3 are widely distributed in the brain and are very similar ((21, 22) and results not shown). The highest expression levels appeared in the neo- and allocortical regions, hippocampus, olfactory bulb, and cerebellum. The mGIRK4/CIR expression was very low in the adult rat brain (not shown). To determine the potential significance of heteromultimeric formation in the brain, a high resolution study obtained by microscopic analysis of emulsion-dipped sections has been performed. Fig. 2b shows an example of the high degree of overlap of the expression patterns of mGIRK1 and mGIRK2 transcripts. More than 80% of the neurons are labeled with the mGIRK1 and mGIRK2 probes in the CA3 pyramidal cell layer of the hippocampus (Fig. 2b). Coexpression of both transcripts in the same neuron type was also observed in most of the other strongly labeled central nervous system areas and was particularly evident in the granule cells of the dentate gyrus, in granular layers of the cerebellum, and in the mitral cells in the olfactory bulb (not shown). No area expressing only one GIRK mRNA could be clearly detected in the whole brain.
Similarly to
I(23) , expressed mGIRK channels are stimulated
by the G-protein
dimer. Although the
requirement for these G-protein subunits was not systematically
observed for heteromultimeric channels (not shown),
were always co-injected in the
following part of the work. Injections of mGIRK2 or mGIRK1 +
mGIRK2 (mGIRK1,-2) into Xenopus oocytes resulted in the
expression of inwardly rectifying currents. The expression of mGIRK2 in
the absence of other mGIRK subunits was successful only in 35% of the
oocyte batches tested. This low expression frequency was nevertheless
sufficient to allow a detailed characterization of the biophysical
properties of the current. The GIRK current expression frequency
reached 100% with combined injections of mGIRK1 and mGIRK2. Fig. 3, a and b, shows superimposed current
traces evoked by voltage steps ranging from -135 to +45 mV
in 30-mV increments from a holding potential of 0 mV (K
equilibrium potential). mGIRK2 (Fig. 3a) and
mGIRK1,-2 (Fig. 3b) currents in response to
hyperpolarizing voltage steps display different kinetics. mGIRK2
currents activate rapidly, in less than 5 ms, and then partially
inactivate with a time constant of 243 ± 15 ms (n = 8) at -130 mV. Activation/inactivation kinetics of
mGIRK1,-2 currents were very different, with a slower time constant (81
± 5 ms at -130 mV, n = 14) and no
inactivation.
Figure 3:
Whole cell properties of mGIRK2 and
mGIRK1,-2 currents. a and b, currents recorded under
the double microelectrode technique in oocytes injected with cRNAs
coding for mGIRK2 (a) or mGIRK1+ mGIRK2 (mGIRK1,-2) (b), and for the G-protein
subunits. The 6-s voltage pulses ranged from -135 mV to +45
mV in 30 mV steps. c, mGIRK2 peak currents recorded in 2, 14,
26, 50, 74, or 98 mM external K
. d,
relationship between the reversal potential of GIRK2 currents and the
external K
concentrations. e, currents
recorded during voltage steps to -120 mV. f, steady
state current-voltage relationship, both in various external
Cs
concentrations. g, currents recorded
during voltage steps to -120 mV. h, steady-state
current-voltage relationship, both in various external Ba
concentrations. In e, f, g, and h, the cation external concentrations were 1 µM (
), 3 µM (
), 10 µM (
),
30 µM (
), 100 µM (
), 300
µM (
), 1 mM (
), and 3 mM (
).
As expected for a K-selective inward
rectifier (24) , the activation potential of mGIRK2 became more
negative as external K
concentration decreased and the
amount of shift (52.6 ± 0.8 mV, n = 6, for a
10-fold change in external [K
] was close to
the K
equilibrium value (59 mV) estimated from the
Nernst equation (Fig. 3, c and d). The shifts
in the threshold of activation for mGIRK1,-2 and mGIRK2 +
mGIRK4/CIR (mGIRK2,-4) were, respectively, 50.9 ± 2.3 mV (n = 4) and 50.6 ± 3.3 mV (n = 3) for a
10-fold change in external [K
] (not shown),
consistent with a predominant K
selectivity for these
channels.
As for the majority of K selective
channels, external application of Ba
or Cs
blocked mGIRK2 currents in a concentration-dependent manner (Fig. 3, e-h). The Cs
block was
voltage-dependent giving rise to typical bell-shaped I/V curves for
potential values negative to -50 mV (Fig. 3f).
The mechanism of Ba
block, for concentrations less
than 1 mM, is probably of the ``open channel block''
type (24) as suggested by the pronounced fast inactivation
component of the resulting current (Fig. 3g). The
IC
values for the Cs
inhibition were 94.2
± 16 µM (n = 3), 94.5 ± 7.6
µM (n = 5), and 94.3 ± 1.3
µM (n = 3) for mGIRK2, mGIRK1,-2, and
mGIRK2,-4, respectively, while the IC
values for the
Ba
inhibition were 94.2 ± 3.8 µM (n = 3), 105.7 ± 6.9 µM (n = 6), and 97.9 ± 1.9 µM (n = 3) for mGIRK2, mGIRK1,-2, and mGIRK2,-4, respectively.
Other K
channels blockers, including
tetraethylammonium (3 mM), 4-aminopyridine (100
µM), clofilium (33 µM), tedisamil (50
µM), RP 98886 (30 µM), RP 62719A (30
µM), glibenclamide (10 µM), or K
channel openers(15) , such as pinacidil and P1060, both
at 100 µM, were without effect on mGIRK2, mGIRK1,-2, and
mGIRK2,-4 currents. On the other hand, verapamil and bepridil, two L-type Ca
channel blockers, partially
inhibited these currents, up to 60% and 40%, respectively, at 100
µM (not shown).
Finally, activation of protein kinase C by the phorbol 12-myristate 13-acetate (30 nM), the diacylglycerol analog, OAG (100 µM), or arachidonate (100 µM), and activation of protein kinase A by forskolin or 8-chloro-cAMP (3 and 300 µM) were without effect on GIRK currents (not shown).
Figure 4:
Single-channel properties of mGIRK1,-2 (a, b, and c) or GIRK1,-2A channels (d, e, and f). a and d,
current traces recorded in the inside-out configuration at -80 mV
and +80 mV in the presence () or the absence (
) of 10
mM Mg
. b and e, mean I-V
curves (n = 10) in symmetrical 140 mM K
. c and f, open time
distribution obtained at -80 mV in 10 mM Mg
. The histograms were fitted with
single-exponential curves with time constants of 0.21 ms for mGIRK1,-2
and 0.16 ms for mGIRK1,-2A. Currents were filtered at 3.5 kHz. g, time course of the effect of 100 µM spermine
on NP
(N = number of
channels, P
= open probability),
calculated every 1 s from mean outward currents, in the absence of
internal Mg
, at +80 mV. h, current
traces recorded at the indicated points in g). i, bar graph indicating mean NP
values as a function of internal spermine concentration,
100% corresponding to mean NP
without
spermine.
Since a highly voltage-dependent block
both by intracellular Mg and by the polyamine
spermine have been shown to underlie strong inward rectification in
cloned inward rectifiers(25, 26, 27) , we
tested the internal spermine dependence of the inward rectification of
our expressed channels. In the experiment illustrated in Fig. 4, g and h, inside-out patches containing mGIRK1,-2
channels maintained at +80 mV were first perfused with a
Mg
-free internal solution leading to an immediate
removal of the inward rectification. Then, application of 100
µM spermine led to a complete blockade of the outward
current which promptly reappeared after spermine removal. The bar
graph (Fig. 4i) shows that the spermine block was
dose-dependent with an IC
of about 10 µM.
Essentially the same spermine effects were obtained on oocytes
co-expressing mGIRK1 and the splice variant mGIRK2A (not shown).
Aspartic acid, a negatively charged amino acid present in the second
transmembrane domain of inward rectifiers which are not regulated by
G-proteins such as IRK1 and BIR10 (positions 172 and 158) has been
shown to be implicated in their Mg and spermine
sensitivities(25, 27, 28, 29) . The
corresponding residue is an aspartate in position 173 in mGIRK1 and a
neutral asparagine in position 185 in mGIRK2. To evaluate the
importance of the charge at this position for the rectification
characteristics of the heteropolymeric G-protein-activated channel
mGIRK1,-2, we took advantage of the presence of an asparagine (instead
of an aspartate) residue in the corresponding position in mGIRK4/CIR
(position 180) as in mGIRK2(13) . The mGIRK2,-4 channel has no
negative charge in the positions that have been considered as crucial
for Mg
- and polyamine-induced inward rectification in
IRK channels. Similarly to mGIRK1,-2, mGIRK2,-4 presents an inward
rectification in the presence of 10 mM Mg
(Fig. 5, a and b). In addition, Fig. 5(d and e) shows that the outward current
recorded in a Mg
-free solution at +80 mV was
totally abolished in the presence of 100 µM spermine. In
symmetrical 140 mM K
, the unitary conductance
was 39 ± 5 pS (n = 5), and the time constant of
the open-time distribution in steady-state conditions at -80 mV
was 0.51 ms (Fig. 5c).
Figure 5:
Single-channel properties and
immunoprecipitation of GIRK2,-4 channels. a, inside-out patch
recording at -80 mV and +80 mV with () or without
(
) 10 mM internal Mg
. b, mean
I-V curves (n = 5) in symmetrical 140 mM K
. c, open time distribution obtained in
the presence of 10 mM Mg
, at -80 mV.
The histogram was fitted with a single-exponential curve with a time
constant of 0.51 ms. d, bar graph indicating the
effect of 100 µM spermine on the mean NP
, 100% corresponding to mean NP
in spermine-free solution. e,
current traces recorded in the absence (1) and in the presence (2) of 100 µM spermine. f,
immunoprecipitation of mGIRK2 and mGIRK4/CIR subunits from
TsA201-transfected cells with pcDNA carrying the indicated GIRK coding
sequences. Corresponding microsomes were solubilized in nondenaturing
conditions and analyzed by immunoblotting directly or after
immunoprecipitation with anti-mGIRK4/CIR rabbit antibodies (lane
RK4). Antibodies used to reveal the Western blots were prepared
from guinea pig (revealing antibodies K2 and
K4).
Figure 6:
Effect
of ATP on GIRK1,-2 activity. a, effect of disodium salt ATP
solution (ATP/2Na). Current traces were recorded at -80 mV in the
inside-out configuration. b-e, time course of the open
probability (NP) calculated every 10 s. b, effect of 10 mM ATP. c, effect on the
rundown of the protein kinase A catalytic subunit (40 units/ml). A
protein kinase A subunit stock solution was prepared before each
experiment at a concentration of 4000 units/ml in the presence of
dithiothreitol (6 mg/ml). d, effect of alkaline phosphatase
(100 units/ml) on the reactivation of GIRK1,-2. A stock solution was
prepared at 2000 units/ml in water. e, effect of the
nonhydrolyzable ATP analogue AMP-PCP (10 mM). f,
current traces recorded at the indicated numbers in e. Channel
open time distribution, calculated from a sample of 1 min in duration,
in 10 mM ATP, 0 ATP, and 10 mM
AMP-PCP.
Surprisingly,
channel activities were maximal when disodium ATP (ATP/2Na) was used
instead of Mg-ATP. Fig. 7a shows that mGIRK1,-2 channel
activity could be partly restored by application of a 20 mM NaCl in an ATP-free internal solution. To reach maximal channel
activity, the simultaneous presence of ATP and Na ions
was required. Channel activity was only 20% of the maximal activity on
application of 10 mM Mg-ATP in Na
-free
solution (Fig. 7b). Fig. 7c presents
mean results from 5 experiments and clearly shows the synergy of action
of ATP and Na
in the restoration of channel activity.
In the presence of ATP, Li
could replace Na
for the activation of mGIRK1,-2 channels but with less efficacy (Fig. 7d). The sensitivity to internal Na
led us to check if the cloned mGIRK channels could be related to
K
channels activated by internal Na
which have been described in cardiac and neuronal
cells(30, 31) . In fact, the only similarity is the
requirement of a high concentration of Na
(>30
mM) to activate these channels. The high unitary conductance
(>100 pS), the impossibility to replace Na
by
Li
for activation, and the specific blockade by R56865
which characterizes the Na
-sensitive K
channel (32) were not found for mGIRK channels (not
shown).
Figure 7:
Requirement of Na and ATP
for the restoration of GIRK1,-2 channel activity in the inside-out
configuration (a-c). In this series of experiments, the
ATP used was the ATP-Mg
salt. GIRK1,-2 activities
were recorded at -80 mV and filtered at 5 Hz. In a and b, initially, patches were internally bathed with an ATP- and
Na
-free solution. a, effect of 20 mM
Na
in the absence of ATP. b, effects of 10
mM ATP and 10 mM ATP + 20 mM Na
. After the Na
removal, note
the instantaneous reduction of activity to the level reached in the
presence of ATP alone. c, bar graph (n = 5) indicating the respective increase of GIRK1,-2
activities in the presence of Na
, ATP, and ATP +
Na
(taken arbitrary as 100% in each experiment). d, effects of 20 mM Li
followed by
20 mM Na
in the presence of 10 mM ATP.
Figure 8:
Expression of a mGIRK2/mGIRK3 chimeric
construct. a, scheme showing the contribution of mGIRK2
sequences (black) in the chimeric construct mGIRK2/3/2. b, bar graph showing the averaged currents recorded
at -130 mV in oocytes injected with mGIRK1 and the
G-protein subunits together with
mGIRK2, mGIRK3, or the mGIRK2/3/2 chimeric assembly (respectively, n = 32, 10, and 12, in 3 to 5 different batches of
oocytes). The vertical bars indicate the
S.E.
Four proteins with structures corresponding to G-protein-gated inward rectifier (11, 12, 13, 14, 16) have been cloned to date. They are designated as mGIRK1, mGIRK2, mGIRK3, and mGIRK4/CIR. mGIRK4/CIR seems to be specific to the heart, and it is not detected in the brain. Conversely, mGIRK2 and mGIRK3 transcripts are specifically present in the brain. mGIRK1 is present at similar levels in heart and brain. In situ hybridization experiments have shown that the distribution of the three mGIRKs is very similar if not identical. Moreover, the colocalization of distinct GIRK transcripts in the same neuronal cells is in agreement with the hypothesis of heteromeric formation. This hypothesis is strongly supported by the tremendous increase of functional expression of GIRK channels when they are co-injected in the same oocyte as compared to single injections.
K channels expressed after the injection of the
mGIRK2 cRNA alone or in combination with mGIRK1 cRNA (mGIRK1,-2) or
with mGIRK4/CIR cRNA (mGIRK2,-4) in the presence of
had the hallmarks of inward
rectifier channels: (i) an activation by hyperpolarization negative to
the reversal potential for K
(E
),
(ii) an activation potential shifting with E
, and
(iii) a blockade by Cs
and Ba
.
However, in voltage-clamp conditions, there were some differences
between expressions of mGIRK2 and mGIRK1,-2. mGIRK2 currents displayed
a rapid activation (<5 ms) and partial inactivation, whereas
mGIRK1,-2 channels had a slow activation and did not inactivate.
Single-channel analysis of mGIRK2, mGIRK1,-2, and mGIRK2,-4 currents
clearly demonstrated that there was only one population of channels
with very similar properties characterized by a unitary conductance of
about 40 pS and a flickering activity with a mean open time duration of
less than 1 ms. The single-channel parameters of mGIRK2 and mGIRK1,-2
were very similar although their activation kinetics at the whole
oocyte level were distinct.
A splice variant of mGIRK2 (mGIRK2A) has also been cloned. It has the same sequence as mGIRK2 but contains 11 additional amino acids in the carboxyl-terminal end. mGIRK2A transcripts are also specifically located in the brain. Electrophysiological results have not shown any significant difference between the two forms. Therefore, it is not easy to suggest any specific new function for mGIRK2A. A first possibility would be that the mGIRK2A subunit could associate with other mGIRKs which are not yet discovered. Another possibility is that the different carboxyl-terminal sequences could serve to impose different cellular localizations. Interestingly, the mGIRK2A terminal sequence SKV is very similar to the microbody targeting signal motif SKL(33) .
How do expressed
neuronal GIRK channels compare with ``native'' channels?
Native GIRK channels recorded in different neuronal cell types have
unitary conductances varying from 38 to 55 pS and a time constant of
their open-time distribution which is of the order of 2 ms (7, 34) . It then appears that their conductances are
similar, but flickering is more rapid for the cloned channels expressed
in Xenopus oocytes. However, it should be noted that detailed
literature describing neuronal GIRK channel properties at the
single-channel level is not yet available. One possibility is that
flickering GIRK channels are difficult to record in neuronal membranes
where numerous other K channel activities might
coexist. Another likely possibility is that some subunit which normally
slows down the gating kinetics in native channels is still missing in
cloned heteropolymeric channels.
It has been shown previously that
the functional cardiac G-protein-activated inward rectifier is in fact
composed of an assembly of rat GIRK1 and GIRK4/CIR(16) . The
K current expression described above suggests that
mGIRK2 can also form heteromultimeric assemblies with mGIRK1 and
mGIRK4/CIR. This was actually directly demonstrated by
immunoprecipitation studies in the case of the mGIRK2,-4 complex.
Experiments using coexpression of mGIRK1 with chimeras of mGIRK3 (which
do not express alone or co-injected with mGIRK1, mGIRK2, or mGIRK4/CIR)
with the amino- and carboxyl-terminal sequences of mGIRK2 also tend to
lead to the same conclusion. The apparent co-localization of mGIRK1 and
mGIRK2 in the brain, particularly in CA3 pyramidal cells, is a strong
indication that the mGIRK1,-2 complex is a major neuronal GIRK channel.
The case of mGIRK3 is not clear. Its lack of expression suggests that
it might need a partner that still has to be discovered. One possible
partner is the sulfonylurea receptor(35) . ATP-sensitive
K
channels are present in the
brain(36, 37, 38) . They have
inward-rectifying properties(23) , are regulated by
G-proteins(39, 40) , and may be constituted by the
assembly of the protein that binds antidiabetic sulfonylureas (35) and an inward rectifier-type K
channel.
After this work was submitted, it was published that the GIRK3 subunit can assemble with GIRK1 and with GIRK2 to either increase (GIRK1) or decrease (GIRK2) their activities(41) . These effects were never seen in our own experiments. These apparently conflicting observations might be explained by assuming that a third, not yet identified, subunit is endogenously present in oocytes and confers the expression properties observed by Kofuji et al.(41) . This component would not be present in our oocytes.
The inward rectification in cloned inward rectifiers (25, 26, 27) is due to a highly
voltage-dependent block by intracellular Mg and by
polyamines. Mutagenesis experiments have strongly suggested that
aspartic acid in position 172 in the inward rectifier IRK1 is pivotal
for the effects of Mg
and spermine on the inward
rectification (25, 26, 27) . This Asp residue
is present at corresponding positions in sequences of a number of
cloned inward rectifier such as IRK1, mGIRK1, and BIR10(29) ,
but this residue is replaced by an asparagine in mGIRK2, mGIRK4/CIR,
and also in ROMK1(42) . Although they lack this Asp residue,
both mGIRK2 and mGIRK4/CIR, when they are expressed independently or
when they co-expressed, possess all the hallmarks of inward rectifiers,
contrary to ROMK1 which also has an Asn in the corresponding position
171 and which presents a quasilinear I-V relationship. The fact that
replacement of Asn-171 by Asp in ROMK1 results in the appearance of a
Mg
-dependent inward rectification (43) would
tend to confirm the important role of an Asp for
Mg
-dependent inward rectification. However, the fact
that the expression of GIRK2,-4, with Asn in the sequences instead of
Asp, also leads to a Mg
-dependent inward rectifier
K
channel pleads for the importance of other residues
and questions the unique role of this Asp for inducing this inward
rectification.
One particularly interesting observation is the
requirement of a high concentration of internal ATP (10 mM) in
excised patches to prevent a fast rundown of both mGIRK1,-2 and
mGIRK2,-4 activities. This ATP dependence would immediately suggest an
important role of phosphorylation. However, results presented in this
paper show that a kinase activity involving ATP hydrolysis is not
implicated as it is for IRK1 and ROMK1
channels(44, 45) . Treatments capable of activating or
inhibiting protein kinase A or protein kinase C activity were without
effect on the rundown and/or the reactivating action of ATP. Moreover,
alkaline phosphatase which would produce a dephosphorylation did not
modify the response to ATP. Finally, the activating effects of the
nonhydrolyzable ATP analog AMP-PCP on channel activity were similar to
if not identical with those of ATP. All these results taken together
show that mGIRK1,-2 and mGIRK2,-4 channels are ATP-regulated channels.
They require ATP binding to be functional, but ATP hydrolysis is not
necessary. ATP binding might occur at the nucleotide-binding site
represented by the consensus Walker type A sequence
G(X)GK(X)
(V/I). This exact
motif is missing in mGIRK sequences, but two motives that share
similarities with the Walker A consensus sequence are present in the
carboxyl-terminal extremities of mGIRK1, mGIRK2, and mGIRK3 subunits.
The I(X)
GK(X)
V motif is
present in mGIRK1 and mGIRK2, the
V(X)
GR(X)
V sequence is
present in mGIRK3. It has been suggested that similar motives could be
implicated in ATP binding(44) . The mGIRK4/CIR sequence does
not possess such an ATP consensus sequence.
This paper also shows
that internal Na is a regulator of the neuronal
mGIRK1,-2 channel activity. This type of property has in fact been
observed before with the inward rectifier K
channel
which is present in starfish eggs (46) . ATP and Na
are synergistic in their activating effects. The ATP and
Na
dependences of neuronal mGIRK activities might be
important in neurological diseases. In ischemic situations, or in
epileptic seizures, the intracellular ATP concentration drops rapidly
while the internal Na
concentration increases
massively. It is then possible that the function of neuronal GIRK
channels will be affected drastically, leading to changes of membrane
polarization that might be an important component in a cascade of
events leading to very deleterious effects.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U33631[GenBank].