From the Department of Physiology, Tokyo Medical and
Dental University, Graduate School and Faculty of Medicine, Bunkyo,
Tokyo 113-8519, Japan and the Departments of § Neurobiology
and ¶ Neurophysiology, Tokyo Metropolitan Institute for
Neuroscience, Fuchu, Tokyo 183-8526, Japan
Received for publication, October 23, 2000, and in revised form, January 31, 2001
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ABSTRACT |
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Two cDNAs that encode the G
protein-coupled inwardly rectifying K+ channel (GIRK,
Kir3) of tunicate tadpoles (tunicate G protein-coupled inwardly
rectifying K+ channel-A and -B; TuGIRK-A and -B) have been
isolated. The deduced amino acid sequences showed ~60% identity with
the mammalian Kir3 family. Detected by whole mount in situ
hybridization, both TuGIRK-A and -B were expressed similarly in the
neural cells of the head and neck region from the tail bud stage to the
young tadpole stage. By co-injecting cRNAs of TuGIRK-A and G protein
The physiological significance of the G
protein-coupled1 inwardly
rectifying K+ (GIRK, Kir3) channels in the regulation of
the heartbeat as well as neuronal excitability has been well known (1).
Upon stimulation of Gi/o-coupled receptors by various
neurotransmitters, these channels open and induce hyperpolarization
(1). Thus, it has been accepted that these channels function as a basic
mechanism for an inhibitory modulation of neuronal excitability.
Since the first cDNA cloning of a subunit of G protein-coupled
inwardly rectifying K+ channels (GIRK1/KGA, Kir3.1) (2, 3),
some members of this channel family have been subsequently isolated and
classified as a Kir3.x subfamily of two transmembrane type Kir
K+ channels (4-6). The members of the Kir3 subfamily were
shown both in vitro and in vivo to multimerize,
namely to comprise functional heteromultimers. It is well known that
the muscarinic K+ channel in the heart is the
heteromultimer of Kir3.1 and Kir3.4 (5) and that the majority of the G
protein-coupled inward rectifiers in the brain are
heteromultimers of Kir3.1 and Kir3.2 (7).
Tunicate is classified into protochordates and regarded as one of the
closest relatives of the vertebrates. Tunicate embryos form a neural
tube through a folding of the neural plate consisting of the dorsal
ectoderm underlined with the dorsal mesoderm (8). Since this process is
highly similar to that occurred in vertebrates, the tunicate embryo is
regarded as a prototype of neural development of mammals (8). Using a
very simple system of cleavage-arrested tunicate embryos, the
developmental changes of the excitable membrane and the mechanism of
neural induction have been studied intensively (9-13). In those
studies, it was shown that the expression level of the simple inward
rectifier K+ channel is correlated with the cell fate
selection of neural/epidermal cells (13). However, the existence of G
protein-coupled inwardly rectifying K+ channels in tunicate
has not yet been reported. It is thus of great interest to know whether
the G protein-coupled regulation of excitability through the control of
the inwardly rectifying K+ channels is conserved or not in
the prototype of vertebrates.
In the present work, we, for the first time, have isolated cDNAs of
two G protein-coupled inwardly rectifying K+ channel
subunits from the tunicate (Halocynthia roretzi) young tadpole. These cDNAs were designated as tunicate G protein-coupled inwardly rectifying K+ channel-A and -B (TuGIRK-A and -B),
respectively. We have shown that these two subunits form
heteromultimers whose electrophysiological properties are different
from the TuGIRK-A homomultimers. The macroscopic conductance of the
TuGIRK-A/B heteromultimer showed a characteristic decline at strongly
hyperpolarized potentials. Furthermore, to reveal the structural
determinant for this property, we have performed a mutagenesis study
and identified a positively charged amino acid at the external mouth of
the channel pore (H5) as a critical residue that was found
uniquely in the TuGIRK-B subunit.
Molecular Cloning--
Unidirectional cDNA library of
tunicate (H. roretzi) young tadpoles was constructed using a
ZAP-cDNA synthesis kit (Stratagene) with oligo(dT)-linker primers.
ZAP-cDNA was purified using the Wizard Lambda Preps DNA
purification system (Promega). It was used as a template for PCR
screening with a degenerative oligonucleotides, which were designed
based on the conserved sequences of the Kir family, QTTIGYG (P1:
5'-CA(A/G)ACIACIAT(T/C/A)GGI TA(T/C)GG-3') and PKKRAET (P2:
5'-IGT(T/C)TCIGCICG(T/C)TT(T/C)TTIGG-3'). Cycling parameters were
94 °C for 1 min, 46 °C for 1 min, and 72 °C for 1 min for a
total of 30 cycles. The PCR products of 159 base pairs in length
were subcloned, and the sequences were determined. For obtaining
full-length cDNA clones, the same cDNA library was screened by
the plaque hybridization method using the two subcloned PCR products as
probes. Inserts of positive phages were excised by plasmid rescue of
pBluescript SK(
For the electrophysiological analysis, cRNAs were transcribed from
XhoI-digested cDNA using methylated cap analog (Amersham Pharmacia Biotech) and T3 RNA polymerase (Stratagene). Point mutants were made using the Sculptor Kit (Amersham Pharmacia Biotech) or
QuickChange Kit (Stratagene). The mutation was confirmed by sequencing
the primer and the surrounding regions.
RT-PCR--
Unfertilized eggs or young tadpoles were rinsed once
in sea water and then transferred to microtubes containing guanidine isothiocyanate solution. After incubation at room temperature for
2 h, RNA was extracted using the acid guanidium-phenol chloroform method (14). 5 µg of the purified RNA was reverse transcribed by
Superscript 2 (Life Technologies, Inc.), and one-twentieth of the
obtained cDNA solution was used as a template for PCR. TuGIRK-A- or
TuGIRK-B-specific PCR primer sets were as follows: for TuGIRK-A,
5'-CGGTCAAGGTATTTATCCG-3' (upstream) and 5'-ATCTGGGCTTCGACTATGTGC-3' (downstream); for TuGIRK-B, 5'-CCACTTTGGTCGATTTACGC-3' (upstream) and
5'-CGATTTGACTATCTTTGCCCG-3' (downstream).
Amplifications were performed by 25 cycles of 94 °C for 1 min,
55 °C for 1 min, and 72 °C for 1 min. The PCR products were separated on agarose gels and visualized with ethidium bromide.
Whole Mount in Situ Hybridization--
Whole mount in
situ hybridization was carried out as described previously (15,
16). The plasmids containing the inserts were linearized with either
XhoI (sense probe) or NotI (antisense probe) for
in vitro synthesis of RNA probes.
Two-electrode Voltage Clamp--
Oocytes were treated with type
1 collagenase (2 mg/ml; Sigma) for 2 h at room temperature to
remove follicle cells and injected with 50 nl of in vitro
transcribed cRNA solution. For the analysis of TuGIRK-A/B
heteromultimers, the concentrations of cRNA were ~50 ng/µl
(TuGIRK-A) and 500 ng/µl (TuGIRK-B), respectively. This concentration
ratio was used to avoid the formation of TuGIRK-A homomultimers. Since
TuGIRK-B homomultimers were confirmed to be nonfunctional, this ratio
of TuGIRK-A/B cRNAs enabled us to analyze a channel pool that consisted
mostly of TuGIRK-A/B heteromultimers. G
Electrophysiological recordings were carried out 2-3 days later under
two-electrode voltage clamp (OC-725B-HV; Warner Co.) at room
temperature (23 ± 2 °C). Data acquisition and analysis were
done on an 80486-based computer using Digidata 1200 and pCLAMP program
(Axon Instruments). Intracellular glass microelectrodes were filled
with 3 M potassium acetate supplemented with 10 mM KCl, and the resistance ranged from 0.2 to 0.8 M ohm. The bath solution contained 90 mM KCl, 3 mM MgCl2, 5 mM Hepes (pH 7.4). To
decrease the K+ concentration, KCl was replaced with
N-methyl-D-glucamine chloride. The chord
conductance (see Figs. 5, C and F, 9C,
and 10, C and F) was initially calculated based
on the EK values assuming intracellular
K+ to be 80 mM. However, the exact values of
[K+]i and EK of each oocyte
are unknown. Therefore, EK used for the calculation
of the chord conductance was adjusted in the range of ±3 mV to yield a
continuous conductance-voltage plot. In the experiments using blockers,
Ba2+ or Cs+ was added simply to the bath
solution. Fractions of the remaining currents were calculated at each
potential in various concentrations of blockers. By fitting the plot of
the dose-block relationship, the half-block concentration
(Ki) was calculated at each potential. The Patch Clamp--
Single channel recordings were carried out in
the cell-attached configuration of the patch clamp (Axopatch-1D; Axon
Instruments) at 22-25 °C. Data were filtered at 1 kHz by a built-in
eight-pole bessel filter, sampled at 5 kHz through the Digidata 1200 (Axon Instruments), and stored in a computer with the pCLAMP program (Axon Instruments). The resistance of the patch pipettes ranged from 3 to 6 M ohm. The patch pipette (extracellular) solution contained 140 mM KCl, 3 mM MgCl2,
and 5 mM Hepes (pH 7.4). The bath (extracellular) solution
contained 140 mM KCl, 3 mM MgCl2, and 5 mM Hepes (pH 7.4). Single channel current amplitudes
at various potentials and mean open times were determined by the pCLAMP
software. The slope conductance was calculated from a straight line
fitted by eye for the plot of the single channel current-voltage relationship.
Primary Structures--
We aimed at the isolation of cDNA for
inwardly rectifying K+ channels (Kir) from tunicates
(H. roretzi) and screened a cDNA library of young
tadpole by PCR using Kir-specific degenerative oligonucleotide primers.
We obtained two kinds of 159-base pair DNA fragments, whose sequences
showed ~60% amino acid identity with G protein-coupled inwardly
rectifying K+ channels of the Kir3 subfamily. These PCR
fragments were subcloned and used as probes to isolate full-length
cDNA clones from the same cDNA library. Finally, we isolated
two cDNA clones, which were designated as TuGIRK-A and -B.
TuGIRK-A and -B were ~2.4 and 2.0 kilobase pairs in size, encoding
612 and 399 amino acid residues, respectively (Fig.
1A). Both the TuGIRK-A and -B
proteins had two putative transmembrane segments (M1, M2) and an H5
pore region, similar to mammalian Kir channels (17, 18). The amino acid
sequence of TuGIRK-A exhibited an identity of ~60% with murine Kir3.2 (4) or Kir3.4 (5) channels. The identity of TuGIRK-B and
mammalian Kir3.2 or Kir3.4 was ~55%. The results of an
oligodendrogram analysis based on the Unweighted Pair-Group Method
using Arithmetic averages package are shown in Fig.
1B, demonstrating the relationship of two TuGIRKs and
members of the mammalian Kir3 channel families.
Members of the Kir family have a conserved sequence GY(or
F)GXR in the H5 pore region. GY(or F)G is shown as
the K+ ion-selective filter (19, 20), and a positively
charged Arg at the external mouth of the pore is known to have a
critical role for permeation and block (21-25). Both TuGIRK-A and -B
had the GYGXR sequence in the H5 pore. The sequence of
TuGIRK-B (GYGKR) was very unusual in that it had two positively
charged amino acids in tandem. It was expected that this unique Lys
residue at 161 might affect the channel properties of TuGIRK-B.
Developmental Changes of the Expression Patterns--
The
distributions of mRNA expression of TuGIRK-A and -B were studied by
RT-PCR and whole mount in situ hybridization. The RT-PCR
analysis was done using H. roretzi unfertilized eggs and young tadpoles. Neither mRNA of TuGIRK-A nor TuGIRK-B could be detected in unfertilized eggs, but both of them were expressed in the
young tadpoles (Fig. 2). As a next step,
we performed the whole mount in situ hybridization analysis
in the embryos of various developmental stages, 64-, 110-, and
117-cell, gastrula, neurula, tail bud, and young tadpole stages. The
expression patterns of TuGIRK-A and -B highly resembled each other
(Fig. 3). They were not expressed up to
the gastrula stage (Fig. 3, A and B
(a, 64-cell stage; b, gastrula stage)). The
transcription of TuGIRK-A and -B mRNA started at the neurula stage
in the putative neural cells of the head region (Fig. 3, A
and B (c)). From the tail bud stage, the
expression was observed additionally in the neural cells of the neck
region (Fig. 3, A and B (d)). These
signals were also observed in young tadpoles (Fig. 3, A and
B (e)).
Electrophysiological Properties--
Electrophysiological
properties of TuGIRK channels were analyzed under two-electrode voltage
clamp using the Xenopus oocyte expression system.
Mammalian Kir3 channels are known to be activated by G
TuGIRK-A/B current differed from TuGIRK-A current in the following
ways. (a) The macroscopic chord conductance of TuGIRK-A/B channels remarkably decreased at strongly hyperpolarized potentials (Fig. 5, D-F). This property was apparently different from
that of TuGIRK-A channels (Fig. 5, A-C). (b)
TuGIRK-A/B current was less sensitive to the block by extracellular
Ba2+ (Fig. 6,
D-F) than TuGIRK-A current (Fig. 6, A-C). The
values of mean and S.D. of Ki at
We examined the single channel properties of TuGIRK-A channels and
TuGIRK-A/B channels expressed in Xenopus oocytes by
the cell-attached patch clamp method with 140 mM
K+ in the patch pipette and in the bath (Fig.
8, A and B). The
kinetics of both TuGIRK-A and -A/B channels exhibited a flickering
shape (Fig. 8, A and B). The values of mean open
time at Functional Significance of Lys161 Residue of TuGIRK-B
Subunit--
To determine the possibility that Lys161 of
TuGIRK-B, an additional positively charged amino acid at the external
mouth of the pore, is the structural determinant for the difference
between the electrophysiological properties of the pore of TuGIRK-A
homomultimers and TuGIRK-A/B heteromultimers, we made a point mutant of
TuGIRK-B, K161T (TuGIRK-B*). When the K141T mutant of TuGIRK-B was
coexpressed with TuGIRK-A and G
To confirm further the functional significance of an additional
positively charged amino acid at the external mouth of the pore, we
introduced a reverse mutation, T173K, in the TuGIRK-A subunit
(TuGIRK-A*). TuGIRK-A* carries two positively charged amino acids at
the external mouth of the pore, similar to the TuGIRK-B subunit. In
TuGIRK-A* homotetramers (eight charges), a slight decrease in the
macroscopic conductance at hyperpolarized potential was observed (Fig.
11, A-C), which was not at
all seen in the TuGIRK-A homomultimer (four charges) (Fig. 5,
A-C). In comparison with the TuGIRK-A/B heteromultimer
(5-7 charges at the pore mouth) (Fig. 5, D-F), the
decrease in the macroscopic conductance at hyperpolarized potential was
more enhanced in TuGIRK-A*/B heteromultimer (eight charges) (Fig. 11,
D-F). The values of the ratio of the conductance at We have isolated two types of cDNA, TuGIRK-A and TuGIRK-B,
from the young tadpole larvae of the tunicate (H. roretzi),
which belong to the Kir3 subfamily. In Xenopus oocytes,
TuGIRK-A showed inwardly rectifying K+ current when
coexpressed with G Corresponding Subunit of Murine Kir--
Among the members of the
Kir3 subfamily, TuGIRK-A exhibited a higher similarity (~60%) with
murine Kir3.2, -3.3, and -3.4 than with Kir3.1, as shown in the
dendrogram of Fig. 1B. From this sequence homology and its
neuron-specific expression pattern, TuGIRK-A was speculated to
correspond to the murine Kir3.2 subunit. The fact that the
homomultimeric channel is functional also supported this speculation.
TuGIRK-B was rather distant from Kir3.2, -3.3, and -3.4 and was closer
to Kir3.1 (Fig. 1B). In the H5 pore region of the Kir3.1 subunit, there is a unique Phe at 137, where all other members have a
conserved Ser. This unique Phe of Kir3.1 was reported to have an
important role in the control of the channel activity (28, 29). In
TuGIRK-B, Tyr, which has similarity with Phe, is present at the
corresponding site. TuGIRK-B is similar to Kir3.1 also in that the
homomultimeric channel is not functional (5, 7). Taken together,
TuGIRK-B was speculated to correspond to the murine Kir3.1 subunit.
Significance in the Tunicate Embryo--
Since a tunicate
tadpole is a simple prototype of vertebrates, it has been used for
various studies of developmental biology. The developmental changes of
ion channel expression during neural differentiation were studied
intensively using cleavage-arrested blastomeres (9). By these studies,
the presence of multiple types of Ca2+ channels (30),
Na+ channels (31), K+ channels (32), and a
simple inward rectifier K+ channel (13) was demonstrated.
It was also shown that the expression level of the simple inward
rectifier K+ channel is dramatically regulated at the
initial stage of neural/epidermal cell fate selection (13).
Furthermore, cDNAs for voltage-dependent Na+ (16), K+ (33), and Ca2+
channels2 were already isolated.
On the other hand, knowledge of G protein-coupled responses in tunicate
is still insufficient. The presence of the G protein-coupled inwardly
rectifying K+ channel is not demonstrated, although it
would be expected to be a common cell response mechanism of animals. By
molecular cloning, we, for the first time, have demonstrated the
presence of G protein-coupled inwardly rectifying K+
channels in tunicate young tadpole. It remains to be elucidated what
kind of receptors link to the regulation of these channels, due to the
lack of information of G protein-coupled receptors in tunicate.
We investigated the developmental changes of TuGIRK-A and -B mRNA
expression by RT-PCR and whole mount in situ hybridization analyses (Figs. 2 and 3). TuGIRK-A and -B mRNA were observed from the neurula stage in presumptive neural cells of the head region. These
expressions were continuously seen in the tail bud stage and in the
young tadpole larvae. From the tail bud stage, they were additionally
expressed in the neural cells of the neck region. During the entire
course of development, the expression patterns of TuGIRK-A and -B were
highly similar. These results show that TuGIRK-A and -B form functional
heteromultimeric channels in neurons in the head and neck region. They
might function to regulate the speed of the fictive locomotion of the
tadpole by regulating the firing rate of neurons that innervate tail muscles.
Structure-Function Relationship--
We compared the
electrophysiological properties of the TuGIRK-A homomultimer and
the TuGIRK-A/B heteromultimer and observed the following
differences. (a) The macroscopic conductance of TuGIRK-A/B
decreased at strongly hyperpolarized potential, but that of TuGIRK-A
did not (Fig. 5). (b) TuGIRK-A/B was less sensitive to the
block by extracellular Ba2+ than TuGIRK-A (Fig. 6).
(c) TuGIRK-A/B was more sensitive to the block by
extracellular Cs+ than TuGIRK-A (Fig. 7). (d)
The single channel conductance of TuGIRK-A/B was smaller than TuGIRK-A
(Fig. 8). What is the structural determinant for these differences?
In Kir channels, there is a highly conserved positively charged amino
acid (Arg148 of Kir2.1) after the K+-selective
filter at the external mouth of the pore. The only exception is the
Kir7.1 subfamily (23, 24). This Arg residue is known to have an
important role for permeation (21, 23, 24), Mg2+ block
(22), and ionic selectivity (25). It is speculated to serve as an
electrostatic barrier for extracellular cations that regulates the
permeation and block of the channel (22). In both TuGIRK-A and -B, the
Arg residue is also conserved at the corresponding sites.
Interestingly, TuGIRK-B has an additional positively charged amino
acid, Lys, just before the conserved Arg. Thus, two positive charges
exist at the external mouth of the pore of TuGIRK-B subunit.
We speculated that this site might be the structural determinant for
the unique macroscopic conductance property of -A/B channel and
introduced a point mutation of K161T to TuGIRK-B (TuGIRK-B*) to
neutralize this additional positive charge. In the TuGIRK-A/B* channel,
the plot of the current-voltage relationship was straight (Fig. 9),
similar to TuGIRK-A channel (Fig. 5, A-C). This result demonstrates that Lys161 of TuGIRK-B is, at least in part,
the structural determinant for the unique macroscopic conductance
property of TuGIRK-A/B channels. This point was further supported by
the data of a reverse mutant of TuGIRK-A, T173K (TuGIRK-A*) (Fig.
11).
The TuGIRK-A/B* channel was more sensitive to the Ba2+
block and less sensitive to the Cs+ block than the
TuGIRK-A/B channel. We analyzed the voltage dependence of
Ki values of Ba2+ block displayed in
Figs. 6, C and F, and 10C. The
calculated Significance of Heteromultimer Formation--
We showed that
TuGIRK-A is sufficient to form a functional G protein-coupled inwardly
rectifying K+ channel. Why then is TuGIRK-B subunit
coexpressed in the same neuron? What is the physiological significance
of the TuGIRK-B subunit?
One possibility may be that the TuGIRK-B subunit functions to determine
the subcellular distribution of the heteromultimeric channel protein.
It is known that all members of the murine Kir3 family have a binding
motif for integrin, RGD, at the extracellular loop between the first
transmembrane region and the H5 pore region (34). It is reported that
the interaction of these subunits with integrin is required for
appropriate localization and function of Kir3 channels. This integrin
binding motif RGD was also present in the TuGIRK-B subunit (amino acids
119-121) but not in TuGIRK-A. It is possible that the TuGIRK-A
homomultimer is not located appropriately or not clustered due to the
lack of integrin binding domain and that the channel cannot exert its
full function, even if it is fully functional in vitro when
coexpressed with G1/
2 subunits (G
) in
Xenopus oocytes, an inwardly rectifying K+
current was expressed. In contrast, coinjection of TuGIRK-B with G
did not express any current. When both TuGIRK-A and -B were coexpressed together with G
, an inwardly rectifying
K+ current was also detected. The properties of this
current clearly differed from those of TuGIRK-A current, since it
displayed a characteristic decline of the macroscopic conductance at
strongly hyperpolarized potentials. TuGIRK-A/B current also differed
from TuGIRK-A current in terms of the lower sensitivity to the
Ba2+ block, the higher sensitivity to the Cs+
block, and the smaller single channel conductance. Taken together, we
concluded that TuGIRK-A and -B form functional heteromultimeric G
protein-coupled inwardly rectifying K+ channels in the
neural cells of the tunicate tadpole. By introducing a mutation of
Lys161 to Thr in TuGIRK-B, TuGIRK-A/B channels acquired a
higher sensitivity to the Ba2+ block and a slightly lower
sensitivity to the Cs+ block, and the decrease in the
macroscopic conductance at hyperpolarized potentials was no longer
observed. Thus, the differences in the electrophysiological properties
between TuGIRK-A and TuGIRK-A/B channels were shown to be, at least
partly, due to the presence of Lys161 at the external mouth
of the pore of the TuGIRK-B subunit.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) by R408 helper phage. The obtained clones were
sequenced using the PRISM sequence reaction kit (Applied BioSystems)
and a DNA sequencer (Applied BioSystems model 377-18).
cRNAs were used at
a concentration of 500 ng/µl to achieve maximal expression level.
values, which reflect the depth of the blocking site in the electric
field, were calculated as described previously (18).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The primary structures of TuGIRK-A and
TuGIRK-B and comparison with members of the GIRK family.
A, the alignment of the deduced amino acid sequences of the
TuGIRK-A and -B channels with mammalian Kir3.1 (2, 3) and 3.2 (4). The
putative two-transmembrane regions (M1, M2) and H5 pore region are
indicated by the boxes. Amino acids identical in four
channels are marked by asterisks. B, an
oligodendrogram to show the relationship of members of the mammalian
GIRK channel families and TuGIRK-A and -B. The sequences used were
Kir3.1 (2, 3), Kir3.2 (4), Kir3.3 (35), and Kir3.4 (5).
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Fig. 2.
Analysis of transcription of TuGIRK-A and -B
in unfertilized eggs and in young tadpoles by RT-PCR. PCR
fragments for TuGIRK-A (A) and TuGIRK-B (B) were
analyzed on agarose gels, respectively. The expression of neither
TuGIRK-A nor TuGIRK-B was detected in unfertilized eggs. Both of them
were expressed in the young tadpole larvae. M, size markers;
lane 1, unfertilized egg; lane
2, young tadpole; lane 3, negative
control (without reverse transcription) of lane
1; lane 4, negative control (without
reverse transcription) of lane 2;
lane 5, negative control (without RNA);
lane 6, positive control using a cloned cDNA
as a template.
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Fig. 3.
Detection of TuGIRK-A and -B transcripts by
the whole mount in situ hybridization method at
various developmental stages. The expression pattern of TuGIRK-A
(A) and -B (B) is shown. The stages are 64-cell
(a), gastrula (b), neurula (c),
tail bud (d), and young tadpole (e). Both
TuGIRK-A and -B exhibited a similar expression pattern. No signal could
be detected until the gastrula stage. From the neurula stage to the
young tadpole stage, both TuGIRK-A and -B RNAs were detected in the
putative neural cells of the head region (arrowheads in
c-e). Furthermore, from the tail bud stage to the young
tadpole stage, the expression was observed also in the neural cells of
the neck region (arrows in d and
e).
, which is
released upon receptor stimulation (1, 26, 27). When TuGIRK-A or -B
cRNAs were injected alone, they did not express any detectable current
(Fig. 4, A (upper
panel) and B), as is generally observed for Kir3
channels (2-5). As a next step, we coinjected them with G
cRNAs.
TuGIRK-A coexpressed with G
showed inwardly rectifying
K+ current (TuGIRK-A current), but TuGIRK-B with G
did not (Fig. 4, A (lower panel) and
B). When TuGIRK-B was co-expressed with TuGIRK-A and
G
, an inwardly rectifying K+ current, whose
electrophysiological properties were different from those of the
TuGIRK-A current as shown as follows, was observed (Fig. 4,
A (lower panel, middle,
TuGIRK-A/B current) and B). These results demonstrate that
TuGIRK-A forms functional homomultimeric G protein-coupled inwardly
rectifying K+ channels and that TuGIRK-A and -B form
functional heteromultimeric G protein-coupled inwardly rectifying
K+ channels. In the case of the Kir3.1 channel, the small
expressed current was attributed not to homomultimer formation but to
heteromultimerization with the endogenous Kir subunit of
Xenopus oocytes (6). However, the expression level of
TuGIRK-A is extremely high (Fig. 4B), in clear contrast with
that of Kir3.1, suggesting that TuGIRK-A forms a functional
homomultimer. In various concentrations of extracellular
K+, both TuGIRK-A and TuGIRK-A/B channels were active below
the equilibrium potential of K+ (EK)
(Fig. 5), as is observed in mammalian Kir
channels.
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Fig. 4.
Current recordings under voltage-clamp from
Xenopus oocytes expressing TuGIRK-A and/or TuGIRK-B
subunits with or without G
subunits. TuGIRK channels were expressed in
Xenopus oocytes and recorded under two-electrode voltage
clamp in 90 mM K+. The holding potential was 0 mV, and step pulses from +50 mV to
160 mV decremented by 10 mV were
applied. A, combinations of subunits shown are TuGIRK-A
(left), TuGIRK-A and -B (middle), and TuGIRK-B
(right). They are expressed without (upper
panels) or with (lower panels)
G
. B, comparison of the amplitudes of the expressed
current at 50 ms from the beginning of step pulses at
160 mV. The
values of mean and S.D. (n = 5-8) are shown.
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Fig. 5.
Current recordings of the TuGIRK-A
homomultimer and TuGIRK-A/B heteromultimer in various extracellular
K+ concentration. A and D,
current traces of TuGIRK-A (A) and TuGIRK-A/B (C)
channels in 90, 45, 20, 10, and 4 mM K+. The
holding potential was 0 mV, and step pulses were applied from +50 mV to
160 mV by 10-mV decrements. B and E,
current-voltage relationships of the peak amplitudes of the traces in
A (B) and D (E). Shown are
K+ concentrations of 90 mM (filled
circles), 45 mM (open
circles), 20 mM (filled
squares), 10 mM (open
squares), and 4 mM (filled
triangles). The voltages used for the plot are the command
potential applied by the computer. C and F,
macroscopic chord conductance-voltage relationships calculated from the
plot in B (C) and E
(F).
120 mV were
52.4 ± 12.5 µM (n = 4) (TuGIRK-A)
and 628.5 ± 89.3 µM (n = 3)
(TuGIRK-A/B). (c) TuGIRK-A/B current was more sensitive to
the block by extracellular Cs+ (Fig.
7, D-F) than TuGIRK-A current
(Fig. 7, A-C). The values of mean and S.D. of
Ki at
120 mV of TuGIRK-A/B were 188.4 ± 88.2 µM (n = 3). In the case of TuGIRK-A, due
to the very low sensitivity, the data point range was insufficient for
reliable curve fitting to obtain Ki values. The
Ki value at
120 mV was estimated to be
approximately 2532.2 ± 916.3 µM (n = 3) and was obviously no less than 1 mM. It is
apparent that the Ki value of TuGIRK-A is
significantly larger than that of TuGIRK-A/B. Thus, it was confirmed
that the TuGIRK-B subunit forms a functional heteromultimer with
TuGIRK-A, whose electrophysiological properties are distinguishable
from those of TuGIRK-A homomultimer.
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Fig. 6.
The sensitivity of the TuGIRK-A homomultimer
and TuGIRK-A/B heteromultimer to the block by extracellular
Ba2+. Channels were expressed in Xenopus
oocytes and recorded under two electrode voltage clamp in 90 mM K+. Shown are the current traces
(A and D), the current-voltage relationships
(B and E), and the dose-block relationships
(C and F) of TuGIRK-A (A-C) and
TuGIRK-A/B channels (D-F), respectively. The recordings
were obtained in 0 µM (filled
circles), 3 µM (open
circles), 30 µM (filled
squares), 300 µM (open
squares), and 3 mM (filled
triangles) extracellular Ba2+. In C
and F, the data points and fitted
lines indicate the data from 70 mV (far
right) to
160 mV (far left).
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Fig. 7.
The sensitivity of the TuGIRK-A homomultimer
and TuGIRK heteromultimer to the block by extracellular
Cs+. A similar presentation to Fig. 6 is shown
(A, B, D-F). The effect of
Cs+ was examined instead of Ba2+. In the case
of TuGIRK-A (A-C), reliable curve fittings to obtain
Ki values could not be done, because the data of
effective doses were insufficient due to the extremely low sensitivity.
In C, the ratios of the current amplitudes in 0 mM Cs+ and in various concentrations of
Cs+ at each membrane potential are presented as a
bar graph instead of a plot for curve fittings as
in F.
120 mV were 0.72 ms (TuGIRK-A) and 1.1 ms (TuGIRK-A/B),
respectively. These properties are characteristic of mammalian Kir3
channels. The slope conductance of the inward current amplitudes of
TuGIRK-A was 24.2 ± 1.0 picosiemens (n = 3), and that of TuGIRK-A/B was 18.3 ± 0.9 picosiemens
(n = 3) (Fig. 8C). These results confirmed the difference between the TuGIRK-A homomultimeric channels and the
TuGIRK-A/B heteromultimeric channels at the single channel level.
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Fig. 8.
Single channel recordings of the TuGIRK-A
homomultimer and the TuGIRK-A/B heteromultimer. Channels were
expressed in Xenopus oocytes and recorded in the
cell-attached configuration of the patch clamp method. The patch
pipette and the bath contained 140 mM K+.
A and B, single channel recordings of the
TuGIRK-A homomultimer (A) and the TuGIRK-A/B heteromultimer
(B). The displayed traces were low pass-filtered at 500 Hz
by a digital filter of the pCLAMP software. The membrane potentials are
shown on beside each recording. The zero current level is
shown by thin lines. C, relationships
of the single channel current amplitude and the voltage. The values of
single channel conductance of the inward current range of TuGIRK-A
(filled circles) and TuGIRK-A/B (open
squares) were estimated to be 24.5 and 18.0 picosiemens, respectively. The lines were fitted by eye.
, the clear decrease in the
macroscopic conductance at hyperpolarized potentials observed in
TuGIRK-A/B (Fig. 5, D-F) disappeared (Fig.
9, A-C). In comparison with
the wild-type -A/B heteromultimers (Fig. 6, D-F), the
sensitivity of TuGIRK-A/B* channels to the block by Ba2+
was clearly increased (Fig. 10,
A-C). The Ki values at
120 mV were
changed by the mutation from 628.5 ± 89.3 µM
(n = 3) (TuGIRK-A/B) to 23.6 ± 1.85 µM (n = 3) (TuGIRK-A/B*). The sensitivity to the block by Cs+ (Fig. 7, D-F) was slightly
decreased (Fig. 10, D-F). The Ki values
at
120 mV changed by the mutation from 188.4 ± 88.2 µM (n = 3) (TuGIRK-A/B) to 269.7 ± 42.1 µM (n = 3) (TuGIRK-A/B*). From these
results, it was shown that the difference between TuGIRK-A homomultimers and TuGIRK-A/B heteromultimers could, at least partly, be
due to the presence of an additional positively charged amino acid at
the external mouth of the pore of the TuGIRK-B subunit.
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Fig. 9.
Current recordings of the
heteromultimeric channel of TuGIRK-A and -B mutant (K161T) in various
extracellular K+ concentration. A similar presentation
to Fig. 5 is shown. The properties of TuGIRK-A/B* were analyzed.
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Fig. 10.
The sensitivity of the heteromultimeric
channel of TuGIRK-A and -B mutant (K161T) to the block by extracellular
Ba2+ or Cs+. A similar presentation to
Fig. 6 is shown. The effects of Ba2+ (A-C) and
Cs+ (D-F) on TuGIRK-A/B* heteromultimer were
analyzed.
160
mV and the peak conductance were 65.8 ± 2.8% (n = 3) (TuGIRK-A*/B) and 78.9 ± 9% (n = 3)
(TuGIRK-A/B). Taken together, the effect of positively charged amino
acid at the external mouth of the pore to the macroscopic conductance was further supported by T173K mutation of TuGIRK-A. In addition, we
unexpectedly observed that TuGIRK-A* caused a decrease in the intensity
of inward rectification (Fig. 11), suggesting a difference in the
contribution of this site between TuGIRK-A and -B subunits.
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Fig. 11.
Current recordings of the homomultimer of
TuGIRK-A mutant (T173K) and the heteromultimer of TuGIRK-A mutant
(T173K) and TuGIRK-B wild type in various extracellular K+
concentrations. A similar presentation to Fig. 5 is shown. The
properties of TuGIRK-A* homomultimer (A-C) and TuGIRK-A*/B
heteromultimer (D-F) were analyzed.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. In contrast, TuGIRK-B did not show detectable
current even in the presence of G
. When TuGIRK-A and -B
together with G
were coexpressed, an inwardly rectifying
K+ current was observed whose electrophysiological
properties were different from those of TuGIRK-A. Thus, it was
demonstrated that TuGIRK-A but not -B can form functional
homomultimeric G protein-coupled channels and that -A and -B can form
functional heteromultimeric G protein-coupled channels.
values (18), which reflect the depth of the binding
site, differed significantly as follows: TuGIRK-A, 0.25;
TuGIRK-A/B, 0.075; TuGIRK-A/B*, 0.32. From these results, it was
suggested that the high sensitivity in TuGIRK-A and TuGIRK-A/B* is due
to a block at a deep site in the electric field. In TuGIRK-A/B, it was
speculated that the positive charge at the external mouth of the pore
inhibited the access of Ba2+ to the deep blocking site, and
the remaining low sensitivity is due to a block at another very shallow
site(s). Taken together, Lys161 was thought to serve not as
a binding site for these pore blockers but as an electrostatic barrier
for the entrance of extracellular cations, similar to
Arg148 of Kir2.1 (22).
. In contrast, the TuGIRK-A/B heteromultimer,
which is supplied an integrin binding site by the TuGIRK-B subunit,
might acquire physiological function by localizing at appropriate sites.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Y. Katsuyama and Y. Okamura for support in the in situ hybridization experiments; Drs. K. Takahashi and Y. Shinoda for encouragement and advice; Drs. L. Guo, T. Misaka, and H. Abe for comments on the manuscript; and Drs. T. Miyashita, M. Kondoh, and N. Kinoshita for discussion.
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FOOTNOTES |
---|
* This work was supported in part by research grants from the Ministry of Education, Science, Sports and Culture of Japan (to Y. K.), from the Japan Society for the Promotion of Science (to Y. K.), and from the Mitsubishi Foundation (to Y. 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB050440 (TuGIRK-A) and AB050441 (TuGIRK-B).
Supported by CREST from the Science and Technology Corporation
of Japan. To whom correspondence should be addressed: Dept. of
Physiology, Tokyo Medical and Dental University, Graduate School and
Faculty of Medicine, D566, Yushima 1-5-45, Bunkyo, Tokyo 113-8519, Japan. Tel.: 81-3-5803-5156; Fax: 81-3-5803-5156; E-mail:
ykubo.phy2@med.tmd.ac.jp.
Published, JBC Papers in Press, February 26, 2001, DOI 10.1074/jbc.M009644200
2 Y. Okamura, personal communication.
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ABBREVIATIONS |
---|
The abbreviations used are:
G protein, GTP-binding protein;
GIRK, G protein-coupled inwardly rectifying
potassium channel;
TuGIRK-A, tunicate G protein-coupled inwardly
rectifying potassium channel-A;
TuGIRK-B, tunicate G protein-coupled
inwardly rectifying potassium channel-B;
PCR, polymerase chain
reaction;
RT-PCR, reverse transcription-PCR;
G, G protein
1
2 subunits;
TuGIRK-A*, TuGIRK-A T173K
mutant;
TuGIRK-B*, TuGIRK-B K161T mutant.
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