From the
We have isolated a novel inward rectifier K
Neuronal excitation causes an increase of extracellular
potassium ions (K
Recent molecular
biological dissection of inward rectifier potassium channels has shown
the basic motif to be a set of two membrane-spanning segments plus an
H5 segment. Until now, cDNAs for an inwardly rectifying ATP-regulated
potassium channel from the outer medulla of rat kidney (ROMK1) (Ho et al., 1993), classical inward rectifier potassium channels
(IRK1, IRK2, and IRK3) (Kubo et al., 1993a; Morishige et
al., 1993, 1994; Koyama et al., 1994; Takahashi et
al., 1994; Périer et al., 1994; Makhina et
al., 1994), a G protein-activated muscarinic potassium channel
(GIRK1/KGA) (Kubo et al., 1993b; Dascal et al.,
1993), and a cardiac ATP-sensitive potassium channel
(K
In
the present study, we have isolated a novel inward rectifier potassium
channel, which is expressed predominantly in glial cells. The clone
identified here has a Walker type-A ATP-binding domain and 53% sequence
identity to ROMK1 and approximately 40% to other potassium channels
with two transmembrane segments. We designate this new clone as
K
Two-electrode voltage clamp experiments were carried out with
a commercially available amplifier (Turbo Clamp TEC 01C, Tamm, Germany)
with microelectrodes, which, when filled with 3 M KCl, had
resistances of
Single-channel recordings were
performed in the cell-attached patch configuration using a patch-clamp
amplifier (EPC-7, List, Darmstadt, Germany). Both pipette and bath
solutions contained 140 mM KCl, 1.4 mM
MgCl
The isolated cDNA encoded 379 amino acids (Fig. 1A). We designated this clone as K
Several independent cDNA clones of inwardly rectifying
potassium channels have been currently identified as two
membrane-spanning segment type potassium channels (Ho et al.,
1993; Kubo et al., 1993a, 1993b; Morishige et al.,
1993, 1994; Dascal et al., 1993; Koyama et al., 1994;
Takahashi et al., 1994; Périer et al., 1994;
Makhina et al., 1994; Ashford et al., 1994). Here we
report a novel cDNA clone, K
The functional roles of
inward rectifier potassium channels in the brain have been studied
extensively in glial cells. A fundamental difference between glial and
neuronal membranes is that glial cells have a much larger resting
conductance, which has been attributed to inward rectifier potassium
channels existing in glial cells (Barres, 1991). Furthermore,
electrophysiological studies including the patch-clamp technique have
clarified glial function of spatial potassium buffering or siphoning
and extracellular potassium accumulation through inward rectifier
potassium channels (see, e.g., Newman et al., 1984;
Brew et al., 1986; Nilius and Reichenbach, 1988; Barres et
al., 1988; Newman, 1993). In salamander Müller cells, a
single population of the inward rectifier potassium channels of 28 pS
with 98 mM [K
At the single-channel level, we identified two distinct
conducting channels derived from a single clone (K
Expression of K
The nucleotide sequence(s) reported in this paper has been
submitted to the GenBank
We thank Paul Berke (Alkermes Inc.) and Ian Findlay
(University of Tours, France) for critical reading of this manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
channel predominantly expressed in glial cells of the central
nervous system. Its amino acid sequence exhibited 53% identity with
ROMK1 and approximately 40% identity with other inward rectifier
K
channels. Xenopus oocytes injected with
cRNA derived from this clone expressed a K
current,
which showed classical in-ward rectifier K
channel
characteristics. Intracellular Mg
ATP was required to sustain
channel activity in excised membrane patches, which is consistent with
a Walker type-A ATP-binding domain on this clone. We designate this new
clone as K
-2 (the second type of inward rectifying
K
channel with an ATP-binding domain). In situ hybridization showed K
-2 mRNA to be expressed
predominantly in glial cells of the cerebellum and forebrain. This is
the first description of the cloning of a glial cell inward rectifier
potassium channel, which may be responsible for K
buffering action of glial cells in the brain.
) at synaptic sites in the central
nervous system, which if uncorrected would result in the loss of
synaptic transmission by depolarizing the membrane. Glial cells, which
surround neuronal cells, are supposed to transport the accumulated
K
from proximal to distal portions of the cells. This
regulatory function of glial cells was first proposed as a spatial
buffering mechanism of K
for astrocytes in the optic
nerves (Orkand et al., 1966) and also termed the siphoning
mechanism of K
for Müller cells of the retina
(Newman et al., 1984). In this hypothesis, K
that accumulates locally due to neural excitation would enter
glial cells wherever the local reversal potential for K
is more positive than the resting potential of the glial cell.
The elevated intracellular K
would then be rapidly
shunted by current flow from a proximal to a more distal region of the
cell. At the distal region of glial cells or endfeet of Müller
cells, the resting potential would be more positive than the
equilibrium potential for K
(E
),
(
)which allows
K
to come out to extracellular fluid. Along with
K
, chloride ions (Cl
) and water will
also move passively across glial cells. This K
movement is considered to occur through inward rectifier
potassium channels in glial cell membranes. Thus, glial cell inward
rectifier potassium channels may be essential to regulate the
extraneuronal environment of ions and solute (reviewed by Barres et
al.(1990) and Sontheimer(1994)). Actually, several types of inward
rectifier potassium channels have been identified
electrophysiologically at the single-channel level from Müller
cells (Brew et al., 1986; Nilius and Reichenbach, 1988;
Newman, 1993), oligodendrocytes (McLarnon and Kim, 1989), glioma cells
(Brismar and Collins, 1989), and Schwann cells.
-1) (Ashford et al., 1994) have been isolated.
All of these channels were found to be expressed in the brain. ROMK1,
IRKs, and GIRK1 were recently shown to exist mainly in neuronal cells
of the brain (Morishige et al., 1993; Kenna et al.,
1994; Karschin et al., 1994).
(
)
-2 (the second type of inward rectifying K
channel with an ATP-binding domain). In situ hybridization shows that K
-2 mRNA is expressed
predominantly in glial cells of cerebellum and forebrain. This clone
will provide, for the first time, a molecular tool to elucidate the
functions of glial cells, which occupy half of the brain volume.
Polymerase Chain Reaction (PCR) and cDNA
Cloning
The procedure was performed as described previously
(Ishii et al., 1993). Briefly, cDNA templated by mRNA isolated
from rat forebrain was used as a DNA template for PCR amplification.
The sequences of the 5`(N1) and 3` primers(N3) were derived from ROMK1
(Ho et al., 1993) and are as follows (N represents A/G/C/T):
N1 (5`-CA(A/G) GTN ACN AT(A/C/T) GGN TA(C/T) GG-3`, the sequence
corresponding to nucleotides 415-434) and N3 (5`-AA NAC NAC
NA(G/T) (T/C)TC (A/G)AA (A/G)TC-3`, nucleotides 868-887),
respectively. PCR amplification was performed according to the
following schedule: five cycles at 94 °C for 1 min, 46 °C for 1
min, 72 °C for 2 min, followed by 26 cycles at 94 °C for 1 min,
55 °C for 1 min, and 72 °C for 2 min. The PCR products were
electrophoresed on a polyacrylamide gel and excised for subsequent
subcloning and sequence determination. Through this procedure, we
identified a new cDNA clone, pA. Using this as a probe, 1.5
10
phage clones of a rat forebrain cDNA library (Moriyoshi et al., 1991) were hybridized for the isolation of full-sized
cDNAs. Thirty independent clones were isolated and selected by the
digestion of internal HindIII sites. A representative clone,
pA11, was analyzed using an oocyte expression system as described
below. Both strands of the cDNA sequences were determined by the chain
termination method (Sanger et al., 1977).
Functional Expression in Xenopus Oocytes and
Electrophysiological Analysis
pA11s6, which is a deleted form of
pA11 (approximately 220 base pairs of the 5`-non-coding region were
deleted from pA11), was transcribed in vitro as described
previously (Takahashi et al., 1994). The transcript was
dissolved in sterile water and injected (50 nl of a 1 µg/µl
solution) into manually defolliculated oocytes. After injection,
oocytes were incubated in a modified Barths' solution at 18
°C, and electrophysiological studies were undertaken 48-96 h
later.
0.5-1.5 megohms. Oocytes were bathed in a
solution, which contained 90 mM KCl, 3 mM MgCl
, 5 mM HEPES (pH adjusted to 7.4 with
KOH), and 150 µM niflumic acid to block the endogenous
chloride current. Oocytes were voltage-clamped at different holding
potentials, and voltage steps of 1.5 s duration were applied to the
cells in 10-mV increments every 5 s.
, and 10 mM HEPES (pH adjusted to 7.4 with
KOH). Electrophysiological experiments were performed at room
temperature (20-22 °C). Data were stored on video tapes using
a PCM converter system (VR-10, Instrutech Corp., New York, NY). For
analysis, the data were reproduced, low pass-filtered at 600 Hz
(-3 dB) by an 8-pole Bessel filter (Frequency devices, Haverhill,
MA), sampled at 3 kHz and analyzed off-line on a computer (Macintosh
Quadra 700, Apple Computer Inc., Cupertino, CA) with a standard program
(EP Analisis, Human Intelligence Inc., Rochester, MN).
RNA Blot Hybridization
The same filter as Koyama et al.(1994) was used for hybridization. Hybridization using a
random primer-labeled probe was performed with a SacI-digested
fragment (0.6 kilobase) of K-2 in 5
SSC, 50%
formamide, 0.08% Ficoll, 0.08% polyvinylpyrolidone, 0.1% SDS, 0.25%
NaH
PO
, and 250 µg/ml denatured salmon sperm
DNA, at 42 °C for 17 h. A glyceraldehyde-3-phosphate dehydrogenase
cDNA probe was used as a control to verify that equivalent amounts of
mRNA had been transferred. Blots were washed at moderate stringency
(0.5
SSC, 0.1% SDS, 55 °C for 15 min) and exposed to Kodak
XAR-5 film (Kodak, Rochester, NY) with an intensifying screen at
-70 °C.
In Situ Hybridization Histochemistry
Seven male
Wistar rats weighing 200 g were anesthetized with sodium pentobarbital
(50 mg/kg body weight) and then decapitated. Brains were dissected,
frozen quickly in powdered dry ice, and then stored at -80
°C. Sections (20 µm) were cut on a cryostat and thaw mounted on
3-aminopropyltriethoxysilane-treated slides. The BstXI-SacI fragment (nucleotide positions 703-1070)
of K-2 cDNA was used as a template.
S-Labeled
antisense and sense riboprobe were synthesized using T7 RNA polymerase
(for antisense probe) or T3 RNA polymerase (for sense probe) in the
presence of [
S]UTP. Hybridization and washing
conditions were used as described previously (Yoshimura et
al., 1993) except that treatment of sections by proteinase K was
carried out for 30 s. For film autoradiography, slides were exposed to
Fuji RX film (Fuji Photo Film Co., Tokyo, Japan) for 1 week. For
emulsion autoradiography, slides were dipped in an emulsion (K5 type,
Ilford Scientific Product, Mobberley, Cheshire, United Kingdom) and
then exposed for 2-4 weeks.
cDNA Cloning of the K
A number of cDNAs of potassium channels with two
transmembrane segments have been cloned recently, and the size of this
family is increasing rapidly (Ho et al., 1993; Kubo et
al., 1993a, 1993b; Morishige et al., 1993, 1994; Dascal et al., 1993; Koyama et al., 1994; Takahashi et
al., 1994; Périer et al., 1994; Makhina et
al., 1994; Ashford et al., 1994; Suzuki et al.,
1994). In an attempt to clone additional members of this family in the
brain, we utilized a PCR approach. We synthesized sets of degenerate
primers corresponding to the conserved amino acid sequences in the
known members of this family and used them to amplify cDNA templated by
rat brain mRNA. Of a total of 30 PCR clones analyzed, one novel clone
was isolated, and its full-length cDNA was identified by screening the
rat brain cDNA library.
-2
Channel
-2
(the second type of inward rectifying K
channel with
an ATP-binding domain; K
-1 corresponds to ROMK1). The
nucleotide sequence surrounding the predicted initiation codon of
K
-2 was in accordance with the consensus sequence (Kozak,
1987). The estimated molecular mass of this protein was 42,477 daltons.
Hydropathicity profile analysis (Kyte and Doolittle, 1982) indicated
two putative membrane-spanning hydrophobic segments (M1 and M2) with a
pore-forming region (H5), a structure common among IRK1, IRK2, IRK3,
ROMK1, GIRK1, and K
-1 (Kubo et al., 1993a,
1993b; Morishige et al., 1993, 1994; Koyama et al.,
1994; Takahashi et al., 1994; Périer et al.,
1994; Makhina et al., 1994; Ho et al., 1993; Dascal et al., 1993; Ashford et al., 1994). We will use for
convenience ``two membrane-spanning segment type channel''
for this type of channels. A potential N-glycosylation site
was seen at Asn in the predicted extracellular domain of the M1-H5
linker, a feature consistent with ROMK1 and GIRK1. A Walker type-A
motif (GX
GKX
(I/V))
representing a phosphate-binding loop and a single putative ATP-binding
site (Walker et al., 1982; Saraste et al., 1990)
occurred at the position corresponding to that of ROMK1 described by Ho et al.(1993). Furthermore, K
-2 also contained
potential cAMP-dependent protein kinase and protein kinase C
phosphorylation sites (Pearson and Kemp, 1991). The sequence of
K
-2 is compared with those of ROMK1, IRK1, and GIRK1 in Fig. 1B. The deduced amino acid sequence of
K
-2 showed 53, 43, and 43% identity with those of ROMK1,
IRK1, and GIRK1, respectively. In the pore-forming region (H5), the
amino acid sequence of K
-2 was 88% identical to those of
ROMK1 and IRK1, and 65% identical to that of GIRK1. These homology data
indicate that this clone is more closely related to ROMK1 than the
other potassium channels with two membrane-spanning segments.
Figure 1:
The nucleotide sequence and deduced
amino acid sequence of the K-2 cDNA (A) and
alignment of the amino acid sequences of the two membrane-spanning
segment type potassium channels (B). A, the amino
acid sequence deduced from the longest open reading frame and the
position of the putative transmembrane domains (M1 and M2) and
pore-forming region (H5) are boxed. The single Walker type-A
motif is underlined; *, a potential N-glycosylation
site;
, potential phosphorylation sites based on consensus motifs
for cAMP-dependent protein kinase (PKA);
, those for
protein kinase C (PKC). B, the sequences of ROMK1,
IRK1, and GIRK1 are those reported by Ho et al. (1993), Kubo et al. (1993a), and Kubo et al. (1993b),
respectively. The amino acid sequences indicated with single-letter
notation are aligned by inserting gaps (-) to achieve maximum
homology. The amino acids identical in all four IRK families are boxed. The two transmembrane segments (M1 and M2) and the
pore-forming region (H5) are displayed above the sequences with bars.
K
Fig. 2illustrates the results obtained from Xenopus oocytes injected with cRNA derived from a
K-2 Is an Inward Rectifier Potassium
Channel
-2 clone. In a bathing solution containing 90 mM K
([K
]
) (toptraces in Fig. 2, A, C, and E), hyperpolarizing voltage steps from a holding potential of
0 mV revealed rapid activation (<10 ms) of large inward currents.
The effect of [K
]
on
the K
-2 current is depicted in Fig. 2(A and B). As [K
]
was lowered from 90 mM to 45, 20, and 10
mM, the slope conductance of K
-2 current
decreased from 17 µS to 13, 10, and 6 µS, respectively. The
reversal potential was in good agreement with E
predicted from the Nernst equation at the different
[K
]
. Outward currents
at potentials positive to E
were considerably less
than those predicted by a linear current-voltage relationship. Thus,
K
-2 current shares this property of the classical type of
inward rectifier potassium channels (Sakmann and Trube, 1984; Kurachi,
1985), such as IRK1-3 (Kubo et al., 1993a; Morishige et al., 1993, 1994; Koyama et al., 1994; Takahashi et al., 1994; Périer et al., 1994; Makhina et al., 1994). External Ba
clearly induced a
time- and voltage-dependent block, in a concentration-dependent manner
(3-30 µM), with a comparatively small effect upon
the instantaneous current, but a marked influence upon the steady-state
current (Fig. 2, C and D). These concentrations
of Ba
slightly reduced the outward currents recorded
by voltage steps to positive membrane potentials. When compared to
Ba
, Cs
exhibited less of a
time-dependent effect upon the K
-2 current; nevertheless,
it showed a clear voltage dependence of the block in a
concentration-dependent manner (30-300 µM) (Fig. 2, E and F).
Figure 2:
Cell currents recorded from Xenopus oocytes expressing the K-2 clone. A, the
effect of varying external K
concentration. The
holding potential was set at the zero current level in each solution, i.e. at 0 mV in 90 mM K
, at
-17.4 mV in 45 mM K
, at -37.9 mV
in 20 mM K
, and at -55.3 mV in 10
mM K
; the values correspond to the
equilibrium potential for K
at each concentration of
external K
, with an assumption that the intracellular
K
concentration of oocytes is 90 mM (Dascal,
1987). Traces elicited by steps from each holding potential to
+50, +20, -10, -40, -70, -100,
-130, and -160 mV are shown. B, current-voltage
relationships in solutions of 90 mM (
), 45 mM (
), 20 mM (
), and 10 mM (
)
K
. K
was substituted with
Na
. The current amplitude 10 ms from the start of
voltage pulses is plotted. C and E illustrate
currents induced by voltage steps from 0 mV to, in descending order,
+50, +20, -10, -40, -70, -100,
-130, and -160 mV in oocytes bathed in 90 mM K
. C, the effect of external
Ba
. D, current-voltage relationships of the
steady-state currents recorded from this oocyte in solutions containing
0 µM (
), 3 µM (
), and 30
µM Ba
(
). E, the effects
of external Cs
. F, current-voltage
relationships of the steady-state currents recorded in solutions
containing 0 µM (
), 30 µM(
), and
300 µM Cs
(
). Arrows indicate the zero current level.
Intracellular ATP Activates the K
Single-channel currents flowing through
K-2
Channel
-2 were recorded in cell-attached configuration using
oocytes that were injected with K
-2 cRNA (Fig. 3).
Surprisingly, Xenopus oocytes injected with K
-2
cRNA expressed channel currents that exhibited two distinct conducting
states as described below (Fig. 3, A and B). In
both cases, currents that passed through these channels were observed
much more prominently in the inward direction than in the outward
direction and, thus, showed a strong inwardly rectifying property (Fig. 3A). The mean slope conductance of inward current
flowing through the high conducting-state channel (Fig. 3A, a and c) was 36 ± 4
pS (mean ± S.D., n = 3) and that of the low
conducting-state channel (Fig. 3A, b and d) was 21 ± 2 pS (mean ± S.D., n = 4). For convenience in the following description, we will
refer to these as the ``36-pS'' and the ``21-pS''
channels. In individual oocytes, either of the two conducting-state
channels was usually expressed, but sometimes both conducting-state
channels were detected in a single patch (Fig. 3B). We
did not observe any smooth transitions between the 36-pS and 21-pS
channels without overlapping of the two. Therefore, the 21-pS channel
is unlikely to be a sublevel of the 36-pS channel.
Figure 3:
Single-channel recordings from
cell-attached (A and B) and inside-out (C)
membrane patches of Xenopus oocytes expressing
K-2. A, a and b, membrane
current traces recorded from two different oocytes at the membrane
potential values indicated to the left of the traces. These
patches each appeared to contain only one inwardly rectified potassium
channel. Below each family of traces are shown the current-voltage
relationships of the 36-pS (c) and 21-pS (d)
channels. B, two types of conductances of membrane currents
recorded in one patch at -60 mV membrane potential. C,
reactivation of channels in an inside-out patch by Mg-ATP. The channels
showed run-down upon excision into an ATP-free bath solution and were
reactivated by the addition of 3 mM Mg-ATP. The membrane
potential was held at -60 mV. The arrows to the left of each of the traces in this figure indicate the patch current
level recorded when all channels were closed.
We examined the
effects of intracellular ATP on channels in inside-out patches (Fig. 3C). The channel activity decreased spontaneously
after patch excision. When 3 mM Mg-ATP was added to the
internal solution, the channel activity resumed. This result
demonstrates that intracellular Mg-ATP is required to sustain
K-2 channel activity, which is consistent with the
presence of a Walker-A motif on this clone.
The Kinetic Analysis of the K
The steady-state open probability (P-2
Channel
) of K
-2 was estimated from
amplitude histograms constructed from 3-min continuous recordings at
potentials between -40 and -100 mV (Fig. 4A). Fig. 4A (a) shows
examples of the amplitude histogram of 36-pS channels at -100 and
-60 mV. The steady-state P
of the channel
was calculated as the ratio of the area under the open peak to the
total area of the histogram. Steady-state P
values
of the 36-pS and 21-pS channels decreased slightly as the membrane was
hyperpolarized from -40 to -100 mV (Fig. 4A, b).
Figure 4:
The kinetic analysis of the
K-2 channel. A, voltage dependence of the open
probability (P
) of K
-2. a,
examples of the amplitude histogram at potentials of -60 and
-100 mV from the 36-pS channel. The ordinate of the
histogram represents the percentage of the number of counts in each bin
of the total counts in the histogram. b, values of the
steady-state P
obtained from amplitude histograms
of K
-2 channels. The left graph represents 36-pS
channels and the right graph 21-pS channels. Different symbols
indicate results from different patches. B, voltage dependence
of the mean open and closed time of K
-2 channels. a, an example of frequency histograms of the open time (upper column) and the closed time (lower column) of
a 21-pS channel. The ordinate of the histogram represents the
percentage of the number of the events in each bin of the total number
of the open or closed events in the histogram. b, time
constants of the mean open time (
), fast
(
), and slow (
) components of
closed time of K
-2 channels are plotted against voltage.
As above, the left graphs represent 36-pS channels and the right graphs 21-pS channels. Different symbols indicate
different patches.
The gating kinetics of the current fluctuations through
single channels of K-2 were analyzed using current records
of patches containing one channel (Fig. 4B). Fig. 4B (a) shows an example of the frequency
histograms of the open and closed times of a 21-pS channel recorded at
-80 mV. The frequency histogram of open time could be fitted by a
single exponential at potentials from -40 to -100 mV (uppergraphs in Fig. 4B, b;
36-pS (left) and 21-pS (right) channels). The time
constants (
) from both channels for the open time
histogram showed little voltage dependence at potentials between
-60 and -100 mV.
of the 36-pS channel was
constant at
100 ms, and that of the 21-pS channel varied from 100
to 200 ms. As the membrane was depolarized to potentials positive to
-40 mV, channel flickerings during bursts increased. This is
probably due to the intrinsic activation gating of these
inward-rectifying K
channels (Kurachi, 1985), which
resulted in the decrease of channel P
at
potentials > -30 mV (data not shown). The activation gating
flickerings of these channels, however, appeared at different
potentials from channel to channel. It appeared at potentials >
-20 mV in the case of Fig. 3A (a) and at
> +20 mV in the case of Fig. 3A (b). On
the other hand, the histogram of the closed time was fitted by a sum of
two exponentials at potentials between -40 and -100 mV (lowergraphs in Fig. 4B (b);
36-pS (left) and 21-pS (right) channels). Both time
constants of the slow components (
, opensymbols) increased as the membrane was hyperpolarized
from -40 to -100 mV, whereas those of the fast components
(
, filledsymbols) remained
essentially constant at these potentials.
The K
The K-2 mRNA Is Predominantly Expressed
in Glial Cells
-2 mRNA size and
distribution were examined by Northern blot hybridization. A
5.5-kilobase mRNA was detected strongly both in forebrain and
cerebellum and less in kidney, but not in heart or skeletal muscle (Fig. 5).
Figure 5:
Distribution of K-2 mRNA in
different tissues shown by Northern blot analysis. The lanes represent
poly(A)
RNA from rat forebrain, cerebellum, atrium,
ventricle, kidney, and skeletal muscle. A SacI digested
fragment (0.6 kilobase) of pA11 was used for a probe. The positions of
RNA size markers are shown at left. Quantities of RNA samples
were standardized by reprobing the same blot with a labeled cDNA for
glyceraldehyde-3-phosphate dehydrogenase (not shown here; see Koyama et al., 1994).
Further distribution of the K-2 mRNA in
the brain was examined in detail with the in situ hybridization technique. K
-2 mRNA was expressed in a
variety of regions throughout the brain. White matter of the
cerebellum, sensory root of trigeminal ganglion, middle cerebellar
peduncle, and corpus callosum contained high amounts of
K
-2 mRNA, suggesting K
-2 mRNA exists in
oligodendrocytes (Fig. 6). A dense cluster of silver grains
originated at the Purkinje cell layer and extended out into the
molecular layer (Fig. 6, E and F). Examination
of this region using high power bright-field microscopy confirmed that
Purkinje cells themselves were unlabeled by K
-2 cRNA probe
(data not shown). Silver grains were clustered over small cells
surrounding the Purkinje cells. The position of these labeled cells
suggests that they are Bergmann glia. Moderate level of
K
-2 mRNA positive signals were also seen in other brain
regions such as hippocampus (CA1-3 and dentate gyrus), thalamus,
inferior colliculus, motor trigeminal nucleus, superior olive, and
facial nucleus (Fig. 6, B and C). Clear
accumulation of the silver grain on these neurons was not found under
microscopic observation, suggesting that the signals may be derived
from glial cells. These results suggest that K
-2 mRNA is
expressed selectively in glial cells of the central nervous system.
Figure 6:
K-2 mRNA expression in the
brain. A-D, x-ray film autoradiographs illustrating
distribution of K
-2 mRNA in sagital sections of rat brain.
Sections were hybridized with antisense probe (A-C) or
sense probe (D) and then exposed to x-ray film. Strong
expression of K
-2 mRNA was observed in cerebellum, and
moderate expression was found in corpus callosum (cc),
hippocampus (CA1-3 and DG), thalamus (T), inferior colliculus (IC), and brain stem. No
hybridization signal was found in D. E-G,
darkfield photomicrographs of sagital sections of cerebellum (E and F) and coronal section of brain stem (G).
K
-2 mRNA was detected in the Purkinje cell layer (Pur) and white matter (wm) of cerebellum (E) and also found in sensory root trigeminal ganglion (s5) and middle cerebellar peduncle (mcp) (G). CA1-3, fields CA1-3 of Ammon's
horn; DG, dentate gyrus; Mo5, motor trigeminal
nucleus; SO, superior olive; 7, facial nucleus. Scale bars: A-D, 2 mm; E, 200 µm; F and G, 100 µm.
-2, which includes a Walker
type-A motif occurring at the corresponding position in the ROMK1
sequence (Ho et al., 1993). As judged from its primary
structure, the protein described here encoded a novel two
membrane-spanning segment type potassium channel. The amino acid
comparison indicates that this clone is more closely related to ROMK1
than other two membrane-spanning type potassium channels. As
illustrated in Fig. 7, four major groups of these potassium
channels may be classified based upon published sequences: 1)
K
, a classical inward rectifier potassium channel (IRK1,
IRK2, and IRK3), 2) K
, a G protein-activated potassium
channel (GIRK1), 3) K
, an ATP-sensitive potassium channel
(K
-1), and 4) K
, an inward rectifier
potassium channel with an ATP-binding domain (K
-1,
originally identified as ROMK1; see Ho et al.(1993)). Thus,
K
-2 may be a member of the subfamily of two transmembrane
type potassium channels with an ATP-binding domain.
Figure 7:
An evolutionary tree of the potassium
channel family with two membrane-spanning domains. The tree was made
using the UPGMA (Unweighted Pair Group Method with Arithmic Mean) Tree
Window in Geneworks (IntelliGenetics, Inc., Mountain View, CA) The
sequences of rat ROMK1, mouse IRK1, mouse IRK2, mouse IRK3, mouse
GIRK1, and rat K-1 are those reported by Ho et al. (1993), Kubo et al. (1993a), Takahashi et al. (1994), Morishige et al. (1994), Morishige et
al.
and Ashford et al. (1994),
respectively.
The
electrophysiological characteristics of K-2 at the whole
cell current level (i.e. the dependence of the slope
conductance on [K
]
,
rapid activation upon hyperpolarizing pulses, and the time- and
voltage-dependent block by Ba
and
Cs
) were the same as those of classical inward
rectifier potassium channels seen in a variety of cell types, including
IRK1-3. The K
-1 (ROMK1) current expressed in oocytes
does not rectify noticeably (Ho et al., 1993). Recent
experiments using site-directed mutagenesis show that aspartic acid (D)
in the second transmembrane (M2) segment of IRK1, corresponding to
asparagine(N) in K
-1 (ROMK1), plays a role both in the
control of polyamine-mediated channel gating and in the blocking by
intracellular Mg
(Stanfield et al., 1994; Lu
and MacKinnon, 1994; Wible et al., 1994; Ficker et
al., 1994; Lopatin et al., 1994). This residue is
glutamic acid (E) in K
-2 where the charge is conserved. As
shown in the mutation D172E in IRK1 (Stanfield et al., 1994),
the K
-2 current expressed in oocytes shows strong
rectification like the IRK1-3 currents. At the level of
single-channel recording, the polyamine-mediated intrinsic activation
gating appeared consistently at potentials positive to -40
-30 mV in IRK1, while it appeared at various potentials in
K
-2. The activation flickerings of K
-2 were
detected at potentials positive to -40 mV in some cases, but only
positive to +20 mV in other cases (see Fig. 3A, a and c). This variability of the voltage dependence
of activation gating of K
-2 might be related to the
glutamic acid residue of the M2 segment.
]
has been identified (Newman, 1993). In rabbit Müller
cells, three distinct types of channels have been identified; weak
inward rectifiers of 360 pS locate mainly at the endfeet, and moderate
and strong inward rectifiers (60 pS and 105 pS, respectively) exist in
the cell bodies (Nilius and Reichenbach, 1988). Barres et
al.(1988) described cultured oligodendrocytes as expressing an
inward rectifying potassium current that is mediated by 30-pS and
120-pS channels. The unitary conductances of the inward rectifying
potassium channels in bovine oligodendrocytes and human malignant
glioma cells are 29 pS and 27 pS, respectively (McLarnon and Kim, 1989;
Brismar and Collins, 1989). The approximately 30-pS channels shown
above might correspond to the 36-pS channel of K
-2. These
multiple channels electrophysiologically identified suggest the further
heterogeneity of two transmembrane segment type potassium channels in
glial cells.
-2).
This might indicate that a single clone could cause the distinct
conduction levels by assembling in different manners and partly explain
previous description of multiple types of inward rectifier potassium
channels in glial cells.
-2 mRNA in the
brain is different from that of other inward rectifying potassium
channels. mRNAs for IRK1, IRK2, IRK3, and GIRK1 were detected in
neurons (Morishige et al., 1993; Kenna et al., 1994;
Karschin et al., 1994),
while K
-2
mRNA was expressed predominantly in glial cells such as
oligodendrocytes and Bergmann glial cells. Immunocytochemistry by
specific antibodies against K
-2, combining double staining
with the glial fibrillary acidic protein antibody, confirms that
astrocytes derived from primary culture cells also express
K
-2.
(
)A recent in situ hybridization study showed that K
-1 (ROMK1) mRNA,
which mainly expresses in kidney, was also expressed in cortex and
hippocampus in the brain but did not appear to be expressed in glial
cells (Kenna et al., 1994). Thus, the K
-2 channel
is identified, for the first time, as a glial cell inward rectifier
potassium channel. Availability of this cloned K
-2 channel
should enable us to understand at the molecular level how inward
rectifier potassium channels play a physiological function in glial
cells.
/EMBL Data Bank with accession
number(s) X86818.
, equilibrium potential for K
;
ROMK, inwardly rectifying ATP-regulated potassium channel from the
outer medulla of rat kidney; IRK, inward rectifier potassium channel;
GIRK, G protein-activated muscarinic potassium channel;
K
, ATP-sensitive potassium channel; K
,
inward rectifying K
channel with an ATP-binding
domain; PCR, polymerase chain reaction; [K
],
external potassium ion concentration; S, siemen(s); P
, open probability;
, time constant.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.