©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Novel ATP-dependent Inward Rectifier Potassium Channel Expressed Predominantly in Glial Cells (*)

Toru Takumi , Takahiro Ishii (3), Yoshiyuki Horio , Ken-Ichirou Morishige (1), Naohiko Takahashi , Mitsuhiko Yamada , Takeshi Yamashita , Hiroshi Kiyama (2), Koichi Sohmiya (3), Shigetada Nakanishi (3), Yoshihisa Kurachi (§)

From the (1)Department of Pharmacology II, Department of Obstetrics and Gynecology, (2)Department of Neuroanatomy, Biomedical Research Center, Faculty of Medicine Osaka University, Suita, Osaka 565, Japan and (3)Institute for Immunology, Kyoto University Faculty of Medicine, Sakyo, Kyoto 606-01, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have isolated a novel inward rectifier K 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 MgATP 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.


INTRODUCTION

Neuronal excitation causes an increase of extracellular potassium ions (K) 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.

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-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).()

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-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.


EXPERIMENTAL PROCEDURES

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.

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 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.

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, 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% NaHPO, 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.


RESULTS

cDNA Cloning of the K-2 Channel

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.

The isolated cDNA encoded 379 amino acids (Fig. 1A). We designated this clone as K-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 (GXGKX(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-2 Is an Inward Rectifier Potassium Channel

Fig. 2illustrates the results obtained from Xenopus oocytes injected with cRNA derived from a K-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-2 Channel

Single-channel currents flowing through K-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-2 Channel

The steady-state open probability (P) 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-2 mRNA Is Predominantly Expressed in Glial Cells

The K-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.




DISCUSSION

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-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.

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] 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.

At the single-channel level, we identified two distinct conducting channels derived from a single clone (K-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.

Expression of K-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.


FOOTNOTES

*
This work was supported in part by research grants from the Ministry of Education, Science, and Culture of Japan, the Terumo Life Science Foundation, the Ichiro Kanehara Foundation, the Yamanouchi Foundation for Research on Metabolic Disorders, the Mochida Memorial Foundation for Medical and Pharmaceutical Research, the Naito Foundation, Ono Pharmaceutical Company (to Y. K.), and the Kanae Foundation of Research for New Medicine (to T. T.). The contributions of the first two authors should be considered as equal. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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 GenBank/EMBL Data Bank with accession number(s) X86818.

§
To whom correspondence should be addressed: Dept. of Pharmacology II, Faculty of Medicine Osaka University, Yamada-oka 2-2, Suita, Osaka 565, Japan. Tel.: 81-6-879-3510; Fax: 81-6-879-3519.

The abbreviations used are: E, 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.

K. Morishige, N. Takahashi, M. Yamada, N. Mori, and Y. Kurachi, unpublished data.

Y. Horio, A. Inanobe, T. Takumi, and Y. Kurachi, unpublished data.


ACKNOWLEDGEMENTS

We thank Paul Berke (Alkermes Inc.) and Ian Findlay (University of Tours, France) for critical reading of this manuscript.


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