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. 1999 Oct 15;520(Pt 2):373–381. doi: 10.1111/j.1469-7793.1999.00373.x

Expression and polarized distribution of an inwardly rectifying K+ channel, Kir4.1, in rat retinal pigment epithelium

Shunji Kusaka *,†, Yoshiyuki Horio *, Akikazu Fujita *, Kenji Matsushita *,†, Atsushi Inanobe *, Takahiro Gotow ‡, Yasuo Uchiyama ‡, Yasuo Tano †, Yoshihisa Kurachi *
PMCID: PMC2269596  PMID: 10523406

Abstract

  1. In the eye, different substances and ions including potassium (K+) are transported between neural retina and choroid via the subretinal space. Inwardly rectifying K+ channels (Kir) on the apical membrane of retinal pigment epithelial (RPE) cells are thought to play an essential role in K+ transport in the subretinal space.

  2. Single-channel recordings from the apical membrane of RPE cells exhibited functional expression of a Kir channel with properties identical to those of Kir4.1, while recordings from the basolateral membrane showed no detectable Kir channel currents.

  3. The expression of Kir4.1 mRNA in RPE cells was confirmed by RT-PCR analysis and in situ hybridization. Furthermore, using immunohistochemistry, we found that Kir4.1 was prominently expressed in RPE cells and localized specifically on the processes on their apical membrane.

  4. Developmental studies revealed that expression of Kir4.1 started to appear 10 days or later after birth in RPE cells, in parallel with the maturation of retinal neuronal activity as represented by the a- and b-waves of the electroretinogram.

  5. These data suggest that Kir4.1 is one of the Kir channels involved in RPE-mediated control of K+ ions in the subretinal space.

The retinal pigment epithelium (RPE) is a monolayer of cells separating the neural retina and the choroid (Newman, 1994). Its apical membrane possesses prominent microvilli and faces the outer segments of photoreceptor cells. The apical membrane of RPE cells and photoreceptor cell outer segments are loosely connected and the space between them is called the subretinal space (Newman, 1994). It is well documented that the extracellular concentration of potassium ions ([K+]o) in the subretinal space decreases transiently upon light-induced excitation of photoreceptors, and this may underlie formation of the c-wave in the electroretinogram (ERG) (Newman, 1994). The decrease in [K+]o in the subretinal space is supposed to be due to closure of cGMP-activated non-selective cation channels in the outer segments of photoreceptor cells, and its return to the dark-adapted level under continuous illumination may be due to extrusion of K+ ions from RPE cells as well as from retinal glial (Müller) cells (Steinberg et al. 1980; Newman, 1994; Newman & Reichenbach, 1996). This recovery of [K+]o in the subretinal space may be important to maintain the activity of the Na+,K+-ATPase and the Na+-K+-Cl− cotransporter in RPE cells, which are localized on the apical membrane (Miller et al. 1978; Adorante & Miller, 1989).

Previous studies have shown that both apical and basolateral membranes of the RPE have relatively high K+ conductances (Miller et al. 1979; Joseph & Miller, 1991; Quinn & Miller, 1992). Whole-cell recordings from isolated RPE cells have revealed that these cells possess two types of K+ channel, a voltage-dependent, delayed rectifying K+ (KV) channel (Wen et al. 1993; Takahira & Hughes, 1997) and an inward rectifying K+ (Kir) channel (Hughes & Steinberg, 1990; Wen et al. 1993; Segawa & Hughes, 1994; Hughes & Takahira, 1996). The activation threshold of the KV channel is approximately -20 to -40 mV, which is far from the physiological membrane potential of RPE cells (approximately -50 to -70 mV) (Hughes & Steinberg, 1990; Wen et al. 1993; Segawa & Hughes, 1994; Hughes & Takahira, 1996). On the other hand, the Kir channel is active at all physiological membrane potentials (Isomoto et al. 1997) and is, therefore, thought to be mainly responsible for the K+ conductance of the RPE at resting potential. Because the ratio of the apical-to-basolateral membrane resistance of RPE cells suggests that the Kir conductance exists mainly in their apical membrane (Segawa & Hughes, 1994), it has been assumed that the maintenance of [K+]o in the subretinal space is at least partly supported by extrusion of K+ ions through the Kir channels. To date, however, the molecular properties and subcellular localization of the Kir channels in RPE cells have not been examined.

In a previous study of Kir4.1 in retinal Müller cells, we also detected prominent expression of mRNA and immunoreactivity of this Kir subunit in the RPE layer (Ishii et al. 1997). In this study, we investigated expression and function of Kir4.1 channels in mammalian RPE using electrophysiology, molecular biology and immunohistochemistry techniques. We found that Kir4.1 was functionally expressed predominantly on the microvilli of the apical membrane of RPE cells. Therefore, Kir4.1 may be the RPE Kir channel which is involved in the homeostasis of K+ ions in the subretinal space.

METHODS

Preparation of sheets of retinal pigment epithelial cells

All experiments were carried out in accordance with the Guidelines for the Use of Laboratory Animals of Osaka University Medical School. RPE cells were isolated as a sheet, for electrophysiological recordings, from eyes of neonatal Long-Evans rats at postnatal days (P) 10-14 (Nippon Doubutsu, Kyoto, Japan). Animals were anaesthetized with an overdose of pentobarbital (100 mg (kg body weight)−1i.p.) and eyes were enucleated. RPE sheets were isolated as described by Sakagami et al. (1995), with slight modifications. In brief, the eyeballs were incubated for 13-15 min at 37°C in 0.1 % proteinase K (Nacalai Tesque, Kyoto, Japan) in Ca2+-Mg2+-free Hanks’ balanced salt solution (HBSS; Gibco). After incubation, the eyeballs were rinsed in HBSS. Under a dissecting microscope, a circumferential incision was made just below the ora serrata, and the retina-RPE complex was carefully extracted. The retina-RPE complex was incubated for 10-20 min at 37°C in a solution containing (mm): 130 NaCl, 5 KCl, 1.8 CaCl2, 0.53 MgCl2, 5.5 glucose, and 5.5 Hepes-KOH, pH 7.4, after which the RPE could be removed from the retina as a sheet.

Electrophysiological recordings

Single-channel recordings from apical and basolateral sides of isolated RPE sheets were performed at room temperature using a patch-clamp amplifier (Axopatch 200A; Axon Instruments Inc., Foster City, CA, USA) and recorded on videocassette tapes using a PCM converter system (VR-10B; Instrutech Corp., New York, NY, USA). The tips of the patch electrodes were coated with Sylgard (Dow Corning) and heat polished. The tip resistance of the electrodes was 5-7 MΩ when filled with pipette solution (see below). A freshly isolated RPE sheet was placed in a recording chamber fixed on an inverted microscope (Axiovert 135; Zeiss). In the recording chamber, the isolated RPE sheet was held in place with a grid of parallel nylon threads (Konnerth et al. 1987). For analysis, data were reproduced, low-pass filtered at 1 kHz (-3 dB) by an 8-pole Bessel filter (Frequency Devices, Haverhill, MA, USA), sampled at 5 kHz and analysed off-line on a computer (Macintosh Quadra 700; Apple Computer Inc., Cupertino, CA, USA) using commercially available software (Patch Analyst Pro; MT Corporation, Nishinomiya, Hyogo, Japan). The bathing solution contained (mm): 145 KCl, 5 EGTA, 2 MgCl2, and 5 Hepes-KOH, pH 7.4; and the pipette solution contained (mm): 145 KCl, 1 MgCl2, 1 CaCl2, and 5 Hepes-KOH, pH 7.4. The pipette solutions containing lower concentrations of K+ were prepared by replacing KCl with equimolar NaCl. The membrane potential of the RPE cells in a sheet was measured under whole-cell current-clamp conditions. It was 0 mV during perfusion with bathing solution containing 150 mm K+ (n= 5 patches). Data were expressed as means ±s.e.m.

PCR amplification of Kir4.1 cDNA

Total RNA of freshly dissociated sensory retina and a sheet of RPE cells were extracted and cDNAs were synthesized. RT-PCR analysis was performed using primers for Kir4.1. Primers for the PCR reaction were located in nucleotides 194-211 and 848-865 of rat Kir4.1 cDNA (Takumi et al. 1995). As the Kir4.1 gene is an intronless gene (Y. Horio & Y. Kurachi, unpublished observation), to eliminate contamination of genomic DNA, RNA samples were treated with DNase I (Takara, Kyoto, Japan) before cDNA synthesis. PCR amplification was performed for 30 cycles at 95°C for 30 s, 55°C for 30 s and 72°C for 90 s, followed by 72°C for 7 min. The products were electrophoresed on a 1 % agarose gel. The nucleotide sequence of the amplified PCR products was determined using the dye-primer method and DNA sequencer (A-381; The Perkin-Elmer Corp., Foster City, CA, USA) after TA cloning (Invitrogen, Carlsbad, CA, USA).

In situ hybridization histochemistry

In situ hybridization was performed on frozen sections of Wistar rat eyes, as described previously (Takumi et al. 1995). In brief, a Wistar rat weighing about 250 g was anaesthetized with an overdose of pentobarbital (100 mg (kg body weight)−1i.p.) and eyes were enucleated. The Bst XI-Sac I fragment (0.37 kb) of rat Kir4.1 cDNA was used as a template. Frozen sections (20 μm) of rat eyes were fixed with 4 % paraformaldehyde and hybridized with [35S]-labelled Kir4.1 cRNA antisense probe or sense probe. After washing with a high-stringency buffer (50 % formamide, 2 × SSC (mm: 150 NaCl, 15 sodium citrate, pH 7.0) and 10 % 2-mercaptoethanol), sections were dipped in emulsion and developed for 2 weeks.

Immunohistochemistry

A polyclonal antibody for Kir4.1 (anti-KAB-2C2) was raised in rabbit against a synthetic peptide corresponding to amino acid residues 366-379 (EKEGSALSVRISNV) in the C-terminal region of rat Kir4.1 (Ito et al. 1996). The rabbits were killed at the end of the experiment by intravenous injection of an overdose of sodium pentobarbital. The antiserum was purified with protein-A Cellulofine (Seikagaku Corp., Tokyo, Japan) and antigenic peptide-coupled Sulfolink resin (Pierce, Rockford, IL, USA). Dark Agouti or Wistar rats weighing about 250 g, and Wistar rats at P1-25, were deeply anaesthetized with pentobarbital (100 mg kg−1i.p.) and perfused with 100 ml phosphate-buffered saline (PBS) followed by 250 ml of ∼4 % paraformaldehyde in 0.1 M sodium phosphate (PA solution), pH 7.4. After perfusion, eyes were enucleated, fixed again with PA solution for 3-48 h, dehydrated with sucrose and frozen. Sections (10 μm) were cut on a cryostat and thaw-mounted on gelatin-coated slides. Samples were washed twice with PBS containing 0.1 % Triton X-100 (PBST) for 5 min each, treated with 1 % (w/v) bovine serum albumin (BSA) in PBS at room temperature for 30 min, and then incubated with anti-KAB-2C2 (0.15 μg ml−1) and mouse monoclonal anti-pan cytokeratin antibody (45 μg ml−1; Sigma) in PBS at 4°C overnight. The sections were washed 5 times with PBS at room temperature for 15 min each and visualized with fluorescein isothiocyanate (FITC)-labelled anti-rabbit IgG (EY Laboratories, San Mateo, CA, USA) and Texas Red-labelled anti-mouse IgG (Protos Immunoresearch, San Francisco, CA, USA). The sections were examined with a confocal microscope (MRC-1024; Bio-Rad, Hertfordshire, UK). For control experiments, anti-KAB-2C2 preabsorbed with excess antigenic peptide (3 g ml−1) was used.

Developmental studies were performed in eyes of Wistar rats at P1-25 according to Yoshida et al. (1996) with the polyclonal antibody for Kir4.1 (anti-KAB-2C2) and an ABC kit (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA, USA).

Electron microscopy

Immunogold electron microscopy was performed in Dark Agouti rat eyes as described previously (Gotow et al. 1995). After fixation, the retinas were dehydrated in 2.3 M sucrose containing 0.1 M sodium phosphate, pH 7.4, and frozen in liquid nitrogen. Cryothin sections were cut upon a microtome equipped with cryo-attachment (OmU4; Reichert, Vienna, Austria) and collected on Formvar carbon-coated grids. The cryothin sections on grids were treated with 1 % BSA in PBS and incubated with anti-KAB-2C2, and then goat anti-rabbit IgG coupled to 5 nm colloidal gold particles (Amersham, Buckinghamshire, UK). The sections were again fixed with 2 % glutaraldehyde and post-fixed with 1 % OsO4, stained with 0.5 % uranyl acetate, dehydrated in ethanol and embedded in London Resin white.

RESULTS

Electrophysiological properties of RPE cell K+ channels

We first examined whether the apical membrane of RPE cells expresses functional inwardly rectifying K+ (Kir) channels, with the single-channel recording technique. To minimize the loss of apical processes of RPE cells during the isolation procedure, we prepared RPE sheets with gentle treatment of the retina because preliminary experiments showed that RPE cells isolated using regular procedures usually lost the apical processes. The cell-attached patch-clamp technique was applied either to the apical or basolateral membrane of the freshly dissociated sheets of RPE (see the inset to Fig. 1A). The apical portion of the cell could be easily identified by the processes projecting from its surface and pigment granules in its cell body. Pipette and bathing solutions both contained 150 mm K+.

Figure 1. Functional Kir channels recorded from the apical side of RPE cells.

Figure 1

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Single-channel recordings (cell-attached mode) from apical membranes of isolated rat RPE cell sheets. A, membrane current traces were recorded at the membrane potential (Vm) values indicated to the left of each trace with either 150 mm K+ (left-hand panel) or 75 mm K+ (right-hand panel) in the pipette solution. B, current-voltage relationships of the Kir channel of isolated rat RPE cells. The single-channel conductance of this Kir channel was 22 pS with 150 mm[K+]pip, 15 pS with 75 mm[K+]pip and 12 pS with 50 mm[K+]pip. The data were obtained from twenty-two patches for 150 mm[K+]pip, five for 75 mm[K+]pip and four for 50 mm[K+]pip. C, voltage dependence of open probabilities of the Kir channel recorded from rat RPE cells with 150 mm[K+]pip. This channel showed high open probability.

Figure 1A depicts examples of the K+ currents recorded from the apical side of a RPE sheet at various membrane potentials with 150 mm K+ (left-hand panel) or 75 mm K+ (right-hand panel) in the pipette ([K+]pip). The currents flowed much more readily in the inward than in the outward direction, thus clearly exhibiting inward rectification. However, small outward currents flowing through the channel could be recorded. The channel openings occurred in bursts and exhibited a prominent open channel noise. Figure 1B shows the unitary current-voltage relationship of the channel with different [K+]pip. The unitary conductance of the channel in the inward direction, determined by the least-squares method, was 22 pS with 150 mm[K+]pip, 15 pS with 75 mm[K+]pip and 12 pS with 50 mm[K+]pip. Thus, the unitary conductance of the channel was approximately proportional to the root value of [K+]pip. The reversal potential of the channel estimated from the current-voltage relationship at negative potentials was 0 mV with 150 mm[K+]pip, -17 mV with 75 mm[K+]pip and -23 mV with 50 mm[K+]pip. Therefore, we concluded that the recorded currents flowed through an inwardly rectifying K+ (Kir) channel at the apical membrane of RPE cells.

The kinetics of the Kir channel was examined with 150 mm[K+]pip. The open time histogram could be fitted with a single exponential with a time constant of ∼100 ms at membrane potentials between -100 and -60 mV, and the closed time histogram could be fitted with the sum of two exponentials with time constants of ∼3 and 20-30 ms (data not shown). The open probability (Po) of the channel was ∼0.7-0.9 at potentials between -120 and -40 mV (Fig. 1C).

The conductance and kinetic characteristics of the Kir channel at the apical side of RPE cells are identical to those of the Kir4.1 channel in its low conductance state when expressed in Xenopus oocytes and human embryonic kidney (HEK293T) cells (Bond et al. 1994; Takumi et al. 1995; Pessia et al. 1996; Horio et al. 1997; Ishii et al. 1997; Tada et al. 1998). We could record only this type of Kir channel current in 31 patches out of 80 successful cell-attached patch recordings from the apical side of RPE sheets, although some types of voltage-dependent K+ channel could also be recorded (not shown). No significant Kir channel activity was recorded from the basolateral side (n= 25).

Expression of Kir mRNAs in RPE cells

To confirm expression of Kir4.1 in RPE cells, we performed RT-PCR analysis of the total mRNA obtained from the isolated RPE sheet and in situ hybridization of the retina. In the RT-PCR analysis (Fig. 2A), a single band of DNA of the expected size (672 bp) was amplified from RPE cDNA as well as from rat Kir4.1 cDNA (positive control). No band was detected when distilled water was used instead of cDNA (negative control). We sequenced the DNA band from RPE cells after subcloning to the TA vector and identified it as Kir4.1. We examined the distribution of Kir4.1 mRNA in a slice of rat retina by in situ hybridization. Kir4.1 mRNA was detected in the RPE layer (Fig. 2B) and also in the inner nuclear layer, where somata of Müller cells are to be found (not shown; see Ishii et al. 1997).

Figure 2. Expression of Kir4.1 mRNA in RPE cells.

Figure 2

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A, RT-PCR amplification of Kir4.1 cDNA from a sheet of RPE cells. RNA was extracted from the sensory retina and a sheet of RPE cells, and cDNAs were synthesized. After the PCR reaction, products were electrophoresed on a 1 % agarose gel. Kir4.1 fragments (672 bp) were amplified from cDNA/mRNAs from the sensory retina (Retina) as well as from RPE cells. -, negative control (distilled water instead of cDNA). +, positive control (rat Kir4.1 cDNA). B, distribution of Kir4.1 mRNA in frozen sections (20 μm) of rat eyes which were fixed and hybridized with Kir4.1 cRNA antisense probe. Grains showing Kir4.1 mRNA were detected in the RPE layer. ONL, outer nuclear layer; OS, outer segment layer; RPE, retinal pigmented epithelial cell layer. Scale bar, 10 μm.

Confocal image and immunogold electron microscopy analyses of Kir4.1 immunoreactivity in RPE cells

Figure 3 depicts the immunostaining of retinal sections from Dark Agouti (pigmented) (Fig. 3A) and Wistar (albino) (Fig. 3B) rats, using anti-KAB-2C2 antibody. In both species, immunoreactivity was detected in the nerve fibre layer (NFL), ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL) and outer nuclear layer (ONL), as reported previously (Ishii et al. 1997). This immunoreactivity was due to the staining of Müller cells. In addition to Müller cells, very strong immunoreactivity was detected in the photoreceptor outer segment layer (OS), where apical processes of RPE cells and photoreceptor outer segments are localized. Figure 3Ab and Bb shows the immunoreactivity to cytokeratin, a marker of epithelial cells. Because the double staining of anti-KAB-2C2 (green) and anti-cytokeratin (red) produced a prominent yellow signal in OS (Fig. 3Ac and Bc), the Kir4.1 immunoreactivity seems to be localized in the apical processes of RPE. A weak immunoreactivity was also detected in the RPE layer in the albino rat (Fig. 3B), while in the pigmented rat no immunostaining was detected in the RPE layer (Fig. 3A), probably because the weak fluorescence was absorbed by the pigments in RPE cells.

Figure 3. Immunohistochemical analysis of Kir4.1 in the retina.

Figure 3

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Ten micrometre sagittal sections of retina from pigmented (A) and albino (B) rats were double-stained with affinity-purified rabbit anti-rat Kir4.1 antibody followed by FITC-conjugated anti-rabbit IgG (Aa and Ba, in green) and monoclonal anti-pan cytokeratin antibody followed by Texas Red-labelled anti-mouse IgG (Ab and Bb, in red). Ac and Bc represent double exposures of both images. Ad and Bd, Nomarski images of the same sagittal sections as in panels a, b and c. Kir4.1 was expressed mainly in the apical processes of RPE cells and also in Müller cells. Kir4.1- and pan cytokeratin immunostaining was observed in the RPE cell layer from albino rat (B), but not from pigmented rat (A). Scale bars in Ad and Bd, 10 μm. NFL, nerve fibre layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; OS, outer segment layer; RPE, retinal pigmented epithelial cell layer.

To examine the subcellular localization of Kir4.1 in RPE cells, an immunogold electron microscopy study of the retina was performed using anti-KAB-2C2 (Fig. 4). Many gold particles were detected on the membrane of apical processes of RPE cells which surrounded the outer segments of photoreceptor cells (Fig. 4A, B and D). On the other hand, no gold particles were found at the basolateral membrane of RPE cells (Fig. 4C). These data clearly indicate that Kir4.1 is localized specifically at the apical processes and not at the basolateral membrane of RPE cells.

Figure 4. Immunogold electron microscopy of Kir4.1 in the RPE.

Figure 4

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Ultrathin sections were stained with anti-Kir4.1 antibody and anti-rabbit IgG coupled to colloidal gold particles. A-D, electron microscopic images of the areas indicated in the lower right schematic drawing. Positive gold particles were detected on the membranes of the apical processes, but not on the basolateral membrane. OS, photoreceptor outer segment; AP, apical process; CA, choroidal capillary. Scale bars represent 200 nm in A, B and D and 400 nm in C.

Developmental expression of Kir4.1 immunoreactivity in the retina

It has been reported that a- and b-waves in the ERG start to appear ∼11 days after birth (P11) (el Azazi & Wachtmeister, 1990), when the photoreceptor cells become functional. In Fig. 5 we show the postnatal development of Kir4.1 immunoreactivity in the retina. No significant immunoreactivity was detected at P1, P5 and P8. At P10, significant Kir4.1 immunoreactivity was detected in RPE cells and the endfoot region of Müller cells. The immunoreactivity became more prominent at P25. ‘String-like’ staining was observed in inner and outer nuclear layers. There was no significant difference in the time course of development of Kir4.1 immunoreactivity between Müller cells and RPE cells. This indicates that, in both RPE and Müller cells, Kir4.1 is not expressed at birth but appears as early as ∼P10, when a- and b-waves start to be detected in the ERG.

Figure 5. Developmental studies of the expression of Kir4.1 in the retina.

Figure 5

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Comparison of developmental change of Kir4.1 expression in the rat retina at various postnatal days. P1-25, postnatal days 1-25. Sagittal sections of rat retina were stained with the ABC-DAB method. NFL, nerve fibre layer; OPL, outer plexiform layer; ONL, outer nuclear layer; OS, outer segment layer; RPE, retinal pigmented epithelial cell layer. Scale bar, 20 μm.

DISCUSSION

The major findings of this study are as follows. (1) Functional Kir channel currents recorded from the apical membrane of RPE cells exhibited identical properties to those of Kir4.1. (2) Kir4.1 immunoreactivity was specifically localized on the apical processes but not on the basolateral membrane of RPE cells. (3) The developmental expression of Kir4.1 in the RPE approximately paralleled the formation of a- and b-waves of the ERG.

Although previous studies have suggested the presence of Kir channel currents on the apical membrane of RPE, this study has shown for the first time that a Kir channel, Kir4.1, is actually expressed and localized on the apical processes of RPE cells. Using cell-attached patch-clamp recordings, we could record only a single type of Kir channel current from the apical side of the RPE, whose conductance and kinetic properties were identical to those of Kir4.1 channels heterologously expressed in HEK293T cells or in Xenopus oocytes. Therefore, it is likely that Kir4.1 is mainly responsible for the RPE apical membrane Kir conductance which has been measured in the RPE-choroid preparation (Miller et al. 1979;Quinn & Miller, 1992) and in intracellular voltage recordings (Joseph & Miller, 1991). However, we cannot completely exclude the possibility that other Kir channels also exist in this preparation.

An inwardly rectifying K+ current with a unique property has been identified in whole-cell studies of isolated RPE cells in a number of species (Hughes & Steinberg, 1990; Wen et al. 1993; Segawa & Hughes, 1994; Hughes & Takahira, 1996). Although the Kir channel current was blocked by Ba2+ and Cs+, its conductance remained nearly the same with different [K+]o. Because a characteristic of many Kir channels, including Kir4.1, is that their conductances are approximately proportional to the root value of [K+]o (Hagiwara et al. 1976; Standen & Stanfield, 1978; Takumi et al. 1995), there is clear discordance between our recording of Kir4.1-like single-channel currents from the apical membranes of RPE sheets and the whole-cell Kir current recorded from isolated RPE cells. Because Kir4.1 channel proteins are specifically localized on the apical processes of RPE cells and the isolated RPE cells have largely lost the processes during the dissociation procedure, the unique whole-cell Kir current may be derived from Kir channels other than Kir4.1. A Kir channel, Kir7.1, which has been cloned recently from brain, does not show a dependence on [K+]o (Krapivinsky et al. 1998; Döring et al. 1998). In fact, we have confirmed the expression of the unique Kir current in the whole-cell measurement of isolated RPE cells and also the expression of Kir7.1 mRNA in those cells by RT-PCR (not shown). The single-channel conductance of Kir7.1 has been estimated to be ∼50 fS, which is below the resolution of our single-channel recording technique but which could contribute to the whole-cell Kir current. Therefore, it may be the case that Kir7.1 would correspond to the [K+]o-insensitive Kir current recorded in acutely enzymatically isolated RPE cells which have lost the greater part of their apical processes.

The Kir4.1 channels on the apical processes of RPE cells may play an important role in the buffering of extracellular K+ ions in the subretinal space. When light stimulates the retina the concentration of K+ ions in the subretinal space decreases (Oakley, 1977; Steinberg et al. 1980). The light-induced decrease in [K+]o in the subretinal space is probably due to hyperpolarization of the rods consequent to closure of cGMP-gated cation channels (Fujimoto & Tomita, 1979; Yau, 1994). This decrease would provoke a negative shift of the equilibrium potential for K+ ions and thus would increase an outward driving force for K+ ions in RPE cells. As a result, K+ ion efflux from the RPE apical membrane via Kir channels would be facilitated and would contribute to the recovery of [K+]o in the subretinal space. The recovered level of extracellular K+ ions in the subretinal space would be essential for maintaining the activity of the RPE cell apical membrane Na+,K+-ATPase and Na+-K+-Cl− cotransporter (Miller et al. 1978; Adorante & Miller, 1989; Quinn & Miller, 1992).

Kir4.1 has also been found to be localized on the basolateral membrane in renal distal tubule epithelial cells and in marginal cells of cochlear stria vascularis (Ito et al. 1996; Hibino et al. 1997). The presumed function of Kir4.1 in these epithelia is to supply K+ ions to the Na+,K+-ATPase, which is abundant on the basolateral membrane of these cells (Ito et al. 1996; Hibino et al. 1997). Therefore, it seems to be a common feature of epithelial cells that Kir4.1 colocalizes with the Na+,K+-ATPase, and that, in co-operation with colocalized Na+,K+-ATPase, Kir4.1 plays a pivotal role in maintaining homeostasis of [K+]o in the microenvironment. Further studies are needed to elucidate the mechanisms controlling the colocalization of Kir4.1 and Na+,K+-ATPase in various epithelial cells.

With regard to the postnatal development of components of the ERG in the infant rat, el Azazi & Wachtmeister (1990) reported that a- and b-waves appear at ∼P10 and P11, and, that thereafter, their amplitudes increase between P17 and P30. Our results showed the expression of Kir4.1 immunoreactivity in Müller cells and RPE cells at P10 or later, which indicates that the expression of Kir4.1 and the functional development of the retina, as represented by the a- and b-wave components of the ERG, are in parallel and may therefore be closely related. The concurrent development of Kir4.1 and the b-wave is of interest, since the b-wave reflects, at least in part, the function of K+ siphoning by Müller cells (Newman, 1994) where Kir4.1 is abundantly expressed (Ishii et al. 1997). Thus, Kir4.1 is likely to play an important role in homeostasis of [K+]o in the retina. On the other hand, information on the development of the c-wave of the ERG, which reflects the [K+]o change in the subretinal space (Newman, 1994), is unavailable. Probably this is due to technical difficulties in obtaining stable recordings of the c-wave in vivo and therefore further studies are necessary to clarify the relation between the expression of Kir4.1 in RPE cells and the development of the c-wave of the ERG.

In conclusion, the present study indicates that functional Kir4.1 channels are localized specifically on the apical membrane of RPE cells, which may play an important role in the homeostasis of K+ ion concentration in the subretinal space.

Acknowledgments

This work was supported partly by grants from the Ministry of Education, Culture, Sports and Science of Japan, from Research for the Future Program (JSPS-RFTF96L00302) of The Japan Society for the Promotion of Science, from the Human Frontier Science Program (RG0158/1997-B) and from the Yamanouchi Foundation for Research on Metabolic Disorders. We thank Dr Ian Findlay (Université de Tours, Tours, France) for critical reading of the manuscript and Ms Keiko Tsuji for secretarial work.

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