Expression and clustered distribution of an inwardly rectifying potassium channel, KAB-2/Kir4.1, on mammalian retinal Müller cell membrane: their regulation by insulin and laminin signals

Abstract

Inwardly rectifying potassium (K+) channels (Kir) in Müller cells, the dominant glial cells in the retina, are supposed to be responsible for the spatial buffering action of K+ ions. The molecular properties and subcellular localization of Müller cell Kir channels in rat and rabbit retinas were examined by using electrophysiological, molecular biological, and immunostaining techniques. Only a single population of Kir channel activity, the properties of which were identical to those of KAB-2/Kir4.1 expressed in HEK293T cells, could be recorded from endfoot to the distal portion of Müller cells. Consistently, Northern blot, in situ hybridization, and RT-PCR analyses indicated expression of Kir4. 1 in Müller cells per se. The Kir4.1 immunoreactivity was distributed in clusters throughout Müller cell membrane. The Kir4.1 expression in Müller cells disappeared promptly after culturing. When the dissociated Müller cells were cultured on laminin-coated dishes in the presence of insulin, Kir4.1 immunoreactivity was detected in a clustered manner on the cell membrane. Because insulin and laminin exist in the surrounding of Müller cells in the retina, these substances possibly may be physiological regulators of expression and distribution of Kir4.1 in Müller cells in vivo.

Fig. 1.

Functional expression of Kir4.1 in rabbit Müller cells. A, Whole-cell recordings of isolated rabbit Müller cells. The holding potential was −70 mV. Traces were recorded with voltage steps from −120 to +40 mV in 20 mV steps (inset). a, Control. b, Effect of 100 μm external Ba²⁺. c, Current–voltage relationship of the steady-state currents in the presence (•) or absence (○) of 100 μmBa²⁺. Ba²⁺ predominantly inhibited inward currents. B, Single-channel recordings from cell-attached membrane patches of isolated rabbit Müller cells. a, Membrane current traces were recorded at the membrane potential values indicated to the left of each trace. The patch contained a Kir channel dominantly, but at depolarized potentials, currents of K⁺ channels with a large conductance were recorded. b, Current–voltage relationship of the Kir channel of isolated rabbit Müller cells. The single-channel conductance of this Kir was 25 pS. C, Single-channel recordings from cell-attached membrane patches of HEK293T cells expressing rat Kir4.1 channel. a, Traces were elicited from the same potentials as Ba.b, Current–voltage relationship of rat Kir4.1. The single-channel conductance of rat Kir4.1 was 23 pS. D, Voltage dependence of open probabilities of the Kir on rabbit Müller cells (a) and rat Kir4.1 (b). Both channels showed very high open probabilities. E, Distribution of the Kir channel on isolated rabbit Müller cells. The isolated cells exhibited typical morphological characteristics of the Müller cell shown in the figure, i.e., distal end (d), soma (s), proximal stalk (p), and endfoot (e) (left panel). The histograms of Kir4.1 channel number per patch in these four different regions are shown (right panel). Each patch contains 0–6 Kir4.1 channels:x-axis, the number of channels in each patch;y-axis, the relative frequency (in percentage) of each number of Kir4.1 channels (the total number of cell-attached patch experiments = 32 in d, n = 12 in s, n = 14 in p, and n = 18 in e). Channels were distributed diffusely and not concentrated in the endfoot region. Scale bar, 10 μm.

Fig. 6.

Immunostaining and whole-cell currents of Kir4.1 in acutely dissociated and 4 d cultured Müller cells from rat retina. A–D, Acutely dissociated rat Müller cells. E–H, Cells cultured for 4 d on cover glasses coated with poly-d-lysine. A, E, Nomarski images. Dissociated rat Müller cells aggregated with each other, which is different from rabbit Müller cells. InA, there were at least four cells. B, F, Immunostaining of Kir4.1 (green). C, G, Immunostaining of vimentin (red). D, H, Whole-cell currents were induced under voltage steps from −100 to +40 mV with 20 mV steps (inset). Cells were bathed in 5 mm K_o⁺. The holding potential was −70 mV. Inward currents of cultured cells were diminished. Scale bar (shown in G), 10 μm.

Fig. 2.

Expression of Kir4.1 mRNA in the retina.A, Northern blot analysis of rabbit Kir4.1 mRNA. Total RNA (20 μg) from a rabbit Müller cell enriched fraction was separated on 1% agarose gel containing formaldehyde and transferred to a nylon membrane. Rat Kir4.1 cDNA was used as a probe. B, Distribution of Kir4.1 mRNA. Frozen sections (20 μm) of rat eyes were fixed with 4% paraformaldehyde and hybridized with Kir4.1 cRNA antisense probe (left panel) or sense probe (right panel). After a high-stringency wash, sections were dipped in emulsion and developed for 2 weeks. Grains showing Kir4.1 mRNA were detected in the inner nuclear layer (INL), where cell bodies of Müller cells exist, and the retinal pigment epithelial layer (RPE). Retinal pigment epithelial cells also expressed Kir4.1. Labels are explained in Figure 3. C, PCR amplification of Kir4.1 cDNA from a single Müller cell. A single rabbit Müller cell was isolated by using a siliconized glass capillary. RNA was extracted from the cell, and cDNA was synthesized. After PCR reaction, products were electrophoresed on a 1% agarose gel. Kir4.1 fragments (672 bp) were amplified with cDNA/mRNAs from a Müller cell enriched fraction (lane 1), a single rabbit Müller cell (lane 2), six rabbit Müller cells (lane 3), and a control rat Kir4.1 cDNA (lane 4).

Fig. 3.

Immunohistochemical analysis of Kir4.1 in the retina. A sagittal section of rat retina (10 μm) was double-stained with affinity-purified rabbit anti-rat Kir4.1 antibody, followed by FITC-conjugated anti-rabbit IgG (A andC, in green), and monoclonal anti-vimentin antibody, followed by Texas Red-labeled anti-mouse IgG (B and C, in red).C, Double exposures of both images. D, Nomarski image of the same sagittal section asA–C. Kir4.1 was expressed in Müller cells and also in retinal pigment epithelial cells. Scale bar (shown in D), 10 μm. NFL, Nerve fiber layer; GCL, ganglion cell layer;IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer;ONL, outer nuclear layer; OS, outer segment of photoreceptor cell; RPE, retinal pigment epithelial cell layer.

Fig. 4.

Whole-mount immunohistochemistry of Kir4.1 in the retina. Whole retina was fixed with 4% paraformaldehyde and stained with anti-Kir4.1 antibody (green) and anti-vimentin antibody (red), as described in Figure 3. The levels of ONL (A–C) and GCL (D–F) were analyzed with confocal microscopy. Note that the expression of Kir4.1 (green) was clustered around ganglion cells. Scale bar (shown in F), 10 μm.

Fig. 5.

Immunogold electron microscopy of Kir4.1 in the retina. Ultra-thin sections were stained with anti-Kir4.1 antibody and anti-rabbit IgG coupled to colloidal gold particles.A–D, The electron microscopic images of the portions, as indicated in the top left schema. Positive gold particles were detected on the membranes of various regions of Müller cells. M, Müller cell;Ph, photoreceptor cell; Pe, pericyte;E, endothelial cell; CL, capillary lumen;BM, basement membrane; N, nucleus;CF, collagen fiber; VB, vitreous body;arrowheads, gold particles; arrows, collagen fibers. Scale bar (shown in D), 0.5 μm.

Fig. 7.

Expression and distribution of Kir4.1 in cultured cells from rat retina. A, Immunostaining of Kir4.1 (green) and vimentin (red) and whole-cell currents of cultured cells. Dissociated cells were cultured for 4 d on poly-d-lysine with 1 μminsulin (a–d), on laminin without insulin (e–h), and on laminin with 1 μm insulin (i–l). In i and k, Kir4.1 clustered on the membrane of cells. Scale bar (shown ink), 20 μm. Whole-cell current recordings were performed in these cells. The holding potential was −70 mV, and traces were elicited with voltage steps from −120 to +40 mV in 20 mV steps (inset). In l, large inward currents were recorded, whereas no inward currents were recorded in dand h. B, Expression of Kir4.1 mRNA in cultured cells. RT-PCR experiments of Kir4.1 were performed as shown in Figure 2C. Lane 1, Acutely dissociated Müller cells; lane 2, cells cultured on poly-d-lysine; lane 3, on poly-d-lysine in the presence of 1 μminsulin; lane 4, on laminin without insulin; lane 5, on laminin in the presence of 1 μm insulin;lane 6, control Kir4.1 cDNA. Insulin induced the expression of Kir4.1 mRNA.