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. 2000 Aug 1;20(15):5733-40.
doi: 10.1523/JNEUROSCI.20-15-05733.2000.

Genetic inactivation of an inwardly rectifying potassium channel (Kir4.1 subunit) in mice: phenotypic impact in retina

P Kofuji  1 , P Ceelen, K R Zahs, L W Surbeck, H A Lester, E A Newman
Affiliations

Genetic inactivation of an inwardly rectifying potassium channel (Kir4.1 subunit) in mice: phenotypic impact in retina

P Kofuji et al. J Neurosci. .

Abstract

The inwardly rectifying potassium channel Kir4.1 has been suggested to underlie the principal K(+) conductance of mammalian Müller cells and to participate in the generation of field potentials and regulation of extracellular K(+) in the retina. To further assess the role of Kir4.1 in the retina, we generated a mouse line with targeted disruption of the Kir4.1 gene (Kir4.1 -/-). Müller cells from Kir4.1 -/- mice were not labeled with an anti-Kir4.1 antibody, although they appeared morphologically normal when stained with an anti-glutamine synthetase antibody. In contrast, in Müller cells from wild-type littermate (Kir4.1 +/+) mice, Kir4.1 was present and localized to the proximal endfeet and perivascular processes. In situ whole-cell patch-clamp recordings showed a 10-fold increase in the input resistance and a large depolarization of Kir4.1 -/- Müller cells compared with Kir4.1 +/+ cells. The slow PIII response of the light-evoked electroretinogram (ERG), which is generated by K(+) fluxes through Müller cells, was totally absent in retinas from Kir4.1 -/- mice. The b-wave of the ERG, in contrast, was spared in the null mice. Overall, these results indicate that Kir4.1 is the principal K(+) channel subunit expressed in mouse Müller glial cells. The highly regulated localization and the functional properties of Kir4.1 in Müller cells suggest the involvement of this channel in the regulation of extracellular K(+) in the mouse retina.

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Figures

Fig. 1.
Fig. 1.
Targeted strategy for Kir4.1 gene interruption. A, Part of the wild-type Kir4.1 locus containing the coding exon (WT Allele), the targeting construct, and the targeted locus (Mutant Allele) are shown. PGKneo, Neomycin resistance gene.MC1tk, Thymidine kinase gene. The sizes of theXhoI fragments predicted to hybridize to the indicated diagnostic probe are shown. Restriction endonucleases:B, BamHI; Bl,BglII; E, EcoRI;H, HindIII; S,SacI; X, XhoI.B, Southern blot analysis ofXhoI-digested genomic DNA from wild-type (+/+), heterozygous (+/−), and homozygous mutant mice (−/−) with the diagnostic probe indicated in A. C, PCR analysis of tail genomic DNA PCR primers amplify a 634 bp fragment in the +/+ allele and a 383 bp fragment in the mutant allele. Notice the 634 bp fragment obtained from PCR analysis using tail DNA from +/+ mouse, the 383 bp fragment using tail DNA from −/− mouse, and both fragments from +/− mouse. As a control (C), the PCR analysis was performed in the absence of mouse genomic DNA
Fig. 2.
Fig. 2.
Analysis of Kir4.1 mRNA in retina. PCR analysis of total RNA extracted from P21 Kir4.1 +/+ and Kir4.1 −/− mice retinas and Kir4.1 +/+ brain. Kir-specific oligonucleotide pairs were used to determine the expression of the various Kir channels subunits in retina and brain. As a control (C), cDNA was replaced by water, and the PCR was performed using Kir5.1-specific oligonucleotide primers.
Fig. 3.
Fig. 3.
Histological analyses of the retinas of the Kir4.1 +/+ and Kir4.1 −/− mice. Cross-sections of mouse retinas from +/+ and −/− mutant mice (P11) were stained with hematoxylin and eosin.GCL, Ganglion cell layer; OPL, outer plexiform layer. Scale bar, 25 μm.
Fig. 4.
Fig. 4.
Immunohistochemistry of Kir4.1 in retinal whole mounts from Kir4.1 +/+ mouse (P18). Retinal whole mount stained with anti-Kir4.1 antibody (green) and anti-GS antibody (red). Confocal images were obtained in the ganglion cell layer (A), inner plexiform layer (B), inner nuclear layer (C), and outer nuclear layer (D). Note that the expression of Kir4.1 was clustered around neurons in the outer nuclear layer and along the blood vessels in the superficial and inner nuclear layer. Immunofluorescence in the blood vessels revealed by Texas Red donkey anti-mouse antibody (C) represents the binding of the secondary antibody to mouse IgG, because this immunoreactivity is not seen in retinas from mice transcardially perfused with PBS to wash out the blood before fixation. Scale bar, 20 μm.
Fig. 5.
Fig. 5.
Immunohistochemical analysis of Kir4.1 in retinal sections of Kir4.1 +/+ (+/+) (A,C, E) and Kir4.1 −/− (−/−) (B, D, F) P18 mice. Sections were double-stained with affinity-purified rabbit anti-rat Kir4.1 antibody, followed by FITC-conjugated anti-rabbit IgG (A, B, green), and monoclonal anti-GS antibody, followed by Texas Red-conjugated anti-mouse IgG (C, D,red). E, F, Superposition of images A, C and B,D, respectively. Scale bar, 20 μm. GCL, Ganglion cell layer; PhL, photoreceptor layer.
Fig. 6.
Fig. 6.
Input resistance of Müller cells in Kir4.1 +/+, Kir4.1 +/−, and Kir4.1 −/− mice. Traces show the displacement of the membrane potential produced by an injected current pulse. The traces are scaled so that the amplitudes of the responses reflect the relative input resistances of the three cells (voltage divided by injected current is equal for alltraces). Note that the membrane time constant of the −/− cell is substantially longer because of its increased membrane resistance. Amplitude of current pulses: 0.5 nA for +/+ and +/−; 0.1 nA for −/−.
Fig. 7.
Fig. 7.
The slow PIII response of the ERG in Kir4.1 +/+ and Kir4.1 −/− mice. Traces show the intraretinal ERG recorded between an electrode in the distal retina and one in the vitreous humor. In a Kir4.1 +/+ mouse, a transient negative b-wave and a slower positive slow PIII response are evident (+/+). When Ba2+ (0.4 mm) is added to the superfusate (+/+ plus Ba2+), the slow PIII response is abolished, whereas the b-wave remains primarily unchanged. Subtracting the trace in Ba2+from the other (+/+ difference) reveals the Ba2+-sensitive component of the ERG, the slow PIII response in isolation. In a Kir4.1 −/− mouse (−/−), the b-wave is present but the slow PIII response is absent. Addition of Ba2+ (−/− plus Ba2+) produces a small increase in the b-wave but no change in the response after decay of the b-wave, as illustrated by the differencetrace (−/− difference). The time course of the light stimulus is shown at the bottom.Dashed lines indicate prestimulus baseline levels.
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