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The Journal of Neuroscience, August 1, 2000, 20(15):5733-5740
Genetic Inactivation of an Inwardly Rectifying Potassium Channel
(Kir4.1 Subunit) in Mice: Phenotypic Impact in Retina
Paulo
Kofuji1,
Paul
Ceelen1,
Kathleen R.
Zahs2,
Leslie W.
Surbeck1,
Henry A.
Lester3, and
Eric A.
Newman1
Departments of 1 Neuroscience and
2 Physiology, University of Minnesota, Minneapolis,
Minnesota 55455, and 3 Division of Biology, California
Institute of Technology, Pasadena, California 91125
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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.
Key words:
Müller cell; inwardly rectifying potassium channel; Kir4.1; retina; null mouse; glia; electroretinogram; slow PIII
response; b-wave; astrocyte
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INTRODUCTION |
Radially oriented Müller glial
cells span the depth of the neural retina from the inner limiting
membrane at the vitreal surface to the subretinal space adjacent to the
photoreceptors (Newman, 1996 ; Newman and Reichenbach, 1996). As in
other glial cells, inwardly rectifying potassium (Kir) channels
constitute the main K+ conductance in the
plasma membrane of Müller cells (Newman, 1984). These channels
have high open probability near the resting membrane potential and
conduct currents better in the inward than in the outward direction
(Newman, 1993). There is extensive evidence that Kir channels in
Müller cells are vital elements for regulation of the
extracellular K+ concentration
([K+]o) in the
retina (Reichenbach et al., 1992; Newman and Reichenbach, 1996).
Although the Müller cell in the retina has served as an important
model for investigations of glial function, the molecular identity of
the Kir channels expressed in these cells is not yet known. Recently,
many Kir subunits have been cloned. Structural comparisons suggest that
they may be subdivided into six or seven subfamilies (Kir1-Kir6),
forming either homo-oligomeric or hetero-oligomeric channels (Doupnik
et al., 1995; Isomoto et al., 1997; Nichols and Lopatin, 1997).
Expression of various Kir subunits in heterologous systems has shown
that Kir2.1-3 and Kir4.1-2 subunits form
K+ channels with biophysical properties
that resemble the native channels in glial cells (Isomoto et al.,
1997).
Recently, Kir4.1 expression in oligodendrocytes and Bergman glia has
been reported (Takumi et al., 1995). In the retina, Kir4.1 is found
mainly in Müller cells (Ishii et al., 1997) with large enrichment
in the endfoot and perivascular processes (Nagelhus et al., 1999). The
expression of Kir4.1 in Müller cells and its restricted
subcellular distribution led to the suggestion that Kir4.1 mediates
[K+]o homeostasis
in the retina (Ishii et al., 1997; Nagelhus et al., 1999). However, the
possibility remains that other Kir subunits are expressed in these
glial cells because Kir4.1 is able to hetero-oligomerize with other Kir
subunits in heterologous expression systems (Fakler et al., 1996;
Pessia et al., 1996).
We have performed the genetic inactivation of the Kir4.1
gene in the mouse to determine the function of Kir4.1 in the retina. We
investigated the effect of lack of Kir4.1 on retinal organization, the
electrical properties of Müller cells, and the electroretinogram (ERG). Collectively, our results indicate that Kir4.1 is the primary K+ conductance of Müller cells, and
therefore it is likely to have an important role in the regulation of
[K+]o in the
mammalian retina.
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MATERIALS AND METHODS |
Preparation and characterization of Kir4.1 antibody.
Rabbit polyclonal antibodies were made against a synthetic peptide
REQAEKEGSALSVRISNV corresponding to the amino acids 362-379 in the
C terminus of mouse Kir4.1. A reactive cysteine was
included at the N terminus of the synthetic peptide to facilitate
its conjugation to keyhole limpet hemocyanin carrier. Affinity
purification of the antiserum was performed using a column with
immobilized Kir4.1 peptide. Bound anti-Kir4.1 antibody was eluted with
100 mM glycine, pH 2.5, and subsequently dialyzed
against PBS.
To determine the specificity of the affinity-purified anti-Kir4.1
antibody, we transfected COS cells as described previously (Doupnik et
al., 1997), with the following Kir subunits cloned into pcDNA3 vector
(Invitrogen, San Diego, CA): mouse Kir2.1 (kindly provided by Dr. L. Jan, University of California, San Francisco, CA), rat Kir3.1 (Dascal
et al., 1993), rat Kir4.1 (kindly provided by Dr. J. P. Adelman, Oregon Health Sciences University, Portland, OR), and rat
Kir6.2 (kindly provided by Dr. S. Seino, Chiba University, Chiba,
Japan). The immunocytochemistry was performed as described below
for the retinal sections.
PCR analysis. Total RNA from mouse retinas was extracted
using the RNaqueous kit (Ambion, Austin, TX) and treated with DNase I
(Ambion) to prevent contamination by genomic DNA. cDNAs were synthesized by priming with oligo-dT and using Superscript Reverse Transcriptase (Life Technologies, Rockville, MD). PCRs were
performed using the following primer pairs: Kir2.1 (GenBank
accession number AF021136), forward 5'-TTCTCCATCGAGACCCAGAC-3' and
reverse 5'-ATCTATTTCGTGAACGATAG-3'; Kir2.2 (GenBank accession number
X80417), forward 5'-TCCACGGCTTCATGGCAGCC-3' and reverse
5'-GTCCAGTGGGATGTACTCAC; Kir2.3 (GenBank accession number U11075),
forward 5'-CATCAAGCCCTACATGACAC-3' and reverse 5'-AACTCGTTCTCATAGCAGAA;
Kir4.1, forward 5'-TACAGTCAGACGACTCAGACA-3' and reverse
5'-GAAGCAGTTTGCCTGTCACCT-3'; and Kir5.1 (GenBank accession number
AB016197), forward 5'-GCTATTACGGAAGTAGCTACC-3' and reverse 5'-GGTGACACAGCGGTAACCGTA-3'. Each of the 35 cycles of PCR consisted of
1 min at 94°C, 1 min at 55°C, and 1 min at 72°C. Expected sizes for the PCR products were as follows: Kir2.1, 419 bp; Kir2.2, 361 bp;
Kir2.3, 461 bp; Kir4.1, 630 bp; and Kir5.1, 415 bp.
For the genotyping, DNA was isolated from mouse tails using
conventional methods (Sambrook et al., 1989), and the following pairs
were used for the PCR amplifications: Kir4.1, forward
5'-TGGACGACCTTCATTGACATGCAGTGG-3' and reverse
5'-CTTTCAAGGGGCTGGTCTCATCTACCACAT-3'; and neomycin resistance gene,
forward 5'-GATTCGCAGCGCATCGCCTTCTATC-3'. Each of the 35 cycles of PCR
consisted of 1 min at 94°C, 1 min at 65°C, and 1 min at 72°C. PCR
primers amplify a 634 bp fragment in the +/+ allele and a 383 bp
fragment in the mutant allele.
Generation of Kir4.1null (Kir4.1 /)
mouse line. The mouse Kir4.1 gene was isolated
(Sambrook et al., 1989) from a commercial mouse genomic
library derived from 129/SvEvTac mice DNA (Stratagene, La Jolla, CA)
using conventional methods. The gene encoding mouse Kir4.1 was cloned
from a mouse 129/SvEvTac genomic library. A 6 kb fragment, which
contained the entire coding sequence exon, was used for the
construction of the genomic targeting vector. The
HindIII-BglII fragment was deleted in the
targeting vector and replaced by the neomycin resistance gene (see Fig.
1A). An additional herpes simplex virus thymidine
kinase was also added for negative selection. The deleted fragment
encodes the amino acids 33-266 in the Kir4.1 deduced primary sequence
and thus contains the putative transmembrane domains and part of the C
terminus of the Kir4.1 polypeptide. For hybridization, we used a
fragment of the rat Kir4.1 cDNA kindly provided Dr. J. P. Adelman.
The targeting vector was electroporated into CJ7 embryonic stem (ES) cells using a Bio-Rad (Hercules, CA) Gene Pulser set at 230 V and 500 µF capacitance. A total of 25 µg of the linearized targeting vector
was used for 30 × 106 ES cells. ES
cells were submitted to G418 and FIAU selection 24 hr after the
electroporation. ES cell lines with targeted disruption of the
Kir4.1 gene were identified by Southern blot analysis of XhoI- or BglII-digested genomic DNA; the probes
used were the 5' flanking regions. Four clones from a total of 576 G418
and FIAU double-resistant colonies contained the desired targeted allele. Chimeric mice were generated by injecting ES cells from two of
these cell lines into C57BL/6J blastocysts and then implanting the
blastocysts into the uteri of pseudopregnant recipients. Mice were
maintained in a mixed Sv129 and C57BL/6 genetic background.
Histological and immunocytochemical analysis. Mice were
deeply anesthetized with intraperitoneal injection of pentobarbital or
exposure to CO2. Eyes were dissected, and the
cornea and lens were removed. For retinal slices, the resulting eyecups
were fixed in a 4% paraformaldehyde-PBS solution, pH 7.4, overnight
at 4°C. Eyecups were sectioned at 10 µm thickness with a cryostat,
placed onto slides coated with gelatin, and stored desiccated at
80°C. Sections were blocked and permeabilized with 5% donkey
serum-0.2% Triton X-100 in PBS for 1 hr at room temperature and then
incubated with the primary antibody for 2 hr. After being washed with
PBS, retinal sections were incubated for 2 hr with FITC-conjugated donkey anti-rabbit IgG and tetramethylrhodamine
isothiocyanate-conjugated donkey anti-mouse IgG (both secondary
antibodies diluted 1:100 in PBS; Jackson ImmunoResearch, West Grove,
PA). Sections were washed again with PBS and coverslipped with
Vectashield (Vector Laboratories, Burlingame, CA). Sections were imaged
with a Leica TCS4D confocal microscope, using a 50× oil
immersion lens. Optical sections were collected at 0.75-1.5 µm
intervals. Data are presented as projections of several optical images
onto a single plane. Metamorph software (Universal Imaging Corp., West
Chester, PA) was used to assign to each pixel in the final image the
maximum intensity value recorded at that x-y location from
among the stack of optical images. All control tissues were imaged with
identical parameters to enable direct visual comparison of staining.
For the retinal whole mounts, the retinas were isolated and fixed in a
4% paraformaldehyde PBS solution, pH 7.4, overnight at 4°C. Blocking
and permeabilization were performed for 2 hr in PBS solution containing
10% donkey serum-1% Triton X-100. Tissue was then incubated for 72 hr at 4°C in primary antibodies diluted in PBS, 5% powdered milk,
1% thimerosal, 0.01% anti-foam, and 1% Triton X-100. After PBS
rinses, the retinas were incubated for 48-72 hr at 4°C
with the secondary antibodies diluted in PBS-0.3% Triton X-100. After
PBS rinses, the retinas were mounted with Vectashield and imaged as
described for the retinal sections. The monoclonal antibodies
anti-glutamine synthetase (GS) and anti-glial fibrillary acid protein
(GFAP) were purchased from Chemicon (Temecula, CA).
Input resistance. Müller cell input resistance was
measured in current-clamp experiments. Recordings were made from
isolated retinal whole mounts as described previously (Zahs and Newman, 1997). Briefly, isolated retinas were digested in collagenase/dispase (2 mg/ml) and DNase (0.1 mg/ml) for 16 min at room temperature to
remove the vitreous humor and the basal lamina at the retinal surface.
Retinas were affixed to a polycarbonate filter membrane by suction
(vitreal side up), mounted in a perfusion chamber, and superfused with
oxygenated bicarbonate-buffered Ringer's solution at room temperature.
Recordings were made from the endfeet of Müller cells at the
vitreal surface of the retina, using patch electrodes in the whole-cell
recording mode. Patch electrodes were filled with a solution containing
Lucifer yellow to identify the cells. Labeled Müller cells were
identified by an endfoot at the retinal surface, a process projecting
distally through the inner plexiform layer (IPL), and a cell
body in the inner nuclear layer (INL). Input resistance was measured by
passing positive current pulses (0.02-0.5 nA) through the recording
pipette and measuring the resulting voltage displacement. A bridge
circuit was used to balance the voltage drop across the recording pipette.
Electroretinogram. The ERG was measured from the eyecup
preparation, as described previously (Newman and Bartosch, 1999). Briefly, the back half of the mouse eye was everted over a dome and
held in place by a thin plastic sheet with a hole in it. The plastic
sheet prevented the retina from detaching and electrically isolated the
vitreal surface of the retina from the sclera. The retinal surface of
the eyecup was superfused with oxygenated Ringer's solution at ~2.5
ml/min at 29°C.
The transretinal ERG was measured between the vitreous humor (positive)
and the sclera with a bandpass of direct current (DC) to 200 Hz.
We found that, at 29°C, the preparation remained stable for far
longer than had been reported previously (Newman and Bartosch, 1999)
for eyecups maintained at 37°C. The transretinal ERG b-wave declined
by only 25 ± 2% after 6 hr (n = 6). The
amplitude of the transretinal b-wave was measured from the trough of
the a-wave to the peak of the b-wave, ignoring oscillatory potentials.
The intraretinal ERG was measured between an intraretinal micropipette
(positive) and the vitreous with a bandpass of DC to 50 Hz. The pipette
(outer tip diameter of ~2.5 µm) was filled with Ringer's solution
and was advanced into the retina at a shallow angle (20°). The
pipette was advanced into the distal retina in which an intraretinal
a-wave was recorded. The pipette was then withdrawn until the a-wave
all but disappeared, indicating that the pipette tip lay near the outer
limiting membrane (Brown and Wiesel, 1961). The amplitude of the
intraretinal b-wave was measured from the prestimulus baseline to the
b-wave peak, ignoring oscillatory potentials. The amplitude of the slow
PIII response was measured from the baseline to the maximal
positive response after decay of the b-wave.
Solutions. The bicarbonate-buffered superfusion solution
used in electrophysiology experiments contained (in
mM): NaCl 117.0, KCl 3.0, CaCl2 2.0, MgSO4 1.0, NaH2PO4 0.5, dextrose 15.0, NaHCO3 32, and L-glutamate
0.01, and was bubbled with 5% CO2 in
O2. The pipette solution for whole-cell recording
contained (in mM): NaCl 5, KCl 120, CaCl2 1, MgCl2 7, EGTA 5, Na2ATP 5, HEPES 5, and 0.2% Lucifer yellow.
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RESULTS |
General phenotype of Kir4.1 null (Kir4.1 /) mice
Heterozygous animals (Kir4.1 +/) were identified by Southern
blots (Fig. 1B) and PCR
analysis (Fig. 1C) and were bred with each other to obtain
homozygous animals. Progeny of the heterozygous animals show no gross
discernable phenotypic differences in the first postnatal week.
Afterward, the homozygous animals were considerably smaller than their
littermates and displayed a higher rate of mortality. In addition, they
developed clear motor coordination deficits, which became obvious ~2
weeks after birth. The animals displayed awkward and jerky movements
and loss of balance and occasionally fell on their side. The general
phenotype of Kir4.1 / and its impact in the CNS will be
described in a separate report. Most of the homozygous mice survived up
to 3 weeks of age, which allowed us to study their retinal physiology
after eyelid opening [postnatal day 13 (P13) and P14]. All of the
experiments were performed with young mice between P16 and P21 unless
otherwise noted.
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Figure 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 the
XhoI 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 of
XhoI-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
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Analysis of Kir4.1 mRNA in retina
Wild-type (Kir4.1 +/+), heterozygous (Kir4.1 +/), and homozygous
(Kir4.1 /) animals were identified by PCR analysis from tail
biopsies. Mouse retinas were dissected, and total RNA was extracted and
subjected to reverse transcriptase-PCR amplification. PCR using
mouse-specific oligonucleotide primers for Kir2.1, Kir2.2, Kir2.3,
Kir4.1, and Kir5.1 showed the amplification of products of expected
sizes for retinas from Kir4.1 +/+ mice. Using brain RNA from Kir4.1 +/+
mice, we could also detect the expression for all tested Kir channel
subunits (Fig. 2). In contrast, Kir4.1 mRNA was not detected in retinas from Kir4.1 / mice using this assay, although other Kir channel subunits were detected after amplification using other Kir-specific oligonucleotide primers (Fig.
2). Thus, mRNA analysis in the retina shows the expected lack of Kir4.1
mRNA expression in retinas from genotyped Kir4.1 / mice.
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Figure 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.
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Cellular organization and distribution of Kir4.1 in retina
Cellular organization and general morphology were assessed in
retinal sections from Kir4.1 / and Kir4.1 +/+ mice stained with
hematoxylin and eosin. Figure 3 shows
light micrographs of retinal sections from P11 Kir4.1 +/+ and
Kir4.1/ mice. The retinas of the Kir4.1 / mice appeared to be
normally organized with no apparent disruption of the normal pattern of
lamination. Comparable results were obtained with older mice
(P18-P21).
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Figure 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.
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We raised an antibody against a peptide corresponding to a sequence in
the C terminus of Kir4.1 to determine the cellular and subcellular
distribution of Kir4.1 in the retina. The specificity of the
affinity-purified anti-Kir4.1 antibody was tested by transient transfection of COS cells with Kir2.1, Kir3.1, Kir4.1, and Kir6.2 cDNAs, followed by immunostaining using the anti-Kir4.1 antibody. As
expected, only cells transfected with Kir4.1 cDNA showed
immunostaining. Furthermore, this labeling was blocked upon
preadsorption of the anti-Kir4.1 antibody with a large excess of the
antigenic peptide (data not shown).
Double-immunofluorescence experiments were performed on retinal whole
mounts from Kir4.1 +/+ mice. Labeling for Kir4.1 was revealed with
secondary antibodies coupled to FITC. Müller cells were labeled
with an antibody against GS and visualized with secondary antibodies
coupled to Texas Red. Figure 4 shows
confocal optical sections in several retinal layers. At the inner
limiting membrane, staining for Kir4.1 was detected along with staining
for GS (Fig. 4A). Particularly intense staining for
Kir4.1 was seen along the superficial blood vessels. In addition, the
cell bodies of ganglion cells clearly showed a lack of labeling for
both antibodies. In the IPL (Fig. 4B), large punctate
staining was apparent for Kir4.1 and GS. These puncta presumably
reflects the expression of both proteins in the stalk of Müller
cells. In the inner nuclear layer (INL) (Fig. 4C),
overlapping expression of Kir4.1 and GS was seen surrounding neuronal
cell bodies and very prominent Kir4.1 clustering along the blood
vessels. Finally, in the outer nuclear layer (ONL) (Fig.
4D), Kir4.1 and GS were concentrated in Müller
cell processes surrounding the photoreceptors. Thus,
double-immunolabeling reveals the overlapping expression of GS and
Kir4.1 proteins, indicating the expression of Kir4.1 in the
Müller glial cell population. Kir4.1 labeling was particularly
intense in Müller cell processes contacting blood vessels. The
labeling pattern for Kir4.1 for the mouse retinas agrees closely with
the results reported for rat retinas (Nagelhus et al., 1999).
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Figure 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.
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Because amphibian astrocytes isolated from the optic nerve have
K+ channels preferentially localized to
their endfeet (Newman, 1986), we also asked whether Kir4.1 is expressed
in retinal astrocytes. In retinal whole mounts, immunofluorescence
labeling for GFAP revealed astrocytes in the superficial layers of
retina. However, we failed to detect the labeling of Kir4.1 in
astrocytes somata and proximal processes (data not shown). We could not
determine whether Kir4.1 is found on the astrocytic terminal processes
adjacent to blood vessels given the strong Kir4.1 immunoreactivity of
adjacent Müller cell processes.
Double-immunolabeling with the anti-Kir4.1 and anti-GS antibodies was
also performed on cross-sections of Kir4.1 +/+ and Kir4.1 /
retinas. In retinal sections from Kir4.1 +/+ mice (Fig.
5A,C,E), both antisera labeled all parts of Müller cells, staining the outer limiting membrane, the vitreal endfeet, and the main processes spanning the retina from the outer to the inner limiting membrane. Ganglion cells were clearly immunonegative for both anti-GS and anti-Kir4.1 antibodies (Fig. 5E). Strikingly
intense signal for Kir4.1 was found at the inner limiting membrane and
near blood vessels in the INL (Fig. 5A), confirming the
immunolabeling pattern obtained in retinal whole mounts. In contrast to
reports for albino rats (Kusaka et al., 1999a), we did not detect
Kir4.1 in the retinal pigment epithelium (RPE) (data not shown).
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Figure 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.
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In age-matched Kir4.1 / retinas, there was no detectable labeling
with the anti-Kir4.1 antibody (Fig. 5B), although the Müller cells appeared morphologically normal as revealed by
immunolabeling with the anti-GS antibody (Fig.
5D,F). Because Müller
cells express GFAP under some conditions, most notably in pathological
states (Bignami and Dahl, 1979), we also stained retinal sections with anti-GFAP antibody. For both the Kir4.1 +/+ and Kir4.1 / mouse retinas, the GFAP immunolabeling was confined to cells located in the
superficial layers in which the retinal astrocytes are located (data
not shown). Thus, the lack of Kir4.1 did not induce the upregulation of
GFAP in the Kir4.1 / Müller cells.
Müller cell membrane potential and input resistance
The electrophysiological properties of Müller cells in
Kir4.1 +/+, Kir4.1 +/, and Kir/ mice were measured using
whole-cell recordings in current-clamp mode. The resting membrane
potential and input resistance of cells are given in Table
1. In Kir4.1 +/+ and Kir4.1 +/ mice,
the resting membrane potential (85 and 88 mV, respectively) was in
the normal range for Müller cells (Newman, 1987). The resting
membrane potential in Kir4.1 / mice was significantly depolarized,
to 13 mV, indicating that Müller cell
K+ conductance was substantially reduced
in these animals. Input resistance measurements confirmed that the
K+ conductance of Müller cells in
mutant mice was reduced (Fig. 6, Table
1). The input resistance in Kir4.1 +/ mice (47 M ) was nearly
double that of Kir4.1 +/+ mice (25 M), whereas the input resistance
in Kir4.1 / mice (231 M) was more than nine times that of Kir4.1
+/+ animals.
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Figure 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 all
traces). 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 /.
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The membrane potential of Kir4.1 / cells was substantially
depolarized, and it is possible that the large input resistance measured in these cells is attributable to this depolarization. To
avoid this complication, we measured the input resistance of Kir4.1
/ cells that were artificially hyperpolarized by injection of a
constant negative current. Kir4.1 / cells hyperpolarized to an
average of 84 mV had an input resistance of 310 M, >12 times that
of Kir4.1 +/+ cells, demonstrating that the increased input resistance
of Kir4.1 / cells was not attributable to cell depolarization.
Electroretinogram
Several components of the ERG, including the slow PIII response,
are believed to be generated by K+ current
flow through Müller cells (Witkovsky et al., 1975; Bolnick et
al., 1979; Newman, 2000). These ERG components should be absent or
greatly reduced in mutants lacking the predominant Müller cell
K+ channel. We tested this prediction by
measuring the slow PIII response in Kir4.1 +/+ and Kir4.1 / mice.
The slow PIII response was monitored with an intraretinal electrode
positioned in the distal retina of the mouse eyecup. In Kir4.1 +/+
mice, a brief light flash evoked a transient negative intraretinal
b-wave followed by a slower positive response, the slow PIII (Fig.
7, +/+). Addition of the
K+ channel blocker
Ba2+ (0.4 mM) eliminated the
slow PIII, as expected, but spared the b-wave (Fig. 7, +/+ plus
Ba2+). When the intraretinal ERG recorded in
Ba2+ was subtracted from the control ERG
(Fig. 7, +/+ difference), the
Ba2+-sensitive component, the slow PIII,
was revealed in isolation.
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Figure 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 difference
trace (/ difference). The time course
of the light stimulus is shown at the bottom.
Dashed lines indicate prestimulus baseline levels.
|
|
The intraretinal ERG recorded from Kir4.1 / mice differed
qualitatively from that of Kir4.1 +/+ mice (Fig. 7, /). Although the b-wave was present, the slow PIII response was absent. After decay
of the b-wave, the ERG did not rise above the level of the prestimulus
baseline. Addition of Ba2+ (Fig. 7, /
plus Ba2+) produced little change in the
ERG, as shown in the difference trace (Fig. 7, /
difference), which is flat after an initial transient
reflecting a small Ba2+-induced change in
the amplitude of the b-wave.
Amplitudes of the intraretinal slow PIII and b-wave, as well as the
transretinal b-wave, are given in Table
2. The intraretinal slow PIII amplitude,
measured from the prestimulus baseline to the peak of the response, was
283 µV in Kir4.1 +/+ mice and 34 µV in Kir4.1 / mice,
demonstrating that the slow PIII response was completely absent in the
mutant. A negative slow PIII amplitude indicates that the response is
not present.
The b-wave response, in contrast, was not eliminated in the mutant
(Table 2). Measured intraretinally, the b-wave was 418 µV in Kir4.1
+/+ mice and 629 µV in Kir4.1 / mice. The larger intraretinal
b-wave measured in Kir4.1 / mice probably arose because of the
absence in these animals of the positive slow PIII, which normally
would offset the negative b-wave. This would explain why b-wave
amplitude in / animals was similar to the amplitude in +/+ animals
treated with Ba2+. Measured
transretinally, the b-wave was 142 µV in Kir4.1 +/+ and 117 µV in
Kir4.1 / mice. The smaller transretinal b-wave in Kir4.1 / mice
most likely reflects the smaller size of the eyes in mutant animals.
| |
DISCUSSION |
Distribution of Kir4.1 in the retina
Phylogenetic analysis shows that Kir channels encompass at least
13 members subdivided into six or seven structurally related subfamilies (Isomoto et al., 1997; Nichols and Lopatin, 1997). The
Kir4.1 cDNA was first isolated from a brain cDNA library and named
BIR10 (Bond et al., 1994). Subsequently, other groups isolated the same
cDNA and named it BIRK (Bredt et al., 1995) and
KAB-2 (Takumi et al., 1995). The widespread
distribution of Kir4.1 mRNA in brain has been noted (Bredt et al.,
1995), but some uncertainty persists as to whether Kir4.1 is expressed
predominantly in neuronal (Ma et al., 1998) or glial (Takumi et al.,
1995) populations. In the periphery, Kir4.1 is localized to several
cell types involved in transport of K+
ions. Thus, Kir4.1 has been shown to be expressed in the marginal (Hibino et al., 1997) or intermediate (Ando and Takeuchi, 1999) cells
in the cochlea stria vascularis. In this tissue, Kir4.1 may be
critically involved in the generation of the endocochlear potential.
Kir4.1 is also found in distal tubules in kidney (Ito et al., 1996) in
which these channels probably secrete K+
ions at the basolateral membrane.
Electrophysiological experiments demonstrated that the Kir channels are
not uniformly distributed on the plasma membrane of salamander
Müller cells (Newman, 1984). Instead, these channels are
concentrated in the proximal endfeet adjacent to the vitreous (Newman,
1984; Brew et al., 1986). Because of such non-uniform distribution of
K+ conductance, it has been hypothesized
that K+ ions released from depolarized
retinal neurons enter Müller cells and exit preferentially via
their endfeet into the vitreous fluid
("K+ siphoning ") (Newman et al.,
1984). In mouse and other species with vascularized retinas, Kir
conductances also cluster in the apical and perivascular processes of
Müller cells, suggesting additional
K+ siphoning to the blood vessels and
subretinal space (Newman and Reichenbach, 1996).
Ishii et al. (1997) were the first to describe the presence of Kir4.1
in rat retina and its restricted localization to Müller cells. In
their study, Kir4.1 was found to be expressed in Müller cells by
in situ hybridization and immunocytochemical techniques. Immunogold techniques were used to demonstrate that Kir4.1 is selectively expressed in the endfoot of the rat Müller cell and in those processes enveloping the blood vessels (Nagelhus et al., 1999). The present study confirms the cellular and subcellular distribution of Kir4.1 in mouse retinas using confocal
immunocytochemical techniques.
Thus, in both rat and mouse, the spatial distribution of Kir4.1 within
Müller cells is in remarkable agreement with the distribution of
K+ conductances determined by
electrophysiological techniques (Newman, 1987) and argues for the
involvement of this Kir subunit in K+
siphoning in the mammalian retina.
The highly restricted distribution of Kir4.1 within Müller cells
raises new questions concerning the molecular mechanisms responsible
for the localized distribution of these channels. It is interesting to
note that Kir4.1 and aquaporin-4 channels primarily colocalize
in Müller cells and that both proteins contain a PDZ
[Postsynaptic density-95 (PSD-95)/Discs large/zona occludens-1] domain recognition sequence that mediates binding to members of the
PSD-95/SAP90 (synapse-associated protein-90) protein family (Nagelhus
et al., 1998). Thus, PSD-95/SAP90-like proteins may play roles
in regulating the abundance and distribution of channels within glial
cells as has been demonstrated for synapses and other neuronal membrane
specializations (Sheng and Wyszynsky, 1997). It is also interesting to
speculate that such large macromolecular structures underlie the
orthogonal arrays of intramembranaceous particles (OAPs). These OAPs
are localized to Müller cell and astrocyte endfeet (Wolburg,
1995) and are thought to arise from aquaporin-4 (Yang et al., 1996). It
is not known, however, whether Kir4.1 also participates in the
formation of these particles. Freeze fracture studies in Kir4.1 /
Müller cells may answer some of these questions.
In retina, Kir4.1 has also been demonstrated in the RPE (Kusaka et al.,
1999a), which has been implicated in the transport of
K+ in the subretinal space (Immel and
Steinberg, 1986; Newman, 2000). The expression of Kir4.1 in the RPE was
demonstrated in albino but not in pigmented rats, possibly
because pigmentation in the RPE prevents the immunodetection of Kir4.1
in these cells (Kusaka et al., 1999a). Because our studies were
performed in pigmented mice, we could not verify the expression of
Kir4.1 to the RPE.
Biophysical and functional properties of Kir4.1
Several biophysical features of Kir4.1, as revealed by
heterologous expression studies, are similar to those reported for Kir
channels in Müller cells. For example, the single-channel conductance for Kir4.1 expressed in HEK 293 cells is 25 pS (Tada et
al., 1998). This value is similar to the conductance of 28 pS in
salamander Müller cells (Newman, 1993) and of 20 pS in monkey
Müller cells (Kusaka and Puro, 1997). In addition, Kir channels
in human Müller cells are activated by intracellular ATP (Kusaka
and Puro, 1997), as are Kir4.1 channels expressed in Xenopus
oocytes (Takumi et al., 1995). These features are, however, shared by
several members of the Kir channel superfamily (Isomoto et al., 1997;
Nichols and Lopatin, 1997), and thus a more detailed comparison of the
single-channel properties and modulation of Kir currents between Kir4.1
and the native Kir channels in Müller cells is warranted. In
particular, it will be interesting to determine whether Kir4.1 currents
in heterologous expression systems are modulated by protein kinases and
serum factors as reported for the native Kir currents in Müller
cells (Schwartz, 1993; Kusaka et al., 1998, 1999b). In rabbit
Müller cells, Kir channels of 60 and 105 pS (Nilius and
Reichenbach, 1988) have also been found, and the molecular identity of
these channels is presently unclear.
In our study, in situ measurements of the input resistance
of Müller cells shows that Kir4.1 / cells have an ~10-fold
higher resistance than Kir4.1 +/+ cells. These results
indicate that Kir4.1 is the predominant channel in mouse Müller
cells, comprising ~90% of the conductance of the cell at the
resting membrane potential. In addition, the input resistance of Kir4.1
+/ cells is almost precisely double that of Kir4.1 +/+ cells,
indicating that there is a gene dosage effect. Only one-half as many
channels are expressed in cells in which one of the two channel genes
is knocked out.
Although these measurements indicate that Kir4.1 underlies the main
conductance of Müller cells, we do not yet know whether other Kir
subunits are expressed in these cells. In particular, it is interesting
that PCR amplification of several Kir channels revealed the presence of
Kir5.1 in the retina. The Kir5.1 subunits are not functional by
themselves, but in heterologous expression systems, these subunits are
able to coassemble with Kir4.1 and modify their biophysical properties
(Pessia et al., 1996). Heteromultimeric Kir4.1/Kir5.1 channels display
a steeper degree of inward rectification and altered gating properties
(Pessia et al., 1996). It is conceivable that channel composition
varies among the different regions of Müller cells to allow
regional specialization of K+ conductance properties.
Electroretinogram
Several components of the ERG are believed to be generated by
Müller cells in response to light-evoked changes in
[K+]o (Newman,
2000). A case in point, the slow PIII response is thought to be
generated by a light-evoked decrease in
[K+]o in the
distal retina arising from photoreceptor hyperpolarization (Witkovsky
et al., 1975; Bolnick et al., 1979; Fujimoto and Tomita, 1979; Dick et
al., 1985). This
[K+]o decrease
establishes a K+ efflux from the distal
end of Müller cells and a K+ influx
into their proximal end (Newman and Odette, 1984). These K+ currents, flowing through Müller
cell K+ channels, establish a dipole
recorded as a positive field potential in the distal retina.
If the slow PIII response is actually generated by
K+ current flow through Müller
cells, one expects its absence in mutant lacking the predominant
Müller cell K+ channel. This proved
to be the case. The slow PIII, present in Kir4.1 +/+ mice, was
completely absent in Kir4.1 / animals. The response was also
missing in Kir4.1 +/+ preparations treated with
Ba2+ to block Müller cell
K+ channels. The results provide strong
support for a Müller cell origin of the slow PIII response.
The origin of another ERG component, the b-wave, has provoked
controversy over the past 50 years (Newman, 2000). The b-wave was
initially believed to be generated by retinal neurons (Tomita and
Funaishi, 1952), then attributed to a K+
current generated by Müller cells (Miller and Dowling, 1970; Newman, 1980; Newman and Odette, 1984), and, most recently, to retinal
neurons once again (Xu and Karwoski, 1994). Our results strongly
support a neuronal origin of the response. The amplitude of the b-wave
recorded in Kir4.1 / mice did not differ from the response in
Kir4.1 +/+ animals. This finding demonstrates conclusively that the
b-wave is not generated by K+ currents
flowing through Müller cells.
Conclusions
Together, our results indicate that Kir4.1 is the principal Kir
channel subtype in Müller cells. The Kir4.1/ mouse will facilitate future studies on the glial regulation of
[K+]o in the
mammalian retina.
| |
FOOTNOTES |
Received April 4, 2000; revised May 12, 2000; accepted May 17, 2000.
This work was supported by National Institute of Health Grants
GM-29836, MH-49176, EY04077, EY10383, and EY12949. We thank S. Pease,
M. Larabee, and T. Wu for expert technical help.
Correspondence should be addressed to Paulo Kofuji, Department of
Neuroscience, University of Minnesota, 6-145 Jackson Hall, 321 Church
Street SE, Minneapolis, MN 55455. E-mail:
kofuj001{at}tc.umn.edu.
| |
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High Susceptibility to Experimental Myopia in a Mouse Model with a Retinal ON Pathway Defect
Invest. Ophthalmol. Vis. Sci.,
February 1, 2008;
49(2):
706 - 712.
[Abstract]
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S. Machida, D. Raz-Prag, R. N. Fariss, P. A. Sieving, and R. A. Bush
Photopic ERG Negative Response from Amacrine Cell Signaling in RCS Rat Retinal Degeneration
Invest. Ophthalmol. Vis. Sci.,
January 1, 2008;
49(1):
442 - 452.
[Abstract]
[Full Text]
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R. G. Gregg, M. Kamermans, J. Klooster, P. D. Lukasiewicz, N. S. Peachey, K. A. Vessey, and M. A. McCall
Nyctalopin Expression in Retinal Bipolar Cells Restores Visual Function in a Mouse Model of Complete X-Linked Congenital Stationary Night Blindness
J Neurophysiol,
November 1, 2007;
98(5):
3023 - 3033.
[Abstract]
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B. Djukic, K. B. Casper, B. D. Philpot, L.-S. Chin, and K. D. McCarthy
Conditional Knock-Out of Kir4.1 Leads to Glial Membrane Depolarization, Inhibition of Potassium and Glutamate Uptake, and Enhanced Short-Term Synaptic Potentiation
J. Neurosci.,
October 17, 2007;
27(42):
11354 - 11365.
[Abstract]
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J. Ruiz-Ederra, H. Zhang, and A. S. Verkman
Evidence against Functional Interaction between Aquaporin-4 Water Channels and Kir4.1 Potassium Channels in Retinal Muller Cells
J. Biol. Chem.,
July 27, 2007;
282(30):
21866 - 21872.
[Abstract]
[Full Text]
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M. R. Metea and E. A. Newman
Signalling within the neurovascular unit in the mammalian retina
Exp Physiol,
July 1, 2007;
92(4):
635 - 640.
[Abstract]
[Full Text]
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M. R. Metea, P. Kofuji, and E. A. Newman
Neurovascular Coupling Is Not Mediated by Potassium Siphoning from Glial Cells
J. Neurosci.,
March 7, 2007;
27(10):
2468 - 2471.
[Abstract]
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A. Wurm, T. Pannicke, I. Iandiev, E. Buhner, U.-C. Pietsch, A. Reichenbach, P. Wiedemann, S. Uhlmann, and A. Bringmann
Changes in Membrane Conductance Play a Pathogenic Role in Osmotic Glial Cell Swelling in Detached Retinas
Am. J. Pathol.,
December 1, 2006;
169(6):
1990 - 1998.
[Abstract]
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I. Iandiev, O. Uckermann, T. Pannicke, A. Wurm, S. Tenckhoff, U.-C. Pietsch, A. Reichenbach, P. Wiedemann, A. Bringmann, and S. Uhlmann
Glial Cell Reactivity in a Porcine Model of Retinal Detachment
Invest. Ophthalmol. Vis. Sci.,
May 1, 2006;
47(5):
2161 - 2171.
[Abstract]
[Full Text]
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L. Y. Marmorstein, J. Wu, P. McLaughlin, J. Yocom, M. O. Karl, R. Neussert, S. Wimmers, J. B. Stanton, R. G. Gregg, O. Strauss, et al.
The Light Peak of the Electroretinogram Is Dependent on Voltage-gated Calcium Channels and Antagonized by Bestrophin (Best-1)
J. Gen. Physiol.,
April 24, 2006;
127(5):
577 - 589.
[Abstract]
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C. Neusch, N. Papadopoulos, M. Muller, I. Maletzki, S. M. Winter, J. Hirrlinger, M. Handschuh, M. Bahr, D. W. Richter, F. Kirchhoff, et al.
Lack of the Kir4.1 Channel Subunit Abolishes K+ Buffering Properties of Astrocytes in the Ventral Respiratory Group: Impact on Extracellular K+ Regulation
J Neurophysiol,
March 1, 2006;
95(3):
1843 - 1852.
[Abstract]
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T. Pannicke, I. Iandiev, A. Wurm, O. Uckermann, F. vom Hagen, A. Reichenbach, P. Wiedemann, H.-P. Hammes, and A. Bringmann
Diabetes Alters Osmotic Swelling Characteristics and Membrane Conductance of Glial Cells in Rat Retina
Diabetes,
March 1, 2006;
55(3):
633 - 639.
[Abstract]
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J. A. Brzezinski IV, N. L. Brown, A. Tanikawa, R. A. Bush, P. A. Sieving, M. H. Vitaterna, J. S. Takahashi, and T. Glaser
Loss of Circadian Photoentrainment and Abnormal Retinal Electrophysiology in Math5 Mutant Mice
Invest. Ophthalmol. Vis. Sci.,
July 1, 2005;
46(7):
2540 - 2551.
[Abstract]
[Full Text]
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D. De Saint Jan and G. L. Westbrook
Detecting Activity in Olfactory Bulb Glomeruli with Astrocyte Recording
J. Neurosci.,
March 16, 2005;
25(11):
2917 - 2924.
[Abstract]
[Full Text]
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S. C. Hebert, G. Desir, G. Giebisch, and W. Wang
Molecular Diversity and Regulation of Renal Potassium Channels
Physiol Rev,
January 1, 2005;
85(1):
319 - 371.
[Abstract]
[Full Text]
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D. K. Binder, M. C. Papadopoulos, P. M. Haggie, and A. S. Verkman
In Vivo Measurement of Brain Extracellular Space Diffusion by Cortical Surface Photobleaching
J. Neurosci.,
September 15, 2004;
24(37):
8049 - 8056.
[Abstract]
[Full Text]
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N. C. Connors, M. E. Adams, S. C. Froehner, and P. Kofuji
The Potassium Channel Kir4.1 Associates with the Dystrophin-Glycoprotein Complex via {alpha}-Syntrophin in Glia
J. Biol. Chem.,
July 2, 2004;
279(27):
28387 - 28392.
[Abstract]
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S. Uhlmann, A. Bringmann, O. Uckermann, T. Pannicke, M. Weick, E. Ulbricht, I. Goczalik, A. Reichenbach, P. Wiedemann, and M. Francke
Early Glial Cell Reactivity in Experimental Retinal Detachment: Effect of Suramin
Invest. Ophthalmol. Vis. Sci.,
September 1, 2003;
44(9):
4114 - 4122.
[Abstract]
[Full Text]
[PDF]
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D. Yang, D. K. MacCallum, S. A. Ernst, and B. A. Hughes
Expression of the Inwardly Rectifying K+ Channel Kir2.1 in Native Bovine Corneal Endothelial Cells
Invest. Ophthalmol. Vis. Sci.,
August 1, 2003;
44(8):
3511 - 3519.
[Abstract]
[Full Text]
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B. Bakall, L. Y. Marmorstein, G. Hoppe, N. S. Peachey, C. Wadelius, and A. D. Marmorstein
Expression and Localization of Bestrophin during Normal Mouse Development
Invest. Ophthalmol. Vis. Sci.,
August 1, 2003;
44(8):
3622 - 3628.
[Abstract]
[Full Text]
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D. M. Menichella, D. A. Goodenough, E. Sirkowski, S. S. Scherer, and D. L. Paul
Connexins Are Critical for Normal Myelination in the CNS
J. Neurosci.,
July 2, 2003;
23(13):
5963 - 5973.
[Abstract]
[Full Text]
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C. Dalloz, R. Sarig, P. Fort, D. Yaffe, A. Bordais, T. Pannicke, J. Grosche, D. Mornet, A. Reichenbach, J. Sahel, et al.
Targeted inactivation of dystrophin gene product Dp71: phenotypic impact in mouse retina
Hum. Mol. Genet.,
July 1, 2003;
12(13):
1543 - 1554.
[Abstract]
[Full Text]
[PDF]
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D. Yang, A. Pan, A. Swaminathan, G. Kumar, and B. A. Hughes
Expression and Localization of the Inwardly Rectifying Potassium Channel Kir7.1 in Native Bovine Retinal Pigment Epithelium
Invest. Ophthalmol. Vis. Sci.,
July 1, 2003;
44(7):
3178 - 3185.
[Abstract]
[Full Text]
[PDF]
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R. G. Gregg, S. Mukhopadhyay, S. I. Candille, S. L. Ball, M. T. Pardue, M. A. McCall, and N. S. Peachey
Identification of the Gene and the Mutation Responsible for the Mouse nob Phenotype
Invest. Ophthalmol. Vis. Sci.,
January 1, 2003;
44(1):
378 - 384.
[Abstract]
[Full Text]
[PDF]
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N. C. Connors and P. Kofuji
Dystrophin Dp71 Is Critical for the Clustered Localization of Potassium Channels in Retinal Glial Cells
J. Neurosci.,
June 1, 2002;
22(11):
4321 - 4327.
[Abstract]
[Full Text]
[PDF]
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D. C. Marcus, T. Wu, P. Wangemann, and P. Kofuji
KCNJ10 (Kir4.1) potassium channel knockout abolishes endocochlear potential
Am J Physiol Cell Physiol,
February 1, 2002;
282(2):
C403 - C407.
[Abstract]
[Full Text]
[PDF]
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C. B. Ransom, J. T. O'Neal, and H. Sontheimer
Volume-Activated Chloride Currents Contribute to the Resting Conductance and Invasive Migration of Human Glioma Cells
J. Neurosci.,
October 1, 2001;
21(19):
7674 - 7683.
[Abstract]
[Full Text]
[PDF]
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C. Neusch, N. Rozengurt, R. E. Jacobs, H. A. Lester, and P. Kofuji
Kir4.1 Potassium Channel Subunit Is Crucial for Oligodendrocyte Development and In Vivo Myelination
J. Neurosci.,
August 1, 2001;
21(15):
5429 - 5438.
[Abstract]
[Full Text]
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P. A. Sieving, M. L. Fowler, R. A. Bush, S. Machida, P. D. Calvert, D. G. Green, C. L. Makino, and C. L. McHenry
Constitutive "Light" Adaptation in Rods from G90D Rhodopsin: A Mechanism for Human Congenital Nightblindness without Rod Cell Loss
J. Neurosci.,
August 1, 2001;
21(15):
5449 - 5460.
[Abstract]
[Full Text]
[PDF]
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E. Solessio, K. Rapp, I. Perlman, and E. M. Lasater
Spermine Mediates Inward Rectification in Potassium Channels of Turtle Retinal Muller Cells
J Neurophysiol,
April 1, 2001;
85(4):
1357 - 1367.
[Abstract]
[Full Text]
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M. Francke, F. Faude, T. Pannicke, A. Bringmann, P. Eckstein, W. Reichelt, P. Wiedemann, and A. Reichenbach
Electrophysiology of Rabbit Muller (Glial) Cells in Experimental Retinal Detachment and PVR
Invest. Ophthalmol. Vis. Sci.,
April 1, 2001;
42(5):
1072 - 1079.
[Abstract]
[Full Text]
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TOP
ABSTRACT
INTRODUCTION
