1 Vision Science Research Center and 2 Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294
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ABSTRACT |
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Some members of the epithelial
Na+ channel/degenerin (ENaC/DEG) family of ion channels
have been detected in mammalian brain. Therefore, we examined the RNA
and protein expression of these channels in another part of the central
nervous system, the rabbit retina. We next sought to demonstrate
physiological evidence for an amiloride-sensitive current in
Müller glia, which, on the basis of a previous study, are thought
to express -ENaC (Golestaneh N, de Kozak Y, Klein C, and Mirshahi M. Glia 33: 160-168, 2001). RT-PCR of retinal RNA revealed
the presence of
-,
-,
-, and
-ENaC as well as acid-sensing
ion channel (ASIC)1, ASIC2, ASIC3, and ASIC4. Immunohistochemical
localization with antibodies against
-ENaC and
-ENaC showed
labeling in Müller cells and neurons, respectively. The presence
of
-ENaC,
-ENaC, and ASIC1 was detected by Western blotting.
Cultured Müller cells were whole cell patch clamped. These cells
exhibited an inward Na+ current that was blocked by
amiloride. These data demonstrate for the first time both the
expression of a variety of ENaC and ASIC subunits in the rabbit retina
as well as distinct cellular expression patterns of specific subunits
in neurons and glia.
epithelial sodium channel; amiloride; retina; reverse transcriptase-polymerase chain reaction; patch clamp
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INTRODUCTION |
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MEMBERS OF
the epithelial Na+ channel/degenerin superfamily (ENaC/DEG)
include the ion-gated, amiloride-blockable channels located in apical
membranes of many salt-reabsorbing epithelia. The cloning of ENaCs has
revealed the identity of (6, 22)-,
-,
(7)-, and
(38)-subunits. Other family
members include the acid-sensing ion channels (ASICs) or brain
Na+ channels (BNaCs) (2, 15, 31, 37, 39),
Caenorhabditis elegans DEGs (10), the
Phe-Met-Arg-Phe-NH2-gated channel (FaNaC) of
Helix aspera (21), as well as Pickpocket (PPK)
and Ripped Pocket (RPK) genes of Drosophila melanogaster
(1).
Characteristic properties of the proteins in this superfamily include two transmembrane domains, a large extracellular loop with numerous glycosylation sites, and cytoplasmic amino and carboxy termini. Although the actual channel stoichiometry is debated, evidence exists in favor of heteromultimers (7, 13, 14).
ENaC expression has been demonstrated in a variety of tissues including
the retina. Matsuo (23) found expression of the -subunit in the retinal pigment epithelium (RPE), ganglion cells, inner nuclear layer (INL), and outer nuclear layer (ONL) of rat retina
by in situ hybridization. Mirshahi et al. (28)
used a polyclonal antibody that recognizes a region of the
-subunit from rat colon and localized it in outer and inner segments of rods and
cones, ganglion cells, inner plexiform layer (IPL), outer plexiform
layer (OPL), INL, ONL, and RPE of rat and human retina. Mirshahi et al.
(26) revealed
-ENaC expression in cultured bovine
retinal pigment epithelium, and Golestaneh et al. (17) demonstrated expression of
-ENaC in cultured rat Müller glia.
Our aim was twofold. First, we wanted to expand on the previous work
that localized the -subunit by determining the pattern of expression
in Müller cells. These cells have distinct patterns of expression
of other ion channels and transporters. For example, inward-rectifying
K+ channels facilitate the exchange of K+
between extracellular retinal space and the vitreous (29). One role of amiloride-sensitive channels is the regulation of Na+ homeostasis. Thus we hypothesized that the subcellular
localization of ENaC in Müller cells might provide evidence to
support the notion that they are involved in Na+
homeostasis in the retina. Because there are no physiological studies
of amiloride-sensitive channels in the retina, we performed whole cell
patch-clamp analysis with cultured Müller cells. We present here
for the first time direct evidence for a functional ENaC channel in
rabbit Müller glia by patch-clamp analysis.
Second, we sought to identify other subunits of this superfamily in the
rabbit retina and to determine which cell types express specific
subunits. Unique polyclonal antibodies against -ENaC and
-ENaC
were used to determine the distribution of these subunits in the rabbit
retina by immunohistochemistry. In addition, double labeling with known
retinal cell markers was used to identify specific retinal cell types
expressing ENaCs. We also confirmed the presence of
-ENaC,
-ENaC,
and ASIC1 proteins by Western blotting. Finally, subunit-specific
primers were used in RT-PCR analysis of rabbit retinal RNA to test the
presence of ENaC and ASIC mRNA.
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EXPERIMENTAL PROCEDURES |
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All animal procedures followed institutional guidelines for the care and use of laboratory animals and the American Physiological Society's "Guiding Principles for Research Involving Animals and Human Beings."
Immunochemicals.
Polyclonal antibodies were raised in rabbit against the peptides
KGNKREEQGLGPE of human -ENaC (
-hENaC), GEKYCNNRDF of human
-ENaC (
-hENaC), and DVKRHNPCESLRGHP of human ASIC1. Antisera were
protein A purified and used at a dilution of 1:100. Mouse anti-tyrosine
hydroxylase (TH) (Incstar, Stillwater, MN) at 1:800 was used as a
marker for dopaminergic amacrine cells, goat anti-calretinin (Chemicon,
Temecula, CA) at 1:800 for ganglion cells and AII amacrine cells, mouse
anti-calbindin (Sigma, St. Louis, MO) at 1:800 for on-cone bipolar
cells and horizontal cells, mouse anti-protein kinase C (PKC; Amersham,
Little Chalfont, UK) at 1:100 for rod bipolar cells, goat anti-choline
acetyltransferase (ChAT; Chemicon) at 1:200 for cholinergic cells, and
mouse anti-vimentin (Dako, Carpinteria, CA) at 1:200 for Müller
cells. Secondary antibodies (Jackson ImmunoResearch, West
Grove, PA) included fluorophore-conjugated donkey anti-rabbit, donkey
anti-mouse, and donkey anti-goat antibodies. Normal rabbit IgG was
purchased from Jackson ImmunoResearch.
Characterization of -hENaC,
-hENaC, and ASIC1 antibodies.
Several experiments were performed to confirm the specificity of our
anti-
-hENaC, anti-
-hENaC, and anti-ASIC1 antibodies. First,
Madin-Darby canine kidney (MDCK) cells expressing rabbit
-ENAC
(rENaC) (R. G. Morris and J. A. Schafer, unpublished observations) were used as a positive control for Western blotting. Cell lysates from either MDCK cells expressing
-rENaC or
nonexpressing parental MDCK cells were subjected to 8% SDS-PAGE and
Western blotting. Blots were probed with 1:200 anti-
-hENaC or
anti-
-hENaC. Second, we used the method of Jovov et al.
(20) for in vitro transcription and translation and
immunoprecipitation of
- and
-rENaC with our anti-ENaC
antibodies. Both total in vitro translated protein and
immunoprecipitates were subjected to SDS-PAGE and autoradiography. To
test the specificity of anti-ASIC1, a lysate of human glioblastoma
cells, U251-MG, was subjected to 7.5% SDS-PAGE and Western blotting.
U251-MG cells express ASIC1 (D. J. Benos, unpublished observation)
and thus were an appropriate positive control. Blots were
probed with either anti-ASIC1 or normal rabbit IgG.
Immunohistochemistry of retinal sections. Eyes were enucleated from rabbits killed by an overdose of pentobarbital sodium (Socumb; Butler, Columbus, OH), and the retinas were quickly isolated. Retinas were fixed by several methods: periodate-lysine-1% paraformaldehyde (1% PLP) for 2 h at room temperature without vitreous, 2% paraformaldehyde at 4°C for 4 h both with and without vitreous, or 0.01% picric acid-2% paraformaldehyde at 4°C for 10 min with a 50-min postfix of 2% paraformaldehyde at 4°C without vitreous. Retinas were then cryoprotected and embedded in 50% optimum cutting temperature (OCT) solution (Tissue-Tek, Torrance, CA) and 50% Aquamount (Lerner Laboratories, Pittsburgh, PA) and cryosectioned in 10-µm slices.
Sections were stained with QuickStain (American Histology MasterTech Scientific, Lodi, CA) as an equivalent hematoxylin and eosin stain to visualize the retinal cellular layers. Conventional immunohistochemistry methods were used. Briefly, sections were blocked with a solution of 10% normal serum from the host of the secondary antibody, PBS, 0.3% Triton X-100, and 2% nonfat dry milk (NFDM) for 1 h at room temperature. All primary antibodies were diluted in PBS-Triton X-100-2% NFDM and 5% normal serum from the secondary antibody with an overnight incubation at 4°C. All secondary antibodies were diluted in PBS-Triton X-100-NFDM with a 1-h incubation at room temperature and protected from light. Slides were washed with PBS between steps and before coverslipping with Permafluor (Immunon, Pittsburgh, PA). Sections were viewed with fluorescence light microscopy or with a Leica TCS SP confocal laser scanning microscope. Double-labeled slides were scanned sequentially to avoid artifactual bleed-through. Images were imported into Adobe Photoshop for figure modification.Müller cell isolation and culture. We followed the protocol of McGillem et al. (24) for the isolation and culture of rabbit Müller cells. Briefly, adult rabbits were killed and the eyes were enucleated and quickly cleaned. The cornea, iris, anterior segment, and vitreous were removed. The retina was hemisected and submerged in DMEM (Life Technologies, Rockville, MA) and EDTA (Sigma) for several minutes. The retinas were then gently teased away from the choroid and incubated in 5 ml of the above solution with 130 U of papain (Worthington, Lakewood, NJ) and 4.5 mg of cysteine (Sigma). After the enzymatic digestion, the retinas were placed in DMEM with 10% fetal bovine serum (FBS; Life Technologies) with 0.1 mg/ml DNase (Sigma) and triturated through a pipette. The dissociated cell preparation was placed in culture flasks or dishes coated with poly-L-ornithine (Sigma) and incubated overnight to allow the Müller cells to settle. The next day, the flasks were washed and exchanged with plating medium (DMEM-10% FBS) and the unattached cells and debris were washed away. The result was a 90-95% pure population of Müller cells. Cells were maintained in a 5% CO2 37°C humidified incubator. Culture medium was changed every 2-3 days, and cells were split 1:2 with 0.25% trypsin-1 mM EDTA (Life Technologies) for immunohistochemistry or mechanically for patch clamping. One percent antibiotic-antimycotic solution (Life Technologies) was used after 1 wk as needed.
Immunohistochemistry of cultured Müller cells.
This procedure was completed at room temperature on cells plated in
12-well plates on coverslips coated with poly-L-ornithine. All incubations except fixation were performed while shaking the plates. Washes with 0.1 M phosphate buffer (PB) occurred between steps.
Culture medium was aspirated, and cells were washed three times with
0.1 M PB and fixed with 2% PLP for 1 h. Permeabilization and
blocking were carried out for 30 min with 0.3% Triton X-100-PBS with
10% normal serum from the host of the secondary antibody. Cells were
incubated in anti--hENaC or anti-vimentin, diluted 1:100 and 1:500,
respectively, plus 5% normal serum for 1 h to overnight.
Fluorophore-conjugated secondary antibodies with 5% normal serum were
used for 1 h and protected from light. Coverslips were mounted on
slides with Permafluor and viewed with fluorescence light microscopy or
confocal microscopy.
Retinal protein extraction and Western blotting. Freshly enucleated rabbit eyes were submerged in ice-cold saline before dissection. The cornea, iris, anterior segment, and vitreous were removed, and the eyecups were placed in Cellgro-free medium (Mediatech, Herndon, VA) for several minutes at 37°C to allow the retina to detach from the RPE. The retinas were teased away and homogenized on ice in radioimmunoprecipitation (RIPA) buffer [1% Triton X-100, 0.1% SDS, 1% deoxycholate, 10 mM sodium phosphate, 150 mM NaCl, 2 mM EDTA containing 1 mM phenylmethylsulfonyl fluoride (Sigma), 10 µg/ml leupeptin (Sigma), and 10 µg/ml pepstatin A (Sigma)]. The homogenate was spun, and the supernatant was precleared with nonimmunized rabbit IgG. Three parts precleared protein sample was incubated in one part SDS sample buffer, boiled at 100°C for 10 min, vortexed, and spun. The supernatant was cooled on ice before loading on the gels.
Standard electrophoresis and blotting protocols were followed. Briefly, protein was run on 5-15% gradient SDS-PAGE mini gels with 4% stacking gels for ~1 h at 150 V in a BioRad Minisubcell apparatus (Hercules, CA). Gels were transferred onto polyvinylidene fluoride (PVDF), treated with 2% NFDM-TBS-Tween, and probed with the appropriate anti-ENaC antibody. An anti-rabbit secondary antibody was conjugated to horseradish peroxidase (Jackson ImmunoResearch), and visualization was performed with chemiluminescent reagents (Amersham Pharmacia Biotech, Piscataway, NJ). Controls included substitution of normal rabbit IgG for primary antibodies and/or omission of the primary antibody.RNA extraction and RT-PCR. Freshly enucleated rabbit eyes were quickly submerged in RNAlater (Ambion, Austin, TX), and the retinas were isolated while minimizing the amount of RPE. Subsequent RNA isolation was performed with Stratagene's Absolutely RNA RT-PCR Miniprep kit (La Jolla, CA), omitting liquid nitrogen freezing. Total RNA was run on a 1% agarose-formaldehyde gel to check the integrity of the RNA.
RT-PCR was performed with a one-step RT-PCR kit from Qiagen (Valencia, CA) with the subunit-specific ENaC primers (Life Technologies) given in Table 1. Each reaction mixture consisted of 1.2 µg of RNA, 400 µM deoxyribonucleotide triphosphates, 5-10 U of RNAsin (Promega, Madison, WI), 2 µl of enzyme solution, and each primer at 0.6 µM. Reactions with water substituted for RNA were used as negative controls. The reactions were amplified with the GeneAmp PCR System 2400 (Perkin Elmer, Boston, MA). Reverse transcription was carried out at 50°C for 30 min followed by amplification of the cDNA at 95°C for 15 min followed by 35 cycles of 94°C for 1 min (denaturing), 52-63°C for 1.5 min (annealing), and 72°C for 2 min (extension) followed by a 72°C final extension for 20 min. The RT-PCR products were run on a 2% agarose gel with ethidium bromide and visualized with Eagle Eye II (Stratagene).
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Sequencing. RT-PCR products were gel purified with the QIAquick gel extraction kit and cloned into pCR2.1-TOPO vector with the TOPO TA cloning kit (Invitrogen, Carlsbad, CA). Subsequent cDNA extraction was performed with Promega Wizard Plus Minipreps DNA extraction kit. Sequencing was performed at the Iowa State University DNA Sequencing Facility.
Whole cell patch-clamp studies.
Primary and first-passage Müller cells were mechanically scraped
from culture dishes, and plating medium was replaced with serum-free
RPMI 1640 (Life Technologies). The cells were then placed in the
perfusion chamber and allowed to adhere to the bottom before recording.
Micropipettes were constructed with a two-stage micropipette puller.
These tips had an internal diameter of 0.5 µm. When they were filled
with a solution containing (in mM) 100 potassium gluconate, 30 KCl, 10 NaCl, 20 HEPES, EGTA, and 4 ATP, with <10 nM free Ca2+,
tip resistance was 1 M. Pipettes were mounted in a holder and connected to the head stage of an Axon 200A patch-clamp amplifier affixed to a three-dimensional micromanipulator system attached to the
microscope. The pipettes were touched to the cells, and slight suction
was given. Seal resistance was continuously monitored (Nicolet model
300 oscilloscope) with 0.1-mV electrical pulses from an electrical
pulse generator. After forming seals of resistance >1 G
, a second
suction pulse was given to form the whole cell configuration.
Typically, capacitance was between 11 and 47 pF.
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RESULTS |
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Figure 1 demonstrates the
specificity of anti--hENaC and anti-
-hENaC antibodies. Figure
1A shows that anti-
-hENaC recognized a glycosylated form
of the protein at 100 kDa in MDCK cells stably expressing
-rENaC but not in untransfected cells (Fig. 1B) by
Western blotting. Figure 1C is the autoradiograph of in
vitro translated
-rENaC, and Fig. 1D is the
anti-
-hENaC immunoprecipitated protein of ~75-80 kDa. Figure
1, E and F, represents the Western blots of
expressing and nonexpressing MDCK cells probed with anti-
-ENaC, which recognized a protein of 100 kDa in expressing cells. Figure 1G is the autoradiograph of in vitro translated
-rENaC,
and the immunoprecipitate of the same protein with anti-
-hENaC
antibody is shown in Fig. 1H. Anti-
-hENaC also pulled
down a protein of ~75-80 kDa.
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Figure 2 includes representative images
of -rENaC MDCK cells immunolabeled with anti-
-hENaC (Fig.
2A), anti-
-hENaC (Fig. 2B), and normal rabbit
IgG as a control (Fig. 2C). The anti-ENaC antibodies showed
brighter labeling at the cell membrane with some filamentous staining.
Normal rabbit IgG did not specifically label these cells.
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Figure 3 shows the Western blot of
U251-MG cells probed with anti-ASIC1 (Fig. 3A), normal
rabbit IgG (Fig. 3B), and the secondary antibody only (Fig.
3C). Anti-ASIC1 recognized a protein of ~71 kDa that is
likely the glycosylated form of ASIC1. This protein was not recognized
by the IgG and secondary antibodies.
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Figure 4 depicts the immunohistochemical
labeling with anti--hENaC and anti-
-ENaC in retinal sections.
Localization of
-ENaC was specific for Müller cells (Fig.
4A), whereas
-ENaC localization occurred at the ganglion
cell layer (GCL) and INL (Fig. 4B). Normal serum did not
show specific labeling (Fig. 4C). Figure 4D is a section stained with QuickStain for comparison.
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The pattern of labeling with anti--hENaC is shown in Fig.
5. Labeling was specific for Müller
cells and was concentrated in, but not restricted to, the inner half of
the cell including the cell body and the endfoot processes (Fig.
5A). Vimentin localized to the entire cell including the
thin processes that extend to the outer limiting membrane (Fig.
5B). As a result, the overlay image clearly demonstrates the
spatial localization of
-ENaC (Fig. 5C). These findings
were consistent regardless of the type of fixation. After ~1 wk in
culture, Müller cells in poly-L-ornithine-coated dishes developed a fibroblast-like morphology. Some cells appeared multinucleate and extended processes to neighboring cells. Despite this
altered phenotype, these cells displayed
-hENaC (Fig. 5D) and vimentin (Fig. 5E) immunoreactivity. Figure
5F shows the overlay image. Vimentin staining appeared
filamentous with nuclear staining, and
-hENaC staining also appeared
filamentous with brighter staining around the cell perimeter. Some cell
nuclei were also stained with this antibody.
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Anti--hENaC showed clear localization in the GCL and diffuse
labeling in the INL. To identify the specific cell types that express
-ENaC, markers including calbindin, PKC, TH, ChAT, and calretinin
were used for double labeling. Anti-
-hENaC colocalized with the
amacrine and ganglion cell markers predominantly in the cell bodies but
not their processes (Fig. 6,
C-E). Double labeling was
sometimes seen with calbindin for on-cone bipolar cells and horizontal
cells and with PKC for rod bipolar cells (Fig. 6, A and
B). Interestingly, ganglion cell and amacrine cell labeling was consistently brighter than bipolar cell labeling.
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Figure 7 shows that on Western blots of
total retinal protein, anti--hENaC specifically recognized proteins
at ~75 and 45 kDa (Fig. 7A), anti-
-hENaC recognized
proteins at 123, 83, 73, 45, and 40 kDa (Fig. 7B), and
anti-ASIC1 recognized 120-, 70-, 43-, and 36-kDa proteins (Fig.
7C). None of these proteins was recognized by normal rabbit
IgG (Fig. 7D) or the secondary antibody (Fig.
7E).
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RT-PCR analysis demonstrated the presence of -,
-,
-, and
-ENaC and ASIC1-4. Primers designed from human sequences were generally successful and gave the anticipated products. However, the
primers for
-ENaC and ASIC3 produced additional nonspecific products
whereas human primers for
-ENaC gave no product. As a result, we
designed rabbit-specific primers for
-ENaC. These primers produced
only the predicted product. Figure 8
shows the agarose gel of the RT-PCR products for each subunit. Direct
DNA sequencing of the cloned RT-PCR products for
-,
-,
-, and
-ENaC and ASIC1-4 confirmed their identities.
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Whole cell patch clamp was used to demonstrate directly
amiloride-sensitive Na+ current consistent with
Na+ current known to be conducted by ENaC (4).
In the basal state, cells exhibited "ragged" inward currents that
were blocked after 100 µM amiloride perfusion. Figure
9 shows a representative recording with
an inward current that was blocked by 100 µM amiloride.
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DISCUSSION |
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Our study provides direct evidence for the existence of
amiloride-sensitive Na+ channels in both neurons and glia
of the rabbit retina. At the RNA level, all known ENaC and ASIC
subunits appear to be expressed in these retinas. Unique antibodies
against - and
-ENaC show distinct localization. We also presented
several lines of evidence demonstrating the specificity of our
anti-
-hENaC, anti-
-hENaC, and anti-ASIC1 antibodies. Furthermore,
we present the first physiological data demonstrating the presence of
an amiloride-sensitive current in Müller glia.
A previous study reported immunohistochemical evidence for the
expression of -ENaC in cultured rat Müller cells
(17). Our anti-
-hENaC antibody also localized to rabbit
Müller cells. In frozen sections, the most prominent staining was
at the inner portion of the cells and their endfeet. This pattern of
ion channel expression is reminiscent of voltage-gated,
inward-rectifying K+ channels whose function is to siphon
K+ from the extracellular retinal space to the vitreous
(29). It may be that ENaC performs a similar function for
Na+. In cultured cells, anti-
-ENaC staining appeared
filamentous, which agrees with Golestaneh et al. (17). As
in vivo, cells expressing
-ENaC also expressed vimentin and thus
confirmed that they were appropriate for patch-clamp analysis. The
colocalization with vimentin can be attributed to the association of
ENaC with cytoskeletal proteins such as actin (3, 8, 17,
35). Our evidence suggests that Müller cells do not
express an ENaC channel composed of
-,
-, and
-subunits.
Müller cells may, in fact, represent the first example of an
-ENaC homomer in vivo, or the channel composition may include the
ASIC subunits or a novel subunit.
Our data differ from the immunolocalization patterns of -ENaC
reported in rat (28). Possible reasons for this include
differences in the
-ENaC antibodies, species differences, or tissue
fixation. We demonstrated that our antibodies were specific. However,
species differences probably do not account for the conflicting
findings, either. We screened several different tissue fixation
protocols, and we believe that paraformaldehyde gave the best labeling
that was above normal IgG background. Because fixations affect the availability of epitopes for antibodies, this is the most likely reason
for the observed differences in localization patterns. We observed
faint labeling with picric acid fixation, although it was not
appreciably above normal rabbit IgG background.
The -subunit may serve
-like functions in the retina because it
is found in brain and the characterization of this subunit revealed
biochemical and physiological similarities with
-ENaC but not
-
or
-ENaC (38). The
-subunit is important in forming the channel pore (32), anchoring the channel complex to
the cytoskeleton (12), and conferring H+
sensitivity (9).
Anti--ENaC labeling was restricted to cell bodies in the GCL and
INL. We observed colocalization of
-ENaC with several amacrine cell
markers and a ganglion cell marker, and
-ENaC also appeared to be
expressed in rod and cone bipolar cells. Although we observed brighter
labeling of amacrine cells and ganglion cells than bipolar cells, the
significance of this is not clear.
Western blotting suggested the presence of -ENaC,
-ENaC, and
ASIC1 proteins in the rabbit retina. Antibodies against these subunits
recognized proteins of molecular masses consistent with theoretical
calculations and published data (34). Theoretical molecular masses for the unglycosylated proteins are 70-75 kDa for
- and
-ENaC and 60 kDa for ASIC1. The
higher-molecular-mass bands on the blots represent glycosylated forms
and aggregated proteins. Degradation products are represented by the
lower-molecular-mass bands.
RT-PCR analysis demonstrated the presence of all known ENaC and ASIC subunits at the RNA level in the rabbit retina. Subsequent DNA sequencing confirmed their identities.
Whole cell patch-clamp data from cultured Müller cells showed
ragged inward currents at hyperpolarizing potentials. This is a
characteristic feature of ENaC in that they tend to exhibit cooperative
opening and closing (5). Inward currents were completely blocked with high concentrations of amiloride; accordingly, this is
consistent with the expression of an ASIC subunit in addition to
-ENaC because the IC50 is higher for ASIC channels
(10-20 µM) than for ENaCs (0.1-1 µM).
Despite extensive characterization of the ENaC/DEG family of channels
in the brain, a specific physiological role has not yet been
established. It is plausible that some proposed functions for these
proteins in brain may also apply to the retina. Electrophysiology indicates that the amiloride-sensitive Na+ current is not
voltage dependent. Therefore, it is unlikely that these channels play a
role in action potential generation. Osmotic pressure has been shown to
regulate -rENaC in Xenopus oocytes (19). Thus this channel may be involved in the regulation
of cell volume in conjunction with
Na+-K+-ATPase in response to changing osmotic
pressure in the retina or vitreous. ASICs may serve as a signal for pH
changes because they are activated by H+. Neuronal activity
generates pH changes, and an extracellular alkaline shift is elicited
by light in the retina (11). ENaC currents are also
regulated by intracellular pH. For example, acidification inhibits
current in oocytes (9) and native tissues (16, 30,
18). Accordingly, these channels could be novel pH sensors in
this system.
Evidence exists for the presence of a mineralocorticoid receptor (MCR) in Müller cells in the ONL, INL, GCL, and RPE of rat retina (27). Mineralocortocoids, including aldosterone, regulate ENaCs by increasing the probability of opening and upregulating transcription of the channels. MCR localization parallels the broad localization of ENaC in the retina and provides a mechanism for control of channel activity in the retina. Growth, development, and differentiation are some functions of corticosteroid hormones in nervous tissue (25), and ENaC/DEG channels may be temporally regulated by such hormones in ocular development. Formation and volume regulation of the neural tube during development involves ENaC (33). Similarly, ENaC may play a role in the development of the water-rich vitreal body.
We have presented several lines of evidence showing the expression of a variety of proteins from the ENaC/DEG family of Na+ channels in the rabbit retina. Functions of these channels in the central nervous system are merely speculative at present. However, the differential expression in Müller glia and neurons is interesting and may subserve unique functions for each cell type. In addition, individual cells may have several varieties of channels made of diverse combinations of ENaC and ASIC subunits. These findings suggest that the combinations of subunits composing amiloride-sensitive channels may be highly tissue specific. In fact, some assumptions about channel compositions may prove tenuous at best.
The amiloride-sensitive family of channels is involved in diseases and potentially some types of neurodegeneration in other tissues. In the future, detailed physiological, biochemical, and molecular characterizations of these channels in the retina should provide further insight into their importance in the visual system.
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ACKNOWLEDGEMENTS |
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We acknowledge Lee Ann McLean and Susan Copeland for technical assistance.
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FOOTNOTES |
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This study was supported by National Institutes of Health Grants EY-07845 and P30 EY-03039 to K. T. Keyser and DK-37206 to D. J. Benos.
Address for reprint requests and other correspondence: L. M. Brockway, Vision Science Res. Ctr., Univ. of Alabama at Birmingham, 924 South 18th St., Worrell Bldg., Birmingham, AL 35294 (E-mail: brockwaylm{at}cs.com).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published February 20, 2002;10.1152/ajpcell.00457.2001
Received 25 September 2001; accepted in final form 18 February 2002.
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