Am J Physiol Cell Physiol 301: C1017–C1026, 2011.
First published July 27, 2011; doi:10.1152/ajpcell.00185.2011.
KCNQ5/Kv7.5 potassium channel expression and subcellular localization in
primate retinal pigment epithelium and neural retina
Xiaoming Zhang,1 Dongli Yang,1 and Bret A. Hughes1,2
Departments of 1Ophthalmology and Visual Sciences and 2Molecular and Integrative Physiology, University of Michigan, Ann
Arbor, Michigan
Submitted 8 June 2011; accepted in final form 25 July 2011
ion channels; patch clamp; M-type current; photoreceptors
PATCH-CLAMP STUDIES on freshly isolated retinal pigment epithelial (RPE) cells from human (14, 47), monkey (47), and bovine
retina (43) have demonstrated a sustained outwardly rectifying
K⫹ current that is relatively insensitive to block by tetraethylammonium (TEA). This current activates at voltages positive
to about ⫺60 mV and, thus, contributes to the resting membrane potential. The kinetics and voltage dependence of this
current (43) resemble those of the neuronal M-current, which
was first described by Brown and colleagues as a neuronal K⫹
current in frog sympathetic ganglion cells that is suppressed by
the stimulation of muscarinic acetylcholine receptors (5, 7). In
the past decade, it has been well established that many M-type
currents are mediated by members of the KCNQ (Kv7) gene
family (6, 38). The molecular correlates and function of the
channel underlying the M-type current in RPE, however, remain unknown and need to be clarified.
The five members of the KCNQ gene family that have been
identified to date encode six-transmembrane domain-spanning
proteins. KCNQ/Kv7 channels are formed by the assembly of
Address for reprint requests and other correspondence: B. A. Hughes, Dept.
of Ophthalmology and Visual Sciences, Univ. of Michigan, W. K. Kellogg Eye
Center, 1000 Wall St., Ann Arbor, MI, 48105 (e-mail: bhughes@umich.edu).
http://www.ajpcell.org
four identical KCNQ ␣-subunits or the coassembly of different
KCNQ proteins (17, 38). The functional properties of KCNQ
channels are altered by their coassembly with various smaller
accessory ␤-subunits from the KCNE family, termed KCNE1–5
(38), and are modulated by multiple signaling pathways. For
example, phosphorylation of KCNQ3, KCNQ4, and KCNQ5
by Src tyrosine kinase causes channel inhibition (11), and
calmodulin confers Ca2⫹ sensitivity to KCNQ2, KCNQ4, and
KCNQ5 channels (10).
Although KCNQ1 contributes to the K⫹ conductance in some
epithelia (40), the expression of KCNQ2–5 is generally associated
with excitable cells (38). Recent studies, however, have described
the expression of members of the KCNQ family in a variety of
other tissues, suggesting that these channels may have wider
physiological roles than has been previously thought (33). Mutations in KCNQ1– 4 underlie inherited cardiac arrhythmias (in
some cases associated with deafness) (35, 46), neonatal epilepsy
(41), and dominant progressive hearing loss (23). To date,
KCNQ5 mutations are not known to be associated with human
disease. KCNQ4 was cloned from a human retina cDNA library
(23), and KCNQ4 splice variants have been detected in rat retina
by PCR analysis (4). Little else is known about the expression and
function of KCNQ channels in the retina.
To clarify the molecular basis of the M-type current in the
RPE, we assessed the expression of KCNQ subunits in native
monkey RPE. We found that, although KCNQ1, KCNQ4, and
KCNQ5 transcripts are expressed in primate RPE, only
KCNQ5 could be detected at the protein level. Transcripts for
all five KCNQ subunits as well as KCNQ4 and KCNQ5
proteins were detected in neural retina. In addition, we detected
KCNQ5 immunoreactivity near the RPE basal membrane as
well as in rod and cone photoreceptor inner segments and
elsewhere in the neural retina. The results suggest that KCNQ5
subunits contribute to M-type channels in the RPE basolateral
membrane, where they likely participate in the transport of K⫹
from the retina to the choroid. KCNQ5 subunits may also
contribute to voltage-gated K⫹ channels in the inner segments
of rod and cone photoreceptors and in retinal neurons.
MATERIALS AND METHODS
Preparation of Monkey RPE and Neural Retina
Monkey tissues were prepared as described previously (52). All
animal procedures were in accordance with the Association for Research in Vision and Ophthalmology (ARVO) “Statement for the Use
of Animals in Ophthalmic and Visual Research.” Monkey (Macaca
fascicularis or M. mulatta) eyes, skeletal muscle, and heart were
obtained from National Primate Centers or Lonza (Walkersville, MD)
at necropsy from protocols not related to the present studies. Tissues
were transported in CO2-independent medium on wet ice and processed within 36 h of being harvested. The globes were hemisected by
a circumferential incision around the pars plana, and the anterior
0363-6143/11 Copyright © 2011 the American Physiological Society
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Zhang X, Yang D, Hughes BA. KCNQ5/Kv7.5 potassium channel
expression and subcellular localization in primate retinal pigment epithelium and neural retina. Am J Physiol Cell Physiol 301: C1017–C1026, 2011.
First published July 27, 2011; doi:10.1152/ajpcell.00185.2011.—Previous
studies identified in retinal pigment epithelial (RPE) cells an M-type
K⫹ current, which in many other cell types is mediated by channels
encoded by KCNQ genes. The aim of this study was to assess the
expression of KCNQ genes in the monkey RPE and neural retina.
Application of the specific KCNQ channel blocker XE991 eliminated
the M-type current in freshly isolated monkey RPE cells, indicating
that KCNQ subunits contribute to the underlying channels. RT-PCR
analysis revealed the expression of KCNQ1, KCNQ4, and KCNQ5
transcripts in the RPE and all five KCNQ transcripts in the neural
retina. At the protein level, KCNQ5 was detected in the RPE, whereas
both KCNQ4 and KCNQ5 were found in neural retina. In situ
hybridization in frozen monkey retinal sections revealed KCNQ5
gene expression in the ganglion cell layer and the inner and outer
nuclear layers of the neural retina, but results in the RPE were
inconclusive due to the presence of melanin. Immunohistochemistry
revealed KCNQ5 in the inner and outer plexiform layers, in cone and
rod photoreceptor inner segments, and near the basal membrane of the
RPE. The data suggest that KCNQ5 channels contribute to the RPE
basal membrane K⫹ conductance and, thus, likely play an important
role in active K⫹ absorption. The distribution of KCNQ5 in neural
retina suggests that these channels may function in the shaping of the
photoresponses of cone and rod photoreceptors and the processing of
visual information by retinal neurons.
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KCNQ5 EXPRESSION IN THE RPE
segment, lens, and vitreous were removed. After the neural retina was
gently peeled away, RPE sheets were isolated from eyecups following
incubation at 37°C with 1% dispase for 30 to 60 min as described
previously (50). Tissues were snap frozen and stored at ⫺80°C until
use. For electrophysiology experiments, RPE cells were isolated
enzymatically from pieces of fresh RPE-choroid using papain as
described previously (14).
RT-PCR
stringent conditions (55°C). Sections were incubated in staining buffer containing 2 ␮l/ml NBT/BCIP (Roche Diagnostics, Indianapolis,
IN), 100 mM Tris-Cl (pH 9.5), 100 mM NaCl, and 50 mM MgCl2 at
room temperature in the dark and checked periodically to evaluate the
development of the alkaline phosphatase enzyme reaction. The reaction was stopped when the appropriate level of staining was achieved
by rinsing sections in 0.1 M Tris-Cl (pH 8.0).
Transfection
In Situ Hybridization
In situ hybridization to frozen monkey retinal sections was carried
out using digoxigenin-labeled cRNA probes prepared as follows. A
1.1-kb PCR product of KCNQ5 was generated from monkey retinal
cDNA samples using hKCNQ5-specific primers (forward: 5=-CAC
AAA ATT GGC CTC AAG TTG-3=; reverse: 5=- CAT CAC ACT
GGC ATC CTT TTT CAT-3=) and cloned to pGEM-T vector (Promega, Madison, WI). The KCNQ5 insert sequence (nucleotides 803–
1,845) was compared with published human KCNQ5_v2 sequence
(NM_001160130). Digoxigenin-labeled antisense and sense riboprobes were synthesized using SP6 and T7 polymerase and a DIG RNA
labeling kit (SP6/T7) (Roche Applied Science, Indianapolis, IN).
Riboprobes were hybridized to retinal sections under moderately
Chinese hamster ovary (CHO-M1WT3) and human embryonic
kidney (HEK 293T) cell lines (both from American Type Culture
Collection, Manassas, VA) were cultured according to the manufacturer’s instructions. CHO-M1WT3 cells were transfected with the
expression constructs pCMV6-XL5/KCNQ4 transcript variant 1
(GenBank accession no. NM_004700.2) or pCMV6-XL5/KCNQ4
transcript variant 2 (GenBank accession no. NM_172163.1) (both
from OriGene Technologies) using Fugene6 Transfection Reagent
(Roche Diagnostics), according to the manufacturer’s protocol. HEK
293T cells were transfected with pCMV6-XL4/KCNQ5 transcript
variant 1 (GenBank accession no. NM_019842.2) (OriGene Technologies) using the method mentioned above. Whole cell extracts were
prepared for Western blot analysis 72 h after transfection. Protein
concentrations were determined by the Bio-Rad protein assay based
on the method of Bradford (Bio-Rad, Hercules, CA).
Preparation of Monkey Heart, Skeletal Muscle, and RPE Plasma
Membrane, and Neural Retina Crude Membrane Proteins
All protein procedures were performed at 4°C. Monkey heart and
skeletal muscle plasma membranes were isolated according to methods previously published (9, 45). Membranes were stored at ⫺80°C
until use.
Table 1. Target gene primer sequences and expected sizes of RT-PCR products
Target Gene
KCNQ1
Forward
Reverse
KCNQ2
Forward
Reverse
KCNQ3
Forward
Reverse
KCNQ4
Forward
Reverse
KCNQ5
Forward
Reverse
KCNQ4*
Forward
Reverse
Forward
Reverse
KCNQ5*
Forward
Reverse
Rhodopsin
Forward
Reverse
GAPDH
Forward
Reverse
Primer Sequence
Expected Size, bp
GenBank Accession No.
5=-CAC GGG GAC TCT CTT CTG GA-3=
5=-CAC GCG GCC TGA CTC GTT CA-3=
427
NM_000218
5=-GTA TGA GAA GAG CTC GGA G-3=
5=-GTC GTT CTC CCC CTT CTC TG-3=
427
XM_028859
5=-GAT GCA CAA GGA GAG GAG A-3=
5=-GTA TTG CTA CCA CGA GGA TTA GA-3=
452
XM_005249
5=-GTT TGA GCA CGT GCA ACG G-3=
5=-ACA GCA GGC ATG ATG TCG-3=
444
AF105202
5=-CAC AAA ATT GGC CTC AAG TTG-3=
5=-GCA ATG GAA ACA GAT TTC TC-3=
717
AF202977
5=-GGC
5=-AGC
5=-CAT
5=-GTC
230
AF105202(v1)
206
NM_172163(v2)
5=-AAC CCA GCT GCC AAC CTC ATT C-3=
5=-CAT CAC ACT GGC ATC CTT TTT CAT-3=
423
396
453
NM_019842(v1)
NM_001160130(v2)
NM_001160132(v3)
5=-CAT CGA GCG GTA CGT GGT GGT GTG-3=
5=-GCC GCA GCA GAT GGT GGT GAG C-3=
577
NM_000539
5=-GTG AAG GTC GGA GTC AAC G-3=
5=-GAG ATG ATG ACC CTT TTG GC-3=
356
AF261085
CCT
TGC
CCT
TCA
CTT
TGC
TCA
GAG
GTT
TTG
GCA
ATG
TGA
GAA
GCC
CCC
GCA
GGA
GGA
GGA
C-3=
C-3=
T-3=
AG-3=
*Primers for splice variants.
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Total RNA was prepared from RPE sheets and neural retina as
described previously (51) and used for conventional reverse transcription
polymerase chain reaction (RT-PCR) analysis. PCR was performed with
primers specific for human (h)KCNQ1–5 and GAPDH (Table 1). PCR
products were obtained by cycling 40 times (35 times for GAPDH) as
follows: 1 min at 94°C, 1 min at 50 –53°C, 1 min at 72°C, followed by
a 7-min extension at 72°C. The PCR products were separated by 1.4%
agarose gel electrophoresis. RPE samples were tested for retinal contamination using primers specific for rhodopsin (Table 1).
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KCNQ5 EXPRESSION IN THE RPE
Antibodies
Primary antibodies used in this study are listed in Table 2. Secondary antibodies used include donkey anti-mouse IgG-horseradish
peroxidase (HRP; catalog no. sc-2314) and donkey anti-rabbit IgGHRP (catalog no. sc-2313) from Santa Cruz Biotechnology (Santa
Cruz, CA) and Alexa Fluor488 goat anti-rabbit (H⫹L) and Alexa
Fluor555 goat anti-mouse (H⫹L) from Invitrogen (Camarillo, CA).
Western Blot Analysis
Western blot analysis was performed using the techniques described previously (54) with minor modifications. Briefly, proteins
were mixed in Laemmli sample buffer [62.5 mM Tris (pH 6.8), 25%
glycerol, 2% SDS, 0.01% bromophenol blue, and 5% 2-mercaptoethanol; Bio-Rad] at room temperature for 20 min and centrifuged at
20,000 g for 10 min. Proteins (10 –20 ␮g) were applied to a 4% to
20% linear gradient Tris·HCl gel (Bio-Rad). After electrophoresis,
proteins were transferred to a polyvinylidene difluoride (PVDF)
membrane (Bio-Rad) at a constant current of 350 mA for 90 min at
4°C in a solution containing 25 mM Tris, 193 mM glycine, and 10%
methanol. The membrane was then incubated at room temperature
first with Tris-buffered saline containing 5% nonfat dried milk and
0.1% Tween 20 and then with primary antibodies at working dilutions
of 1:1,000 (anti-Na-K-ATPase, anti-CD29), 1:500 (anti-KCNQ1, antiKCNQ4), or 1:750 (anti-KCNQ5). Immune complexes were detected
with HRP-conjugated secondary antibodies at a dilution of 1:2,500
and enhanced with chemiluminescent substrate (Pierce). In experiments testing antibody specificity, antibodies plus 20-fold excess
antigenic peptide were prepared at 4°C 1 day before use. Blots probed
with antibody alone and with antibody preabsorbed with antigenic
peptide were processed in parallel.
Immunohistochemistry
Pieces of monkey retina-RPE-choroid were fixed by immersion for
1 h in freshly prepared 4% paraformaldehyde in 0.1 M phosphate
buffer (PB) and then washed in chilled PB (3⫻, 20 min). To cryoprotect before freezing, tissues were incubated in successive 1-h
incubations in 5% and 10% sucrose solutions in PB, then in 20%
sucrose in PB overnight at 4°C. Tissues were embedded in optimal
cutting temperature embedding medium (Tissue-Tek; Sakura Finetek,
Torrance, CA) and frozen in liquid nitrogen. Cryosections (10 or 20
␮m) were cut, collected on glass slides, dried at room temperature,
and stored at ⫺80°C until use. For immunofluorescence labeling,
cryosections were blocked with phosphate-buffered saline (PBS)
containing 10% normal goat serum, 1% BSA, and 0.3% Triton X-100
and then incubated overnight at 4°C in PBS containing primary antibodies (KCNQ1, 1:100 dilution; KCNQ4, 1:100 dilution; KCNQ5, 1:500
dilution; CD29, 1:100 dilution) plus 2% normal goat serum and 0.2%
Triton X-100. Sections were washed eight times and incubated at
room temperature in the dark for 1 h with two mixed secondary
antibodies (Alexa Fluor488 goat anti-rabbit and Alexa Fluor555 goat
anti-mouse) diluted in 0.2% Triton X-100 and 2% normal goat serum
in PBS to a final dilution of 1:500. After being washed eight times in
the dark, sections were covered in mounting medium (Gel Mount;
Bio-Meda) and secured with a coverslip.
Specimens were analyzed on a scanning laser confocal microscope
(Leica SP5, Mannheim, Germany). Digital images were collected at
16-bit resolution in 0.29-␮m z-sections and analyzed by image analysis
software (Leica LAS AF). The confocal immunofluorescence images in
Fig. 6 are three-dimensional projections generated from four to eight
consecutive z-sections. Files were exported for additional processing by
Photoshop CS2 (Adobe, San Jose, CA). In experiments testing antibody
specificity by preabsorption with antigenic peptide, laser settings, photomultiplier tube gain, exposure time, and brightness and contrast settings
were identical for both experimental and control images.
Electrophysiology
Enzymatically dissociated monkey RPE cells were plated on the
bottom of a recording chamber that was constantly perfused with
HEPES-buffered Ringer solution consisting of (in mM) 135 NaCl, 5
KCl, 10 Na2HEPES, 10 glucose, 1.8 CaCl2, 1 MgCl2, pH 7.4 (290
mosM). Whole cell currents were recorded at room temperature from
single RPE cells using the whole cell configuration of the patch-clamp
technique as described previously (14). Pipettes were filled with
pipette solution containing (in mM) 30 KCl, 83 K-gluconate, 10
K2HEPES, 5.5 K2EGTA, 0.5 CaCl2, 4 MgCl2, 4 mM K2ATP, 0.5 mM
Table 2. Primary antibodies
Antibody
Source
Catalog No.
Epitope
Type
Na-K-ATPase ␣-1 subunit
CD29
KCNQ1
Abcam
Biolegend
Santa Cruz Biotechnology
Rabbit
Human
COOH terminus human
Mouse monoclonal
Mouse monoclonal
Goat polyclonal
KCNQ4
Santa Cruz Biotechnology
NH2 terminus human
Goat polyclonal
KCNQ4
Santa Cruz Biotechnology
COOH terminus rat
Goat polyclonal
KCNQ5
Santa Cruz Biotechnology
aa 727-896 Human
Rabbit polyclonal
KCNQ5
ABR
Ab7671
303002
sc-10646
C-20
sc-9385
N-20
sc-20882
G-14
sc-50416
H-170
PA1-941
aa 880-897 Human
Rabbit polyclonal
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The plasma membranes of monkey RPE were isolated as previously described (27) with some modifications. Briefly, RPE sheets
from 30 monkey eyes were homogenized in 100 ml of 0.25 M sucrose
plus homogenizing buffer [0.025 M Tris-acetate pH 7.0, 1 mM
EDTA, 1 mM 2-mercaptoethanol, and complete protease inhibitor
cocktail (Pierce, Rockford, IL)] and then centrifuged at 27,000 g for
20 min. The supernatant was then centrifuged at 150,000 g for 1 h.
The resulting pellet, homogenized in 5 ml of 57% sucrose containing
homogenizing buffer, was placed at the bottom of clear tube and
overlaid with 5 ml of 34% sucrose and 5 ml of 8.5% sucrose in
homogenizing buffer. The sucrose gradient was centrifuged for 20 h
at 75,500 g. Two fractions between the 8.5% and 57% overlayers were
collected, diluted with homogenizing buffer, and centrifuged again at
150,000 g for 1 h. The pellets were resuspended with 200 ␮l of
homogenizing buffer and stored at ⫺80°C until use.
Crude membranes of monkey neural retina were prepared as
previously described with slight modifications (42). Three frozen
monkey neural retinas were homogenized in 10 ml homogenizing
buffer (20 mM Tris-Cl pH 7.4, 1 mM EDTA, 50 mM NaCl, 1 mM
2-mercaptoethanol, and complete protease inhibitor cocktail). Cell
debris and nuclei were removed by centrifugation at 1,000 g for 15
min. The supernatant was separated into a soluble fraction and crude
membrane protein pellet by centrifugation at 100,000 g for 30 min.
The crude neural retina membrane protein pellet was suspended with
350 ␮l of homogenizing buffer, the protein concentration was determined, and the pellet was stored at ⫺80°C until use.
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KCNQ5 EXPRESSION IN THE RPE
Na2GTP, pH 7.2. Series resistance (Rs) was normally ⬍5 M⍀ and was
uncompensated. Patch electrodes were pulled from 1.65 mm OD glass
capillary tubing (Warner Instruments, Hamden, CT) using a multistage programmable microelectrode puller (model P-97, Sutter Instruments, San Rafael, CA) and had an impedance of 2–5 M⍀ after fire
polishing. Signals were amplified with an Axopatch 200 amplifier
(Molecular Devices, Sunnyvale, CA).
Data acquisition and analysis were carried out using pClamp 9
(Molecular Devices). Statistical analysis and curve fitting were performed using Excel (Microsoft) and SigmaPlot 10 (Systat Software,
San Jose, CA). Tail current analysis was used to determine the M-type
conductance, as described previously (43).
RESULTS
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M-Type Current in the RPE Cells Is Sensitive to Block by
XE991
Previous studies on freshly isolated human and bovine RPE
cells identified an outwardly rectifying K⫹ current with kinetic
properties similar to those of M-type currents in excitable cells
(14, 43). To determine whether the M-type current in the RPE
is mediated by KCNQ channels, we tested the effects of
XE991, a blocker that is specific against KCNQ/Kv7 channels
when used in the micromolar range (6, 30). Figure 1 shows the
effects of 3 ␮M XE991 on whole cell currents in an isolated
monkey RPE cell. Under control conditions (Fig. 1A, top),
membrane hyperpolarizations from a holding potential of ⫺10
mV elicited current relaxations resulting from the deactivation
of M-type channels and repolarizations to the holding potential
produced characteristic tail currents reflecting channel activation (43). Superfusion of the cell with 3 ␮M XE991 eliminated
the time-dependent current (Fig. 1A, middle) and outwardly
rectifying current voltage relationship (Fig. 1B), unmasking an
inwardly rectifying K⫹ current (Kir) likely mediated by Kir7.1
channels (50). Similar results were obtained in other cells; in
three cells, 3 ␮M XE991 blocked 93 ⫾ 2% (means ⫾ SE) of
the M-type conductance and in four others, 10 ␮M XE991
blocked the M-type conductance by 99 ⫾ 3%. These results
provide strong evidence that KCNQ channels are the basis for
the M-type current in the RPE.
KCNQ mRNA Expression in RPE and Neural Retina
As a first step toward determining the specific KCNQ
subunit(s) that compose the M-type channel in the RPE, we
performed conventional RT-PCR analysis using mRNA isolated from monkey RPE and KCNQ subunit-specific primers
(Table 1). Reactions using mRNA from neural retina served as
positive controls. In monkey RPE, KCNQ1, KCNQ4, and
KCNQ5 transcripts were detected, whereas in monkey retina,
transcripts for all five KCNQ subunits were identified
(Fig. 2A). Positive PCR fragments were sequenced, and a
comparison of sequenced products with published human sequences confirmed their identities.
Alternative splicing of KCNQ genes contributes to diversity
in KV7 channel function and expression. There are two physiologically relevant splice variants of KCNQ1, a long isoform
(KCNQ1_v1) that contributes to voltage-gated K⫹ channels
underlying slowly inactivating current (IKs) in heart, and a
shorter isoform (KCNQ1_v2) with a truncated NH2 terminus
that is nonconductive and dominant negative when coexpressed with KCNQ1_v1 (19). Because KCNQ1 protein could
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Fig. 1. M-type current in monkey retinal pigment epithelial (RPE) cells is
blocked by the KCNQ channel blocker XE991. A: whole cell currents in the
absence (top) and presence (middle) of 3 ␮M XE991. The XE991-sensitive
component of whole cell current (bottom) shows the isolated M-type current.
Currents were evoked from a holding potential of ⫺10 mV by voltage steps
ranging from ⫺120 mV to 40 mV, as indicated. Horizontal lines indicate the
zero-current levels. B: current-voltage relationship of the steady-state currents
depicted in A. Note that XE991 specifically blocked the outwardly rectifying
component of whole cell current.
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KCNQ5 EXPRESSION IN THE RPE
not be detected in either the RPE or neural retina (see below),
we did not investigate KCNQ1 transcripts in further detail.
Two splice variants have been reported for human KCNQ4 (4)
and three splice variants have been reported for KCNQ5 (39).
Using splice variant-specific primers (Table 1), PCR of monkey
retina cDNA produced appropriately sized bands for KCNQ4
variants 1 and 2 (_v1 and _v2), whereas a single band corresponding to KCNQ4_v1 was produced from monkey RPE cDNA (Fig.
2B). KCNQ5 has in the region coding for the COOH terminus a
splice site that generates three variants that vary in size (39). To
clarify the expression of KCNQ5 splice variants in monkey retina
and RPE, we designed a single primer set that spans this variable
region. PCR of monkey retina cDNA produced two bands corresponding to KCNQ5_v1 and v2, whereas only a single band
corresponding to KCNQ5_v2 was produced when monkey
RPE cDNA was used as template (Fig. 2C).
KCNQ Protein Expression in RPE and Neural Retina
To examine the expression of KCNQ1, KCNQ4, and
KCNQ5 subunits in the RPE and neural retina at the protein
level, we performed gel electrophoresis and Western blot
analysis. In preliminary experiments using commercially available antibodies against KCNQ1, KCNQ4, and KCNQ5 (Table
2), we were unable to detect any of these subunits in crude
membrane proteins from RPE, indicating that, if present, they
were a relatively small fraction of total membrane proteins or
that the antibodies were not sensitive enough to detect KCNQ
subunit proteins. Consequently, we performed subsequent
Western blot analysis using plasma membrane proteins pooled
from RPE sheets collected from 30 monkey eyes.
To evaluate the quality of the RPE plasma membrane protein
sample, we performed Western blot analysis using anti-Na-KATPase ␣-subunit (50) and anti-CD29 antibodies (28) as markers of the apical and basolateral membranes, respectively. The
anti-Na-K-ATPase and anti-CD29 antibodies labeled bands of
the appropriate size (not shown), confirming that the sample
contained both apical and basolateral membrane proteins.
KCNQ1. We used a goat polyclonal anti-KCNQ1 antibody
(Santa Cruz C-20) that has been previously characterized and
AJP-Cell Physiol • VOL
shown to label appropriately sized bands in various tissues and
in mammalian cell lines transfected with human KCNQ1 (12,
37). To determine the reactivity of the anti-KCNQ1 antibody in
monkey, we tested it in Western blots of monkey heart plasma
membrane protein and found that it labeled a single band of
⬃78 kDa (data not shown), the expected molecular mass of a
KCNQ1 monomer. Preabsorption of the anti-KCNQ1 antibody
with antigenic peptide reduced the degree of immunolabeling
of the 78 kDa band, confirming the specific reactivity of the
antibody with monkey KCNQ1. In blots of crude membrane
proteins from monkey neural retina or plasma membrane
proteins from monkey RPE, however, KCNQ1 protein was not
detected (data not shown).
KCNQ4. We characterized a goat polyclonal antibody raised
against the NH2 terminus of human KCNQ4 (Santa Cruz N-20) in
Western blots of whole cell lysates of CHO cells transfected with
recombinant human KCNQ4 splice variant 1 (_v1) or 2 (_v2). The
antibody labeled appropriately sized bands (⬃77 kDa) in blots of
lysates from both KCNQ4_v1-transfected (Fig. 3A, lane 2) and
KCNQ4_v2-transfected cells (Fig. 3A, lane 3), but failed to label
Fig. 3. KCNQ4 protein expression in neural retina and RPE. A: anti-KCNQ4
antibody immunolabeled a ⬃77 kDa band in Chinese hamster ovary (CHO)
cells transfected with KCNQ4_v1 (lane 2) or KCNQ4_v2 (lane 3), but not in
untransfected cells (lane 1). B: in retina crude membrane protein, anti-KCNQ4
antibody labeled ⬃77 and ⬃154 kDa bands (left), which were diminished after
the antibody was preabsorbed with antigenic peptide (right). C: KCNQ4 was
not detected in blots of RPE plasma membrane. Results are representative of
more than 3 separate experiments.
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Fig. 2. KCNQ subunit mRNA expression in monkey neural
retina and RPE. A: total RNA isolated from macaque neural
retina (ret) and RPE was used in conjunction with KCNQ
subunit-specific primers (Table 1) for RT-PCR analysis of
KCNQ subunit transcript expression. Neural retina expresses all five KCNQ channels, whereas the RPE expresses
KCNQ1, KCNQ4, and KCNQ5. B: KCNQ4 splice variant
expression in monkey neural retina and RPE. Both
KCNQ4_v1 and _v2 are expressed in neural retina, whereas
only KCNQ4_v1 is expressed in the RPE. C: KCNQ5 splice
variant expression in monkey neural retina and RPE.
KCNQ5_v1 and v2 are expressed in the neural retina,
whereas only KCNQ5_v2 is expressed in the RPE. All
bands were sequenced to confirm their identities.
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a band in lysates from untransfected cell samples (Fig. 3A, lane 1).
Our results indicate that this KCNQ4 antibody can detect both
human KCNQ4_v1 and _v2.
In neural retina, the anti-KCNQ4 antibody recognized a
band of ⬃77 kDa and a broad smear between ⬃100 and 250
kDa (Fig. 3B). The presence of the ⬃77 kDa band is consistent
with the labeling of a KCNQ4 monomer, but the similar
molecular masses of KCNQ4_v1 and _v2 (⬃77 kDa and ⬃71
kDa, respectively) do not allow us to specify which splice
variant is expressed. Preabsorption of the antibody with antigenic peptide reduced the labeling of the ⬃77 kDa (Fig. 3B),
indicating that the antibody recognized monkey KCNQ4 protein. It is clear that the higher molecular mass smear is not
KCNQ4 because antigen preabsorption of the antibody did not
decrease the band. The smear bands of 100 to 150 kDa may be
caused by aggregation of KCNQ4 with other proteins or a
KCNQ4 dimer since these bands were lightly eliminated by
antigen preabsorption. In contrast, the anti-KCNQ4 antibody
did not label any band in the RPE (Fig. 3C). Similar results in
neural retina and RPE were obtained using a goat polyclonal
Localization of KCNQ5 Transcripts in Monkey Retina
To determine the cellular distribution of KCNQ5 in the
retina, we performed in situ hybridization on frozen adult
monkey retina sections using an antisense probe recognizing a
1,042-bp segment of the coding sequence. Figure 5 shows a
differential interference contrast (DIC) photomicrograph of a
monkey retina section hybridized with antisense probe. A
strong hybridization signal was observed in the outer nuclear
layer (ONL), which contains the cell bodies of rod and cone
photoreceptors, in the inner nuclear layer (INL), which includes the cell bodies of bipolar, amacrine, horizontal, and
Müller cells, and also in some of the cell bodies located in the
ganglion cell layer (GCL; Fig. 5A). Despite our detection of
KCNQ5 in purified RPE sheets by RT-PCR (Fig. 2), a hybrid-
Fig. 5. Localization of KCNQ5 message in monkey retina. In situ hybridization to sections of
monkey retina with antisense (A) and sense (B)
digoxigenin-labeled monkey KCNQ5 riboprobes.
Specific expression is seen in the ganglion cell
layer (GCL), inner nuclear layer (INL), and outer
nuclear layer (ONL). Melanin may have obscured
KCNQ5 signal in the RPE. Scale bar, 50 ␮m.
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Fig. 4. KCNQ5 protein expression in neural retina and RPE. A: anti-KCNQ5
antibody recognized an appropriately sized band of ⬃100 kDa (expected
molecular mass, 103 kDa) in lysates from human embryonic kidney (HEK
293T) cells transfected with KCNQ5 (lane 2), but not in untransfected cells
(lane 1). B: anti-KCNQ5 antibody immunolabeled a ⬃100 kDa band in
monkey skeletal muscle plasma membrane (left), as well as ⬃75 and ⬃50 kDa
bands. Immunoreactivity of the ⬃100 and ⬃50 kDa bands was greatly reduced
after preabsorption of the antibody with antigenic peptide (right). C: KCNQ5
antibody immunolabeled ⬃100 kDa bands in monkey neural retina crude
membrane and RPE plasma membrane proteins. Results are representative of
more than 3 separate experiments.
antibody raised against a polypeptide in the rat KCNQ4 COOH
terminus (G-14, Santa Cruz Biotechnology; data not shown).
KCNQ5. We characterized a rabbit polyclonal anti-KCNQ5
antibody raised against the human KCNQ5 NH2 terminus
(PA1-94-1, ABR) in Western blot analysis using lysates from
HEK 293T cells transfected with recombinant human
KCNQ5_v1 and found that it labeled an appropriately sized
band of ⬃100 kDa (Fig. 4A). In blots of plasma membrane
proteins from monkey skeletal muscle, where KCNQ5 expression has been documented (39), the anti-KCNQ5 antibody
labeled ⬃50, ⬃75, and ⬃100 kDa bands (Fig. 4B, left lane).
Preabsorption of the antibody with antigenic peptide diminished or eliminated the 50 and 100 kDa bands (Fig. 4B, right
lane), indicating reactivity of the antibody with monkey
KCNQ5 protein. As the expected molecular mass of a KCNQ5
monomer is ⬃100 kDa, the ⬃50 kDa band is likely a protein
degradation product. Figure 4C shows that the anti-KCNQ5
antibody also detected bands of ⬃100 kDa in neural retina and
RPE. Another rabbit polyclonal antibody raised against the
COOH terminus of human KCNQ5 (H-170, Santa Cruz Biotechnology) also labeled ⬃100 kDa bands in retina and RPE
(results not shown), but the identification of these bands as
KCNQ5 was not possible due to the lack of an antigenic
peptide.
In summary, our results show that both KCNQ4 and
KCNQ5 proteins are expressed in the monkey neural retina and
that in the RPE, only KCNQ5 is expressed at a detectable level.
KCNQ5 EXPRESSION IN THE RPE
ization signal could not be seen in the RPE, perhaps because it
was obscured by melanin. In situ hybridization with the complementary sense probe demonstrated no detectable signal
(Fig. 5B). Hence, KCNQ5 transcript appears to be present in
most retinal neurons and the RPE as well.
Immunofluorescence Localization of KCNQ5 Protein in
Monkey Retina
segments. Diffuse KCNQ5 immunoreactivity was also observed in the inner (IPL) and outer (OPL) plexiform layers, in
some cell bodies in the INL and GCL, and in occasionally in
retinal blood vessels (not shown). Immunolabeling in both the
RPE and neural retina was reduced or eliminated when the
antibody was preabsorbed with antigenic peptide (Fig. 6C). At
higher magnification, diffuse as well as punctate immunoreactivity was observed near the RPE basal membrane (Fig. 6, E
and F). The basal membrane also exhibited diffuse CD29
immunostaining (Fig. 6, E and F), but, interestingly, KCNQ5
immunostaining appeared to lie on the cytoplasmic side of the
CD29 signal, perhaps reflecting KCNQ5 expression in the basal
infoldings (36). A rabbit antibody against human KCNQ5 NH2
terminus (a gift of A. Villarroel, Leioa, Spain) produced a
similar staining pattern of rod and cone photoreceptor inner
segments and RPE (data not shown).
DISCUSSION
Previous studies identified an M-type K⫹ current in mammalian RPE cells (14, 43, 47). However, the subunit composition of
the underlying channel has remained unknown. In the present
study, we demonstrated that the M-type current in monkey RPE
cells is inhibited by the specific KCNQ channel blocker XE991
and investigated the expression of KCNQ subunits in monkey
RPE and neural retina at the mRNA and protein levels by
molecular, biochemical, and immunohistochemical approaches.
This is the first comprehensive survey of KCNQ expression in the
Fig. 6. Immunofluorescence localization of KCNQ5 protein in monkey retina. A and B: differential interference contrast (DIC, A) and confocal immunofluorescence image (B) of a monkey neural retina-RPE-choroid (CH) cryosection labeled with anti-KCNQ5 antibody (green). Anti-KCNQ5 antibody labeled the inner
plexiform layer (IPL) and outer plexiform layer (OPL), cone (single asterisk), and rod (double asterisk) photoreceptor inner segments (IS), and punctate structures
near the RPE basal membrane (block arrows). DAPI-labeled cell nuclei are shown in blue, as is Bruch’s membrane, which exhibits autofluorescence in the blue
emission channel. Scale bars, 50 ␮m. C: specificity of anti-KCNQ5 antibody: confocal immunofluorescence image of a cryosection from the same tissue as that
depicted in A and B, but processed with anti-KCNQ5 antibody preabsorbed with antigenic peptide. Immunofluorescence labeling in the neural and RPE was
reduced or eliminated. Scale bar, 50 ␮m. D–F: subcellular localization of KCNQ5 in the distal retina: DIC (D) and confocal immunofluorescence image (E) of
a cryosectioned neural retina-RPE-choroid from a different monkey double-labeled with anti-KCNQ5 (green) and anti-CD29 (red) antibodies. DAPI-labeled cell
nuclei are shown in blue. Single and double asterisks indicate the location of cone and rod photoreceptor inner segments, respectively, and solid arrows point
to regions of punctate KCNQ5 staining. OS, outer segments. Scale bar, 25 ␮m. F: inset indicated by the box in E showing KCNQ5 distribution in the RPE at
higher magnification. KCNQ5 immunolabeling is present near the basal membrane (ba) above Bruch’s membrane (Br, blue). Note that much of KCNQ5 does
not colocalize with CD29 (red), perhaps reflecting its distribution in basal infoldings. Lipofuscin appears light purple. ap, Apical membrane. Scale bar, 5 ␮m.
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To localize KCNQ1, KCNQ4, and KCNQ5 proteins in RPE
and neural retina, we performed confocal immunofluorescence
microscopy on frozen sections of monkey retina using the
anti-KCNQ antibodies that we characterized by Western blot
analysis (Figs. 3 and 4). Sections were double-labeled with
antibodies against CD29, a member of the integrin family
(␤1-integrin), which has been localized to the RPE basolateral
membrane as well as to retinal and choroidal blood vessels
(28, 34).
Neither the anti-KCNQ1 antibody (Santa Cruz C-20) nor the
anti-KCNQ4 antibodies (N-20, G-14) labeled the neural retina,
RPE, or choroid (not shown), indicating that the proteins were
in low abundance or that these antibodies do not work in fixed
monkey tissue. On the other hand, the anti-KCNQ5 antibody
consistently immunolabeled structures in both the RPE and
retina. Figure 6B shows KCNQ5 immunolabeling of puncta at
the basal aspect of the RPE (block arrows) as well as strong
immunolabeling of rod (indicated by the double asterisks) and
cone photoreceptor (indicated by the single asterisk) inner
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mass consistent with a KCNQ1 monomer and because this
immunolabeling was eliminated by preincubation of the antibody with antigenic peptide. We cannot exclude, however, the
possibility that KCNQ1 protein is expressed in the neural retina
and RPE at levels too low to be detected with the antibody we
used. Likewise, the antibody against KCNQ4 recognized monkey KCNQ4 protein in Western blots, as it labeled an appropriately sized band in neural retina that was diminished after
preabsorption with antigenic peptide; it failed, however, to
reveal the presence of KCNQ4 protein in the RPE.
Our immunofluorescence studies on frozen sections of monkey retina also failed to reveal KCNQ1 or KCNQ4 protein
expression in monkey neural retina or RPE, perhaps because
the antibodies used do not recognize the epitopes in fixed
monkey tissue. We conclude that KCNQ5 protein is expressed
in monkey RPE and that KCNQ1 and KCNQ4, if expressed,
are present at levels below detection. Additional studies investigating the expression of KCNQ1 and KCNQ4 in the RPE and
neural retina are warranted.
In situ hybridization experiments to sections of monkey
retina indicated the presence of KCNQ5 transcripts in the
perinuclear regions of photoreceptors as well as some cell
bodies in the inner nuclear layer and ganglion cell layer.
KCNQ5 transcript expression in the RPE could not be determined by this method due to the presence of melanin, but our
PCR results using cDNA from isolated RPE sheets established
that KCNQ5 is expressed in this tissue.
Immunofluorescence experiments using antibodies against a
peptide sequence in the human KCNQ5 COOH terminus revealed a more limited expression pattern for KCNQ5 protein in
monkey retina. KCNQ5 immunoreactivity was prominent in
rod and cone photoreceptor inner segments and was more
weakly expressed in the inner and outer plexiform layers. In
the RPE, diffuse KCNQ5 immunostaining was observed near
the basal membrane. The staining pattern of KCNQ5 in the
RPE was similar to but not identical to that of CD29 (Fig. 6F),
a marker of RPE basal membrane (28, 29). CD29 staining
appeared to be limited to a region adjacent to Bruch’s membrane, whereas KCNQ5 immunoreactivity extended somewhat
deeper into the cytoplasm. The reason for this difference is
unclear, but it may reflect expression of the integrin at regions
of contact between the RPE basal membrane and Bruch’s
membrane on the one hand and the distribution of KCNQ5 in
the basal infoldings on the other. In addition to diffuse staining,
the anti-KCNQ5 antibody immunolabeled punctuate structures
near the RPE basal membrane. It is tempting to speculate that
structures represent the aggregation of KCNQ5 channels in
specialized membrane domains, but more detailed experiments
are needed to test this idea.
Previously, we and others identified a sustained outward K⫹
current in acutely dissociated human and monkey RPE cells
that is kinetically similar to the M-current (14, 47). The M-type
current in primate RPE has a half-maximal activation voltage
(V0.5) of ⫺37 mV and is blocked by barium but is relatively
insensitive to block by extracellular TEA (14). In the present
study, we found that low doses of the KCNQ/Kv7 channel
blocker XE991 eliminated the M-type current in freshly isolated monkey RPE cells. Although this result provides strong
evidence that the current is mediated by KCNQ channels, it
does not indicate which subunits expressed in the RPE are
involved, as XE991 exhibits minimal selectivity against chan-
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RPE or neural retina of any species. A major finding of this study
is that KCNQ5 protein is expressed in primate RPE and neural
retina and that it is localized near the RPE basal membrane as well
as in cone and rod photoreceptor inner segments.
As a first step toward defining the expression of KCNQ
channels in the retina, we performed RT-PCR analysis on
monkey neural retina and isolated RPE sheets. This revealed
the expression of KCNQ1-Q5 in the neural retina and KCNQ1,
KCNQ4, and KCNQ5 transcripts in the RPE.
Six alternative splice variants of human KCNQ1 have been
reported that differ depending on the use of exons in the 5=-end
of the gene, but only splice variants 1 and 2 are thought to be
physiologically relevant: KCNQ1_v1 coassembles with the
␤-subunit KCNE1 to form IKs channels in the heart or with
KCNE2 or KCNE3 to form constitutively active K⫹ channels
that depend linearly on voltage in certain epithelia (40, 44).
KCNQ1_v2 lacks 127 amino acids in the NH2 terminus, is
nonconducting, and has dominant-negative effects on KCNQ1_v1
channels (19). We did not determine which KCNQ1 splice
variants are expressed in the RPE or neural retina, but this issue
should be explored in future studies.
Using splice variant specific primers, we determined that the
RPE expresses KCNQ4_v1 transcript alone, whereas the neural
retina expresses both KCNQ4_v1 and _v2 transcripts. Human
KCNQ4_v1 and _v2 are identical except that KCNQ4_v2 lacks
exon 9, resulting in the deletion of 54 amino acids in a membrane
proximal region of the COOH terminus. Although originally
thought to have restricted expression in the central and peripheral
auditory system (22, 23), KCNQ4 is now known to have a wider
tissue distribution: in mouse, mKCNQ4_v1 is expressed in a
variety of tissues, whereas the mouse ortholog of hKCNQ4_v2
(mKCNQ4_v4) is found predominantly in excitable tissues, including the neural retina (4). Patch-clamp studies have shown that,
compared with mKCNQ4_v1 channels, mKCNQ4_v4 channels
activate at more negative voltages and are expressed at higher
levels (48). It is currently not known whether the human
KCNQ4_v1 and _v2 also differ in terms of voltage sensitivity and
surface expression.
We also investigated which of the three known splice variants of KCNQ5 are expressed in the RPE and neural retina
using primers for KCNQ5 that span a splice site in the region
coding for the COOH terminus. We found that the RPE
expresses KCNQ5_v2 and that the neural retina expresses
KCNQ5_v1 and _v2. Initial studies identifying KCNQ5 indicated that it is expressed mainly in brain and skeletal muscle
(25, 39). KCNQ5_v1, which encodes a protein 932 amino
acids long, apparently is the only variant expressed in brain
(39, 53), whereas in skeletal muscle, KCNQ5_v2, which translates into a protein with a truncation of 9 amino acids in the
vicinity of calmodulin and phosphatidylinositol 4,5-bisphosphate (PIP2) binding domains, and KCNQ5_v3, which contains
19 additional amino acids, are also expressed (53).
We performed Western blot analysis of monkey RPE plasma
membrane and retinal crude membrane proteins for the three
KCNQ subunits that were detected in the RPE by RT-PCR. In
the neural retina, we detected KCNQ4 and KCNQ5 proteins,
but not KCNQ1, whereas in the RPE we detected only KCNQ5
protein. The failure to detect KCNQ1 protein in neural retina
and RPE was not due to a lack of antibody reactivity with
monkey protein because in Western blots of monkey heart
protein the antibody stained a prominent band with a molecular
KCNQ5 EXPRESSION IN THE RPE
AJP-Cell Physiol • VOL
express a sustained outwardly rectifying current called IKx (3),
which serves to set the dark resting potential and accelerate the
photoresponse to dim light. IKx resembles the M-current kinetically (24), leading to speculation that the underlying channel
is composed of KCNQ subunits, but there is other evidence
suggesting that the current is mediated at least in part by
ether-a-go-go (EAG) channels (8). Patch-clamp studies of
macaque cone inner segments have identified outwardly rectifying K⫹ currents that were partially blocked by 20 mM
extracellular TEA (49). It remains to be determined whether
KCNQ5 channels contribute to voltage-gated K⫹ currents in
rod or cone inner segments or in retinal neurons.
ACKNOWLEDGMENTS
The authors thank Anuradha Swaminathan for contribution to preliminary
experiments, Dr. Peter Hitchcock and Laura Kakuk-Atkins for help with in situ
hybridization, Mitchell Gillette for assistance with retinal cryosectioning, and
Dr. Stephen Lentz for assistance with confocal microscopy.
GRANTS
This work was supported by National Institutes of Health Grant EY08850
and Core Grant EY07003, the Foundation Fighting Blindness, and Research to
Prevent Blindness Lew R. Wasserman Merit Award (to B. A. Hughes).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
REFERENCES
1. Adorante JS. Regulatory volume decrease in frog retinal pigment epithelium. Am J Physiol Cell Physiol 268: C89 –C100, 1995.
2. Bal M, Zhang J, Zaika O, Hernandez CC, Shapiro MS. Homomeric
and heteromeric assembly of KCNQ (Kv7) K⫹ channels assayed by total
internal reflection fluorescence/fluorescence resonance energy transfer and
patch clamp analysis. J Biol Chem 283: 30668 –30676, 2008.
3. Beech DJ, Barnes S. Characterization of a voltage-gated K⫹ channel that
accelerates the rod response to dim light. Neuron 3: 573–581, 1989.
4. Beisel KW, Rocha-Sanchez SM, Morris KA, Nie L, Feng F, Kachar B,
Yamoah EN, Fritzsch B. Differential expression of KCNQ4 in inner hair
cells and sensory neurons is the basis of progressive high-frequency
hearing loss. J Neurosci 25: 9285–9293, 2005.
5. Brown DA, Adams PR. Muscarinic suppression of a novel voltagesensitive K⫹ current in a vertebrate neurone. Nature 283: 673–676, 1980.
6. Brown DA, Passmore GM. Neural KCNQ (Kv7) channels. Br J Pharmacol 156: 1185–1195, 2009.
7. Constanti A, Brown DA. M-currents in voltage-clamped mammalian
sympathetic neurones. Neurosci Lett 24: 289 –294, 1981.
8. Frings S, Brull N, Dzeja C, Angele A, Hagen V, Kaupp UB, Baumann
A. Characterization of ether-a-go-go channels present in photoreceptors
reveals similarity to IKx, a K⫹ current in rod inner segments. J Gen Physiol
111: 583–599, 1998.
9. Galante P, Mosthaf L, Kellerer M, Berti L, Tippmer S, Bossenmaier
B, Fujiwara T, Okuno A, Horikoshi H, Haring HU. Acute hyperglycemia provides an insulin-independent inducer for GLUT4 translocation in
C2C12 myotubes and rat skeletal muscle. Diabetes 44: 646 –651, 1995.
10. Gamper N, Li Y, Shapiro MS. Structural requirements for differential
sensitivity of KCNQ K⫹ channels to modulation by Ca2⫹/calmodulin. Mol
Biol Cell 16: 3538 –3551, 2005.
11. Gamper N, Stockand JD, Shapiro MS. Subunit-specific modulation of
KCNQ potassium channels by Src tyrosine kinase. J Neurosci 23: 84 –95,
2003.
12. Horikawa N, Suzuki T, Uchiumi T, Minamimura T, Tsukada K,
Takeguchi N, Sakai H. Cyclic AMP-dependent Cl⫺ secretion induced by
thromboxane A2 in isolated human colon. J Physiol 562: 885–897, 2005.
13. Hougaard C, Klaerke DA, Hoffmann EK, Olesen SP, Jorgensen NK.
Modulation of KCNQ4 channel activity by changes in cell volume.
Biochim Biophys Acta 1660: 1–6, 2004.
14. Hughes BA, Takahira M, Segawa Y. An outwardly rectifying K⫹
current active near resting potential in human retinal pigment epithelial
cells. Am J Physiol Cell Physiol 269: C179 –C187, 1995.
301 • NOVEMBER 2011 •
www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.1 on October 1, 2016
nels formed by various KCNQ subunits (30). On the other
hand, the voltage dependence and TEA insensitivity of the
M-type current are inconsistent channels composed of KCNQ1
subunits (18), but are compatible with homomeric KCNQ4_v1
and KCNQ5_v1 channels, which have V0.5 values of ⫺27 mV
and ⫺31 mV (26), respectively, and low sensitivities to block
by TEA (2). KCNQ5_v2, the KCNQ5 variant expressed in the
RPE, has biophysical and pharmacological properties similar
to those of KCNQ5_v1 (53), and although its TEA sensitivity
has not been determined, the truncation of nine amino acids in
its COOH terminus is not likely to affect sensitivity to this
blocker (2). It has been suggested that KCNQ5v_2 may differ
from other KCNQ5 splice variants in terms of its regulation by
Ca-calmodulin or PIP2 (53), and it will be interesting to
determine how KCNQ5_v2 channels and native M-type channels in the RPE are modulated by these signaling pathways. If
KCNQ4 and KCNQ5 subunits are expressed in the same RPE
cells, they may exist as KCNQ4/Q5 heteromeric channels, as
studies have demonstrated that these subunits are capable of
coassembling (2). Future studies should investigate this possibility as well as the potential contributions to the channel(s) by
␤-subunits encoded by the KCNE gene family.
Mutations in KCNQ1 and KCNQ4 genes underlie a number
of inherited diseases in humans, including cardiac arrhythmia
and nonsyndromal deafness (38), but no disease has been
linked to mutations of KCNQ5. Interestingly, no visual disorder has been linked to KCNQ1 or KCNQ4 mutations that cause
channelopathies in other tissues. This suggests that if they are
expressed, KCNQ1 and KCNQ4 subunits likely play minor or
redundant roles in RPE and neural retina physiology.
The active transport of K⫹ across the RPE in the apical-tobasal direction plays an important role in the regulation of
subretinal K⫹ concentration and solute-linked fluid absorption.
K⫹ absorption across the RPE involves the influx of K⫹ across
the apical membrane via the Na-K pump and Na-K-2Cl
cotransporter and its efflux across the basolateral membrane
via a K⫹ conductance (20, 31) composed of Ba2⫹-sensitive K⫹
channels (15, 21, 32). Our findings that KCNQ5 is expressed in
monkey RPE and is localized near the basal membrane suggest
that KCNQ5 channels may contribute to the basolateral membrane K⫹ conductance. K⫹ channels in the RPE basolateral
membrane have also been implicated in regulatory volume
decrease (1). KCNQ4 (13) and KCNQ5 (16) channels are
activated by increases in cell volume and thus may contribute
to regulatory volume decrease in the RPE following cell
swelling.
The activity of KCNQ channels is dependent on the levels of
PIP2 in the plasma membrane and the stimulation of certain
Gq/11-coupled receptors leads to the inhibition of M-type and
KCNQ channels via activation of phospholipase C and depletion of membrane PIP2. Although a number of G proteincoupled receptors have been identified in the RPE apical
membrane, it is not known whether this class of receptor is
located in the basal membrane where the hydrolysis of membrane PIP2 secondary to their activation could potentially
inhibit KCNQ5 channels.
An unexpected finding of this study was the strong KCNQ5
immunolabeling of cone and rod photoreceptor inner segments.
This immunoreactivity appears to be specific for KCNQ5
because labeling was eliminated when the antibody was preabsorbed by antigenic peptide. Vertebrate rod photoreceptors
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36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
novel mutation in the potassium channel gene KVLQT1 causes the Jervell
and Lange-Nielsen cardioauditory syndrome. Nat Genet 15: 186 –189,
1997.
Nilsson SE. Ultrastructural organization of the retinal pigment epithelium
of the Cynomolgus monkey. Acta Ophthalmol (Copenh) 56: 883–901,
1978.
Peroz D, Dahimene S, Baro I, Loussouarn G, Merot J. LQT1-associated mutations increase KCNQ1 proteasomal degradation independently
of Derlin-1. J Biol Chem 284: 5250 –5256, 2009.
Robbins J. KCNQ potassium channels: physiology, pathophysiology, and
pharmacology. Pharmacol Ther 90: 1–19, 2001.
Schroeder BC, Hechenberger M, Weinreich F, Kubisch C, Jentsch TJ.
KCNQ5, a novel potassium channel broadly expressed in brain, mediates
M-type currents. J Biol Chem 275: 24089 –24095, 2000.
Schroeder BC, Waldegger S, Fehr S, Bleich M, Warth R, Greger R,
Jentsch TJ. A constitutively open potassium channel formed by KCNQ1
and KCNE3. Nature 403: 196 –199, 2000.
Singh NA, Charlier C, Stauffer D, DuPont BR, Leach RJ, Melis R,
Ronen GM, Bjerre I, Quattlebaum T, Murphy JV, McHarg ML,
Gagnon D, Rosales TO, Peiffer A, Anderson VE, Leppert M. A novel
potassium channel gene, KCNQ2, is mutated in an inherited epilepsy of
newborns. Nat Genet 18: 25–29, 1998.
Stohr H, Stojic J, Weber BH. Cellular localization of the MPP4 protein
in the mammalian retina. Invest Ophthalmol Vis Sci 44: 5067–5074, 2003.
Takahira M, Hughes BA. Isolated bovine retinal pigment epithelial cells
express delayed rectifier type and M-type K⫹ currents. Am J Physiol Cell
Physiol 273: C790 –C803, 1997.
Tinel N, Diochot S, Borsotto M, Lazdunski M, Barhanin J. KCNE2
confers background current characteristics to the cardiac KCNQ1 potassium channel. EMBO J 19: 6326 –6330, 2000.
Trumble WR, Sutko JL, Reeves JP. ATP-dependent calcium transport
in cardiac sarcolemmal membrane vesicles. Life Sci 27: 207–214, 1980.
Wang Q, Curran ME, Splawski I, Burn TC, Millholland JM, VanRaay TJ, Shen J, Timothy KW, Vincent GM, de Jager T, Schwartz
PJ, Toubin JA, Moss AJ, Atkinson DL, Landes GM, Connors TD,
Keating MT. Positional cloning of a novel potassium channel gene:
KVLQT1 mutations cause cardiac arrhythmias. Nat Genet 12: 17–23,
1996.
Wen R, Lui GM, Steinberg RH. Whole-cell K⫹ currents in fresh and
cultured cells of the human and monkey retinal pigment epithelium. J
Physiol 465: 121–147, 1993.
Xu T, Nie L, Zhang Y, Mo J, Feng W, Wei D, Petrov E, Calisto LE,
Kachar B, Beisel KW, Vazquez AE, Yamoah EN. Roles of alternative
splicing in the functional properties of inner ear-specific KCNQ4 channels. J Biol Chem 282: 23899 –23909, 2007.
Yagi T, Macleish PR. Ionic conductances of monkey solitary cone inner
segments. J Neurophysiol 71: 656 –665, 1994.
Yang D, Pan A, Swaminathan A, Kumar G, Hughes BA. Expression
and localization of the inwardly rectifying potassium channel Kir7.1 in
native bovine retinal pigment epithelium. Invest Ophthalmol Vis Sci 44:
3178 –3185, 2003.
Yang D, Swaminathan A, Zhang X, Hughes BA. Expression of Kir7.1
and a novel Kir7.1 splice variant in native human retinal pigment epithelium. Exp Eye Res 86: 81–91, 2008.
Yang D, Zhang X, Hughes BA. Expression of inwardly rectifying
potassium channel subunits in native human retinal pigment epithelium.
Exp Eye Res 87: 176 –183, 2008.
Yeung SY, Lange W, Schwake M, Greenwood IA. Expression profile
and characterisation of a truncated KCNQ5 splice variant. Biochem
Biophys Res Commun 371: 741–746, 2008.
Zhang XM, Kimura Y, Inui M. Effects of phospholipids on the oligomeric state of phospholamban of the cardiac sarcoplasmic reticulum. Circ
J 69: 1116 –1123, 2005.
301 • NOVEMBER 2011 •
www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.1 on October 1, 2016
15. Immel J, Steinberg RH. Spatial buffering of K⫹ by the retinal pigment
epithelium in frog. J Neurosci 6: 3197–3204, 1986.
16. Jensen HS, Callo K, Jespersen T, Jensen BS, Olesen SP. The KCNQ5
potassium channel from mouse: a broadly expressed M-current like
potassium channel modulated by zinc, pH, and volume changes. Brain Res
Mol Brain Res 139: 52–62, 2005.
17. Jentsch TJ. Neuronal KCNQ potassium channels: physiology and role in
disease. Nat Rev Neurosci 1: 21–30, 2000.
18. Jespersen T, Grunnet M, Olesen SP. The KCNQ1 potassium channel:
from gene to physiological function. Physiology (Bethesda) 20: 408 –416,
2005.
19. Jiang M, Tseng-Crank J, Tseng GN. Suppression of slow delayed
rectifier current by a truncated isoform of KvLQT1 cloned from normal
human heart. J Biol Chem 272: 24109 –24112, 1997.
20. Joseph DP, Miller SS. Alpha-1-adrenergic modulation of K and Cl
transport in bovine retinal pigment epithelium. J Gen Physiol 99: 263–290,
1992.
21. Joseph DP, Miller SS. Apical and basal membrane ion transport mechanisms in bovine retinal pigment epithelium. J Physiol 435: 439 –463,
1991.
22. Kharkovets T, Dedek K, Maier H, Schweizer M, Khimich D, Nouvian
R, Vardanyan V, Leuwer R, Moser T, Jentsch TJ. Mice with altered
KCNQ4 K⫹ channels implicate sensory outer hair cells in human progressive deafness. EMBO J 25: 642–652, 2006.
23. Kubisch C, Schroeder BC, Friedrich T, Lutjohann B, El-Amraoui A,
Marlin S, Petit C, Jentsch TJ. KCNQ4, a novel potassium channel
expressed in sensory outer hair cells, is mutated in dominant deafness. Cell
96: 437–446, 1999.
24. Kurenny DE, Barnes S. Proton modulation of M-like potassium current
(IKx) in rod photoreceptors. Neurosci Lett 170: 225–228, 1994.
25. Lerche C, Scherer CR, Seebohm G, Derst C, Wei AD, Busch AE,
Steinmeyer K. Molecular cloning and functional expression of KCNQ5,
a potassium channel subunit that may contribute to neuronal M-current
diversity. J Biol Chem 275: 22395–22400, 2000.
26. Li Y, Gamper N, Shapiro MS. Single-channel analysis of KCNQ K⫹
channels reveals the mechanism of augmentation by a cysteine-modifying
reagent. J Neurosci 24: 5079 –5090, 2004.
27. Mata NL, Villazana ET, Tsin AT. Colocalization of 11-cis retinyl esters
and retinyl ester hydrolase activity in retinal pigment epithelium plasma
membrane. Invest Ophthalmol Vis Sci 39: 1312–1319, 1998.
28. McLaughlin BJ, Fan W, Zheng JJ, Cai H, Del Priore LV, Bora NS,
Kaplan HJ. Novel role for a complement regulatory protein (CD46) in
retinal pigment epithelial adhesion. Invest Ophthalmol Vis Sci 44: 3669 –
3674, 2003.
29. Menko AS, Philip NJ. Beta 1 integrins in epithelial tissues: a unique
distribution in the lens. Exp Cell Res 218: 516 –521, 1995.
30. Miceli F, Soldovieri MV, Martire M, Taglialatela M. Molecular pharmacology and therapeutic potential of neuronal Kv7-modulating drugs.
Curr Opin Pharmacol 8: 65–74, 2008.
31. Miller SS, Edelman JL. Active ion transport pathways in the bovine
retinal pigment epithelium. J Physiol 424: 283–300, 1990.
32. Miller SS, Steinberg RH. Passive ionic properties of frog retinal pigment
epithelium. J Membr Biol 36: 337–372, 1977.
33. Moser SL, Harron SA, Crack J, Fawcett JP, Cowley EA. Multiple
KCNQ potassium channel subtypes mediate basal anion secretion from the
human airway epithelial cell line Calu-3. J Membr Biol 221: 153–163,
2008.
34. Mullins RF, Kuehn MH, Faidley EA, Syed NA, Stone EM. Differential
macular and peripheral expression of bestrophin in human eyes and its
implication for best disease. Invest Ophthalmol Vis Sci 48: 3372–3380,
2007.
35. Neyroud N, Tesson F, Denjoy I, Leibovici M, Donger C, Barhanin J,
Faure S, Gary F, Coumel P, Petit C, Schwartz K, Guicheney P. A