Mice with altered KCNQ4 K+ channels implicate sensory outer hair cells in human progressive deafness.

Abstract

KCNQ4 is an M-type K+ channel expressed in sensory hair cells of the inner ear and in the central auditory pathway. KCNQ4 mutations underlie human DFNA2 dominant progressive hearing loss. We now generated mice in which the KCNQ4 gene was disrupted or carried a dominant negative DFNA2 mutation. Although KCNQ4 is strongly expressed in vestibular hair cells, vestibular function appeared normal. Auditory function was only slightly impaired initially. It then declined over several weeks in Kcnq4-/- mice and over several months in mice carrying the dominant negative allele. This progressive hearing loss was paralleled by a selective degeneration of outer hair cells (OHCs). KCNQ4 disruption abolished the I(K,n) current of OHCs. The ensuing depolarization of OHCs impaired sound amplification. Inner hair cells and their afferent synapses remained mostly intact. These cells were only slightly depolarized and showed near-normal presynaptic function. We conclude that the hearing loss in DFNA2 is predominantly caused by a slow degeneration of OHCs resulting from chronic depolarization.

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Mice with altered KCNQ4 Kþchannels implicate
sensory outer hair cells in human progressive
deafness
Tatjana Kharkovets1, Karin Dedek1,4,
Hannes Maier2, Michaela Schweizer1,
Darina Khimich3, Re ´gis Nouvian3,
Vitya Vardanyan1, Rudolf Leuwer2,
Tobias Moser3and
Thomas J Jentsch1,*
1Zentrum fu ¨r Molekulare Neurobiologie, ZMNH, Universita ¨t Hamburg,
Hamburg, Germany,2Department of Otolaryngology,
Universita ¨tsklinikum Hamburg-Eppendorf, Hamburg, Germany and
3Department of Otolaryngology, Center for Molecular Physiology of the
Brain, University of Go ¨ttingen, Go ¨ttingen, Germany
KCNQ4 is an M-type Kþchannel expressed in sensory hair
cells of the inner ear and in the central auditory pathway.
KCNQ4 mutations underlie human DFNA2 dominant
progressive hearing loss. We now generated mice in
which the KCNQ4 gene was disrupted or carried a
dominant negative DFNA2 mutation. Although KCNQ4 is
strongly expressed in vestibular hair cells, vestibular
function appeared normal. Auditory function was only
slightly impaired initially. It then declined over several
weeks in Kcnq4?/?mice and over several months in mice
carrying the dominant negative allele. This progressive
hearing loss was paralleled by a selective degeneration
of outer hair cells (OHCs). KCNQ4 disruption abolished the
IK,ncurrent of OHCs. The ensuing depolarization of OHCs
impaired sound amplification. Inner hair cells and their
afferent synapses remained mostly intact. These cells were
onlyslightlydepolarizedandshowednear-normalpresynaptic
function. We conclude that the hearing loss in DFNA2
is predominantly caused by a slow degeneration of OHCs
resulting from chronic depolarization.
The EMBO Journal (2006) 25, 642–652. doi:10.1038/
sj.emboj.7600951; Published online 26 January 2006
Subject Categories: neuroscience
Keywords: capacitance; channelopathy; KCNQ5; ribbon
synapse; vestibular organ
Introduction
Hearing loss is the most frequent inherited sensory defect
in humans and increases dramatically with age (Petit et al,
2001). Inherited hearing loss may be syndromic (associated
with other abnormalities) or nonsyndromic. Nonsyndromic
deafness may be inherited as X-linked, autosomal dominant
or/and autosomal recessive traits. In general, autosomal
recessive deafness has an early onset and is very severe,
whereas autosomal dominant deafness often develops slowly
over decades.
We had cloned KCNQ4 as a hitherto unknown member of
the KCNQ Kþchannel family, have mapped its gene to the
DFNA2 deafness locus and have identified KCNQ4 mutations
in patients with DFNA2 (Kubisch et al, 1999). These muta-
tions suppressed currents of coexpressed wild-type (WT)
subunits in a dominant negative manner. Like other KCNQ
channels,inparticular
(Schroeder et al, 1998; Wang et al, 1998), KCNQ4 mediates
M-type Kþcurrents (Kubisch et al, 1999). M-currents may
regulate neuronal excitability and are characterized by their
modulation through neurotransmitters, their voltage depen-
dence and their pharmacological profile (Jentsch, 2000).
Most KCNQ proteins (KCNQ2 to KCNQ5) can form homo-
and heteromeric channels. KCNQ3 is able to associate with
all KCNQ proteins except for KCNQ1. The carboxy-terminal
domain mediating subunit-specific interactions has been
mapped (Schwake et al, 2003). KCNQ4 can form functional
heteromers with KCNQ3 in heterologous expression systems
(Kubisch et al, 1999), but it is unknown whether this occurs
in vivo. In the inner ear, KCNQ4 mRNAwas localized to outer
hair cells (OHCs) of the organ of Corti (Kubisch et al, 1999).
Immunocytochemistry revealed the presence of the KCNQ4
protein in plasma membranes of OHCs and type I vestibular
hair cells (Kharkovets et al, 2000). In the first few days after
birth, KCNQ4 was expressed in the entire basal and lateral
membrane of OHCs (Boettger et al, 2002), but after the onset
of hearing (P12–14), it localized exclusively to the basal pole.
This localization suggested (Kharkovets et al, 2000) that
KCNQ4 might serve to extrude Kþions that enter OHCs
through apical mechanosensitive channels, which may be
encoded by TrpA1 (Corey et al, 2004). After their exit from
OHCs, Kþions are taken up by supporting cells, presumably
using K–Cl cotransport (Boettger et al, 2002, 2003). KCNQ4 is
also quite specifically expressed in several nuclei and tracts of
the central auditory pathway in the brainstem (Kharkovets
et al, 2000).
In order to understand the mechanism leading to DFNA2
deafness and to elucidate the role of KCNQ4 in hearing, we
now generated a constitutive knockout (KO) of KCNQ4 and
a knock-in (KI) of a dominant negative KCNQ4 mutation
we had identified in DFNA2 patients (Kubisch et al, 1999).
Our results show that deafness is due to a slow degeneration
of cochlear OHCs. This degeneration is faster in the total KO
than in animals heterozygous for the dominant negative
mutation. Our study identifies the native Kþcurrents that
are mediated by KCNQ4 and provides animal models for
human progressive deafness.
KCNQ2–KCNQ3heteromers
Received: 29 July 2005; accepted: 19 December 2005; published
online: 26 January 2006
*Corresponding author. Zentrum fu ¨r Molekulare Neurobiologie, ZMNH,
Universita ¨t Hamburg, Falkenried 94, Hamburg 20246, Germany.
Tel.: þ49 40 42803 4741; Fax: þ49 40 42803 4839;
E-mail: Jentsch@zmnh.uni-hamburg.de
4Present address: Carl-von-Ossietzky-Universita ¨t Oldenburg, Carl-von-
Ossietzky-Strasse 9-11, 26111 Oldenburg, Germany
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Results
Generation of KCNQ4 mouse models
Using homologous recombination in embryonic stem (ES)
cells, we created a mouse line with a deletion of Kcnq4 exons
6–8 and a KI mouse strain carrying the equivalent (G286S)
of the human G285S mutation that was identified in a
patient with DFNA2 and that exerted strong dominant
negative effects (Kubisch et al, 1999) (Figure 1A–C and
Supplementary Figure S1).
The elimination of exons 6–8 yields a non-functional
protein by truncating it at the beginning of the pore-forming
P-segment (Figure 1C). The deletion of a carboxy-terminal
module that is essential for subunit oligomerization (Schmitt
et al, 2000; Schwake et al, 2003) prevents dominant negative
effects on KCNQ3 and KCNQ4 subunits. The dominant nega-
tive G286S mutation (Figure 1B), which changes the first
glycine of the conserved GYG motif in the P-segment,
abolishes channel activity not only in homomeric mutant
channels, but also when present in a heteromer with WT
subunits (Kubisch et al, 1999). This allele is called Kcnq4dn.
Mice homozygous for either genotype were obtained at
approximately Mendelian ratio. Staining for the KCNQ4
protein revealed its presence at the base of WT OHCs
(Figure 1D), as described previously (Kharkovets et al,
2000). In contrast, KCNQ4 could not be detected in OHCs
from Kcnq4?/?mice (Figure 1E), a result that also confirmed
the specificity of the antibody. Because the expression of the
dominant negative allele might be changed by the intronic
loxP sites (Figure 1B), mutant mRNA levels were determined
by quantitative PCR from brainstem, which also expresses
KCNQ4 (Kharkovets et al, 2000). Its abundance was indis-
tinguishable from that of the WT message (Supplementary
Figure S1C).
Both KCNQ4 mouse models lacked a visible phenotype and
displayed normal growth and development. Although KCNQ4
is expressed in type I vestibular hair cells (Kharkovets et al,
2000), the vestibular function of KO mice appeared normal.
They lacked a circling behaviour and performed equally well
in rotarod tests. In histological analysis, the vestibular organ
appeared normal (Figure 5H and data not shown). As KCNQ4
is also weakly expressed in the heart (Kubisch et al, 1999),
electrocardiogram (ECG) analysis was performed. It did not
reveal obvious abnormalities (data not shown).
Evaluation of hearing
We examined auditory function by measuring auditory brain-
stem responses (ABR) to clicks and tone bursts and oto-
acoustic emissions. ABR reflect the synchronized electrical
activity of the ascending auditory pathway and thus provide
information on the sequential processing of acoustical signals
in the inner ear and brainstem. Otoacoustic emissions, on the
other hand, reflect OHC activity. The nonlinear properties of
the cochlear amplifier, which depends on the electromotility
of OHCs, leads to emission of multiple frequencies like 2f1?f2
when the cochlea is exposed simultaneously to two frequen-
cies f1and f2. These can be measured as distortion product
otoacoustic emissions (DPOAEs) (Robles and Ruggero, 2001).
Auditory brainstem responses to clicks with an upper
frequency limit of 5.5kHz were used to determine hearing
thresholds of mice of different genotypes from P14 (14 days
after birth) up to the age of 60 weeks (Figure 2A and B). WT
animals had a hearing threshold of about 57dB pe SPL (peak
equivalent sound pressure level) that remained stable over
time. At P14 (mice begin to hear at P10–11), Kcnq4?/?,
Kcnq4þ/dnand Kcnq4dn/dnanimals had a higher hearing
threshold than controls. However, after an additional week
(P21), their hearing ability at these frequencies approached
that of WT controls. At that time, ABR latencies and inter-
peak intervals of waves I–V were normal (no significant
differences (o5%)) (Supplementary Figure S2). As these
peaks originate in distinct brainstem regions involved in
auditory processing, the loss of KCNQ4 expression in these
tracts and nuclei (Kharkovets et al, 2000) apparently had no
major effect.
Both Kcnq4?/?and Kcnq4dn/dnanimals thereafter devel-
oped a significant hearing loss. In heterozygous dominant
negative mice (Kcnq4þ/dn), the hearing loss was variable and
progressed more slowly. Both Kcnq4þ/dnand Kcnq4dn/dn
animals displayed a threshold shift of about 50–60dB pe
SPL (compared to WT of the same age) at 42 weeks
(Figure 2B). In old mice (412 weeks), the average hearing
loss of Kcnq4dn/dnmice (n¼37) was somewhat more pro-
nounced than that of Kcnq4?/?mice (n¼28, DB11dB;
Figure 1 Generation of KCNQ4-deficient mice. (A) Scheme of
mouse genomic Kcnq4 sequence (exons shown as white boxes)
with an insertion of a loxP site in intron 5 and a neomycin selection
cassette (NEO) that was flanked by loxP sites in intron 8. A point
mutation introduced into exon 6 causes the G286S amino-acid
exchange that is equivalent to a mutation found in a patient
(Kubisch et al, 1999). After homologous recombination in ES
cells, treatment with Cre-recombinase yielded two types of clones
that were selected for injection into blastocysts: (B) a clone carrying
the G286S mutation and two intronic loxP sites (it encodes a
channel carrying the dominant mutation in the pore, as indicated
by the red ball in the topology model below); (C) a clone in which
deletion of exons 6–8 leads to a truncated, non-functional protein
schematically shown below. Immuncytochemistry on the organ of
Corti of WT (D) and Kcnq4?/?(E) mice revealed the KCNQ4 protein
(green) at the base of WT but not KO OHCs. Supporting Deiters’
cells (DC), located at the base of OHCs, are stained for the KCl
cotransporter KCC4 (Boettger et al, 2002) (red). Nuclei are stained
with TOTO (blue). Scale bar: 29mm (D).
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Po0.001; Figure 2A). No hearing impairment was detected in
Kcnq4þ/?animals.
To investigate whether the lack of KCNQ4 affects hair cell
function at 3 weeks, when low-frequency clicks revealed only
slight, nonsignificant differences in hearing ability and when
OHCs had not yet degenerated (see below), we measured
ABR at a higher frequency and determined otoacoustic emis-
sions. In response to a 16kHz stimulation (that excites the
region of high sensitivity in mouse cochlea), the ABR thresh-
old of 3-week-old Kcnq4?/?mice was shifted by about 35dB
pe SPL compared to controls (62.971.8dB pe SPL (n¼7) for
KO versus 27.872.8dB pe SPL (n¼9) for WT).
DPOAEs could be recorded from 3-week-old Kcnq4?/?
mice, albeit with lower amplitude (Figure 2C). Consistent
with the ABR findings, the average input–output function of
DPOAEs showed a shift of about 20dB (4 KO versus 6 WT
mice, f2¼16kHz; Figure 2D). At 8–10 weeks of age, DPOAEs
were not distinguishable from the noise floor in Kcnq4?/?
mice, but were readily observed in WT littermates (data not
shown).
Morphological analysis of the inner ear
The morphology of the inner ear was assessed by haematox-
ylin–eosin (HE) staining of paraffin sections. First, abnorm-
alities were observed in Kcnq4?/?animals at an age of about
4 weeks, whereas they became detectable in Kcnq4þ/dn
animals only later (Figure 3A–L and Supplementary Table
1). We observed a selective degeneration of OHCs of the basal
(high frequency) turn, whereas inner hair cells (IHCs) and
the neurons of the spiral ganglion were apparently not
affected. The degeneration, which displayed considerable
variability between individual animals, progressed over
time. Examination of 1-year-old animals revealed a nearly
complete loss of OHCs in the basal turn of both Kcnq4?/?and
Kcnq4þ/dngenotypes (Figure 3J–O and Supplementary Table
1), whereas OHCs of the apical (low frequency) turn were
apparently preserved over the entire lifespan. Even at 1 year
of age, the degeneration nearly exclusively affected OHCs,
while leaving IHCs largely intact. These results were con-
firmed by staining OHCs with an antibody against the motor
protein prestin (Weber et al, 2002) and IHCs for calretinin
(Dechesne et al, 1994) in basal (Figure 3M–O) and apical
(Figure 2P–R) turns. A few old (41 year) Kcnq4?/?and
Kcnq4þ/dnanimals also displayed a loss of IHCs of the basal
turn that was associated with a neurodegeneration in the
spiral ganglion. However, even in WT animals, some IHC
degeneration was occasionally seen in basal turns.
The position of Reissner’s membrane, which delimits the
scala media, was normal in all genotypes, indicating that salt
and fluid secretion by the stria vascularis was not signifi-
cantly impaired. Although KCNQ4 is expressed in type I
vestibular hair cells (Kharkovets et al, 2000), histological
examination revealed no degeneration in the vestibular
organ. We could neither detect morphological changes in
the cochlear nucleus, a relay station in the central auditory
pathway that expresses KCNQ4 (Kharkovets et al, 2000).
The ultrastructure of the organ of Corti was investigated
by electron microscopy (Figure 4). At 10 weeks of age, OHCs
and IHCs of apical turns were well preserved in the KO
(Figure 4B) as compared to WT (Figure 4A). In basal turns,
however, OHCs showed cytoplasmic vacuolization that was
not observed in the WT (Figure 4C and D). In spite of these
degenerative changes in OHCs, the cuticular plate and the
tight junctions, which are important for maintaining the
electrochemical gradient between the scala media and
the perilymph bathing the basolateral membranes of hair
cells, appeared intact (Figure 4E and F). High-power electron
Figure 2 Hearing ability of Kcnq4 genotypes as a function of age.
(A) Time course of ABR thresholds (low-frequency clicks): Kcnq4þ/þ
(WT) and Kcnq4þ/?animals have thresholds of B57dB pe SPL,
which remain constant after the onset of hearing at B2 weeks. After
improving from postnatal day 14 (P14) to BP21, Kcnq4?/?(KO)
hearing deteriorates rapidly to a constant hearing loss of B51dB pe
SPL after 8 weeks. (B) ABR thresholds of homo- and heterozygous
animals carrying the dominant negative G286S Kcnq4dnallele
compared to WT littermates. In Kcnq4dn/dnmice, hearing loss
progresses as in KO mice to reach B62dB pe SPL. Kcnq4þ/dn
mice mimicking heterozygous DFNA2 patients develop deafness
much more slowly. (C) Power spectrum of the microphone signal
recorded from representative 3-week-old Kcnq4?/?(red) and
Kcnq4þ/þ(black) mice showing the 2f1?f2 distortion product
otoacoustic emission (DPOAE at 10.7kHz), as well as the primary
tones (f1¼13.3kHz and f2¼16kHz); mutant data are laterally offset
for better visibility. (D) Input–output functions for DPOAEs (noise
floor subtracted 2f1?f2DPOAE, f2¼16kHz, six ears for WT (black)
and four ears for KO (red)). Dashed lines represent linear approx-
imations of the suprathreshold part of the input–output functions.
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Figure 3 Degeneration in the organ of Corti. Basal turns of the cochlea of WT, Kcnq4þ/dnand Kcnq4?/?(KO) mice (left, centre and right
panels, respectively) at ages stated at left were either stained with HE (A–L) or with an antibody against prestin (green) and against calretinin
(red) (M–O). As the time course of degeneration varied, representative pictures are shown. No changes were seen in mutated cochleae at P14
(B, C) or P28 (E, F) compared to WT of the same age (A and D, respectively). At 7 weeks (G–I), OHCs of KO mice (I) showed severe
degeneration, whereas those of Kcnq4þ/dnmice (H) remained largely intact. At 1 year of age (J–L), OHCs of both mutants had disappeared,
whereas IHCs appeared normal. This was confirmed by staining for the OHC marker prestin and for calretinin, which labels IHCs, in a basal
cochlear turn of 1-year-old mice of the three genotypes (P–R). Similar staining of apical turns from 1-year-old mice reveals the presence of both
OHCs and IHCs. For a statistical analysis of hair cell degeneration, see Supplementary Table 1. Scale bar: 30mm (M).
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microscopical pictures of IHCs and OHCs of apical turns of
10-month-old animals did not reveal differences between WT
and KO (Figure 4G–J). Except for occasional double ribbon
synapses in the KO (Figure 4M), which are commonly inter-
preted as a sign of immaturity (Sobkowicz et al, 1982), the
synaptic zones of KO IHCs did not differ morphologically
from WT controls of the same age (4 months) (Figure 4K
and L).
As the impact of a loss of KCNQ4 may depend on hetero-
merization with KCNQ3 or on a coexpression with other
KCNQ channels, we investigated their cochlear expression
by immunocytochemistry. In the cochlea, KCNQ1 is found
exclusively in the apical membranes of marginal cells of the
stria vascularis (Neyroud et al, 1997). An antibody against
KCNQ2 (Cooper et al, 2001) failed to label hair cells, but
stained apparently unmyelinated segments of axons innervat-
ing these cells (Figure 5A and B). An antibody against KCNQ3
(Cooper et al, 2001) did not label cochlear structures,
although it stained brain sections under the same conditions
(Devaux et al, 2004) (data not shown). Whereas KCNQ5 was
found in vestibular hair cells, where its expression over-
lapped with KCNQ4 (Figure 5C–E), no specific KCNQ5 signal
was detected in the organ of Corti with our own antibody or
that of Villarroel and co-workers (Yus-Najera et al, 2003).
Kþcurrents of outer and inner hair cells
Patch-clamp studies of hair cells from the first apical turn
were performed on animals from various genotypes at P12–
14, that is, before degenerative changes became visible.
Resting potentials VRof OHCs were measured at zero current
in the current clamp mode of the whole-cell configuration
with an extracellular solution containing 5.8mM Kþ.
Kcnq4þ/þand Kcnq4þ/?animals showed similar values of
Figure 4 Semithin sections of apical (A, B) and basal turns (C–E)
of 10-week-old WTand Kcnq4?/?mouse cochleae. Sensory cells in
(B) are well preserved, whereas OHCs in (D) show vacuolization of
the cytoplasm. Note the presence of the reticular lamina and
cuticular plate (cp) in an ultrathin section from a basal turn of a
10-week-old Kcnq4?/?organ of Corti (E). (F) Higher magnification
of the boxed area in (E), with normal tight junction (arrow). (G–J)
High-power transmission electron micrographs of 10-week-old (G,
H) and 4-month-old (I, J) OHCs and IHCs, respectively, from WT
and KO mice in the apical cochlear turn. Even after 4 months, when
KO OHCs of basal turns have degenerated, they are morphologically
unaltered in the KO. (K–M) Ultrastructural analysis of synaptic
zones of IHCs from 4-month-old WT and KO mice. In addition to
mature single ribbon synapses (K, L), double ribbon synapses
typically found in immature IHCs were found in the KO (M).
(r: synaptic ribbon; A: afferent dendrite). Scale bars: 20mm (A–E),
1mm (F), 2.5mm (G–J) and 0.5mm (K–M).
Figure 5 Expression of KCNQ2 and KCNQ5 in the inner ear.
(A) KCNQ2 (green) in afferent fibres of IHCs that are identified
by calretinin staining (red). DC: supporting Deiters’ cells. (B) Counter-
staining for myelin-associated glycoprotein (MAG, red) shows
that KCNQ2 (green) is present in unmyelinated segments of the
nerve. No KCNQ2 staining is detected in IHCs or OHCs. (C) KCNQ5
is expressed in a subset of vestibular hair cells of the crista ampullaris,
which also express KCNQ4 (red) (D) as shown in the overlay (E).
(F–H) Vestibular expression of KCNQ5 in WT (F), Kcnq4þ/?
(G) and Kcnq4?/?(H) mice. Scale bars: 29.4mm.
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