Audiological Medicine, 2009; 00: 1–6

Inner ear connexins, intercellular signalling and deafness

FABIO MAMMANO1-3 & fabio anselmi2

1Departmentof Physics ‘G.Galilei’, University of Padova, Padova, 2Venetian Institute of Molecular Medicine, Foundation for
Advanced Biomedical Research, Padova, and 3CNR Neuroscience Institute, Padova, Italy

Abstract
The genes GJB2 and GJB6, respectively encoding transmembrane proteins connexin 26 (Cx26) and connexin 30 (Cx30)
are found within 50 kb of each other in the DFNB1 complex deafness locus on chromosome 13q12. Up to 50% of all
patients with autosomal recessive non-syndromic prelingual deafness in different populations present with mutations or
deletions in this locus. Cx26 and Cx30 are abundantly expressed in the inner ear and, in recent years, have been shown to
form hemichannels that release ATP from the endolymphatic surface of supporting and epithelial cells of the organ of Corti
(OoC), as well as gap junction (GJ) channels that allow the concomitant intercellular diffusion of Ca21 mobilizing second
messengers. Released ATP in turn activates G-protein coupled P2Y2 and P2Y4 receptors, PLC-dependent generation of IP3,
release of Ca21 from intracellular stores, ensuing in the regenerative propagation of intercellular Ca21 signals (ICS) across
these coupled cells. The range of ICS propagation in the OoC is sensitive to the concentration of extracellular divalent
cations and to ectonucleotidase activity. Strictly related oscillations of the intracellular free Ca21 concentration ([Ca21]i) in
cochlear supporting cells are also evoked by nanomolar concentrations of ATP on the endolymphatic surface of the OoC.
ICS and [Ca21]i oscillations are of great interest in relation to the responses evoked by damaging stimuli delivered to hair
cells, and may play a crucial role in development of the OoC and the acquisition of hearing.

Key Words: cochlea, hearing, DFNB1, mouse models, development, organotypic cultures, CELAbs

Cochlear GJ networks
All cells providing mechanical support to cochlear
hair cells are designated as supporting cells (1). In
the mature OoC, these include inner phalangeal
cells, inner and outer pillar cells, outer phalangeal
cells (also known as Deiters’ cells), as well as Hens-
en’s, Böttcher’s and Claudius’ cells. The inner pha-
langeal cells completely surround the inner hair cells
(IHCs). The outer phalangeal cells form cups hold-
ing the synaptic poles of the outer hair cells (OHCs)
and send fine processes, or phalanges, to the reticular
lamina (RL). This stiff cytoplasmic plate is composed
of a mosaic of apposing phalangeal processes of outer
pillar cells and outer phalangeal cells, both of which
seal the endolymphatic poles of the hair cells, extend-
ing from the innermost row of OHCs to the Hensen’s
cells. Thus, only stereociliary bundles of OHCs
emerge above the RL (1).
The OoC is part of the tubular cochlear duct
epithelium in which cells are connected by a network
of tight junctions and adherens junctions near their
surface facing the scala media, thus providing separa-
tion of the endolymph from the perilymph (2). Non-
sensory cells in the cochlear duct are also massively
interconnected by intercellular GJ channels (3–5),
which mediate the transfer of ions, metabolites and
second messengers between cells (6–8). The develop-
ment of the cochlear GJ system precedes the func-
tional maturation of the murine inner ear (2), which
takes place between the second and third postnatal
week (9).
The connective tissue GJ network starts to develop
around birth and comprises interdental cells and
fibrocytes in the spiral limbus, fibrocytes of the spiral
ligament, basal and intermediate cells of the SV. The
epithelial GJ network forms around embryonic day
16 (E16) and connects all supporting cells in the
OoC as well as adjacent epithelial cells (2). In the
hearing cochlea, the epithelial GJ network apparently
subdivides further in two separate, medial and lat-
eral, buffering compartments, which are thought to
be individually dedicated to the homeostasis of IHCs
and OHCs (10).
In the so-called potassium recycle hypothesis (3),
the epithelial GJ network is presumed to intervene in
the cycling of K1 following activation of the mecha-
notransduction process in hair cells (11). Indeed,
several classes of supporting cells in the sensory epi-
thelium express glial fibrillary acidic protein (GFAP)
(12), a classic marker for astrocytes, and are thought

Correspondence: F. Mammano, VIMM, Via G. Orus 2, 35129 Padova, Italy. Tel/fax: +39 049 7923 231/250. E-mail: fabio.mammano@unipd.it

ISSN 1651-386X print/ISSN 1651-3835 online © 2009 Informa UK Ltd. (Informa Healthcare, Taylor & Francis AS)
DOI: 10.3109/16513860903444311
2 F. Mammano

to perform similar spatial buffering of K1 (3,5). postnatal day 12–13, and thereafter decreased sig-
Although the widespread localization of cochlear GJ nificantly in parallel with the appearance of epithe-
channels (4), K/Cl cotransporters (13), and aqua- lial breaches that compromised the integrity of the
porins (14) could provide a passive mechanism of endolymphatic compartment. Endolymphatic K1
K1 buffering and osmotic homeostasis, it is worth concentration was also significantly lower in adult
emphasizing that currently there is no direct evidence Cx26OtogCre mice compared to controls. Likewise,
for the potassium recycle hypothesis. expression of the Cx26R75W dominant negative
mutation in mice, obtained by injection of the trans-
gene into fertilized mouse eggs (28) (also called
Connexins, deafness and mouse models pronucleus injection), resulted in deafness associ-
ated with significant histological abnormalities
Most cell−cell channels in the cochlear GJ networks
within the inner ear including degeneration, by
are composed of two types of integral transmem-
postnatal week 7, of the OHCs. In a conditional (c)
brane proteins subunits, namely Cx26 and Cx30
Cx26 null mouse model (29) (obtained by crossing
(15), which share 77% amino acid identity and may
Cx26loxP/loxP mice with R26cre-ERT mice in
assemble to form heteromeric and heterotypic chan-
which Cre is activated in the presence of the syn-
nels (16). The genes encoding Cx26 (GJB2) and
thetic oestrogen 4-hydroxytamoxifen), the earliest
Cx30 (GJB6) are found within 50 kb of each other
cell death occurred around postnatal day 14 in
in the DFNB1 complex deafness locus. DFNB1
OHCs and their surrounding supporting cells in the
mutations, which are almost as frequent as those
middle cochlear turn (CT). Hence, death rapidly
causing cystic fibrosis, account for around 50% of
spread to the basal CT so that all types of cells had
genetic cases of severe to profound non-syndromic
disappeared from the OoC of cCx26 null mice a few
hearing loss in various ethnic groups (17). For both
months after birth. Unlike Cx26OtogCre mice, in
connexins, expression is evident in several sub-com-
cCx26 null mice peripheral nerve fibres and soma
partments of the cochlear duct already one week
of SGNs at corresponding cochlear locations were
before the onset of hearing (18,19), in keeping with
completely degenerated as well, whereas hair cells
the notion that intercellular connections by GJ pro-
in the apical CT were relatively preserved (29).
teins are crucial for the maturation of different tis-
In Cx30(–/–) mice (30) (obtained by deletion of
sues (20). In particular, immunolabelling with
the Cx30 coding region) the cochleae were morpho-
selective antibodies highlighted the expression of
logically indistinguishable from those of Cx30(1/–)
connexins at point of contacts between supporting
and wt mice up to postnatal day 17. However,
and epithelial cells of the OoC on both sides of the
Cx30(–/–) mice failed to develop any measurable EP,
hair cell region (HCR), as well as in basal and inter-
and adults had significantly decreased endolymphatic
mediate cells of the stria vascularis (SV). Further-
K1 levels. Auditory thresholds worsened from 84dB
more, both Cx26 and Cx30 showed marked
at postnatal day 17–18 to more than 100dB in adult
immunoreaction in the spiral limbus and the spiral
Cx30(–/–) mice; hence, hearing loss was more severe
ligament (10,18,19,21–25).
in Cx30(–/–) mice than in Cx26OtogCre mice. Corre-
Mouse models with defective expression of Cx26
spondingly, the apoptotic process in Cx30(–/–) mice,
or Cx30 confirmed that they are essential for hear-
albeit delayed by about four days relative to Cx26O-
ing. Thus, targeted ablation of Cx26 in the epithelial togCre mice, affected ultimately both IHCs and OHCs.
GJ network of Cx26OtogCre mice (26) (obtained by
Transgenic expression of extra copies of the Cx26
crossing Cx26loxP/loxP mice with Otog-Cre mice line
gene from a modified bacterial artificial chromosome
expressing the Cre gene under the control of the
(BAC) in a Cx30(–/–) background restored cochlea
murine Otog promoter) ensued in cell death accom-
development and hearing (31).
panied by epithelial breaches shortly after the onset
of hearing (which, in mice, occurs at postnatal day
12 (27)), along with progressive and significant
Permselectivity of GJ channels
hearing loss ranging from 30dB to 70dB. The apop-
totic process affected first the two supporting cells Although mouse models confirmed that Cx26 and
that surround the IHCs and later extended to OHCs Cx30 are essential for auditory function, as well as
and supporting cells around them. IHCs were pre- survival and development of the OoC (26,28–30),
served in adult Cx26OtogCre mice, and cell death was the physiopathological mechanisms leading to deaf-
not detected at any stage either in spiral ganglion ness when connexins are absent or mutated remain
neurons (SGNs), in the fibrocytes of the spiral lim- unclear (15). Thus, deafness and lack of EP in
bus and spiral ligament or in the SV. Endochlear Cx30(–/–) mice correlate with: 1) disruption of the
potential (EP) values developed normally up to endothelial barrier of the capillaries supplying the SV

before EP onset; 2) significant down-regulation of
Bhmt; and 3) local increase in homocysteine, a known
factor of endothelial dysfunction (32), with no obvious
link to GJ channel function. Likewise, the K1 recycle
hypothesis is challenged by the identification of Cx26
recessive deafness mutants, e.g.V84L (33), which retain
partial channel function (34) and are as permeable to
K1 as the wt channels. However, the transfer of the
Ca21-mobilizing second messenger IP3 (and possibly
other signalling molecules) is impaired between the
cells expressing these mutant proteins (35).
The concept that each GJ channel is unique in
terms of permselectivity is supported by a large num-
ber of studies (36) and suggests that permeability to
larger metabolites, rather then small inorganic ions,
plays an important role in the development, physiol-
ogy and aetiology of connexin related diseases
(7,8,37). This is a crucial issue, for it has been dem-
onstrated that many disease leading mutations cause
changes in the permeability of the channel to signal-
ling molecules (7). Furthermore, several lines of
experiments showed that the effect of single point
mutations can be subtle, discriminating between
molecules having the same net charge but different
charge distribution; however, the molecular mecha-
nisms remain unclear (7). The recent publication
of the structure of the Cx26 at 3.5 Å resolution
(38) will undoubtedly pave the way to the study,
at atomic level, of connexin channel function and
dysfunction.
Connexin hemichannels, ATP release and
cell–cell signalling
While the exact function of inner ear connexins
remains unclear (15), we note that Cx26 and Cx30
also form unpaired connexons (39), i.e. non-junc-
tional connexin hemichannels (40,41). Using anti-
bodies against Cx26 extracellular loop peptides
(CELAbs) (42), immunofluorescence signals cor-
responding to unpaired connexons (i.e. connexin
hemichannels) were detected in the plasma mem-
brane of cells facing the endolymphatic surface of
the OoC, except the hair cells, from inner sulcus
(IS) cells across the RL to outer sulcus (OS) cells
(43). Using organotypic cultures of the cochlea
from mice with defective expression of pannexin 1
(Px1), P2X7 receptors, Cx30 or Cx26, Anselmi et
al. (43) further demonstrated that, in response to
activation of a P2Y/PLC/IP3/Ca2 signalling cas-
cade, hemichannels formed by these connexins
release ATP from the endolymphatic surface of
cochlear supporting and epithelial cells, whereas GJ
channels allow the concomitant diffusion of Ca2
mobilizing second messengers across these coupled
Inner ear connexins 3
cells. Nanomolar levels of ATP on the endolym-
phatic surface of the OoC, which have been linked
to sound exposure (44), in turn activate G-protein
coupled P2Y2 and P2Y4 receptors, PLC-dependent
generation of IP3 and release of Ca2 from intracel-
lular stores, ensuing in the regenerative propagation
of ICS across the networks of cochlear supporting
and epithelial cells (43,45–47). The range of ICS
propagation is sensitive to the concentration of
divalent cations in the extracellular medium and is
controlled by ATP degradation (43) due to ecto-
nucleotidases expressed at the endolymphatic sur-
face of the OoC (48,49). By manipulating the
bathing medium one can either reduce the rate of
ATP degradation, by inhibiting ectonucleotidases
with ARL67156, or else increase this rate, e.g. by
supplementing soluble apyrase. ICS propagation is
blocked altogether by membrane permeable block-
ers of connexin channels, such as CBX, whereas
inhibitors of the P2Y receptors, such as suramin, or
blockers of connexin hemichannels, such as La3,
confine ICS propagation to cells directly coupled
to the stimulated cells through GJ channels
(35,43,45,46).
The mechanisms underlying the spreading of ICS
have been analysed in detail and shown to entail
release of ATP from connexin hemichannels at the
endolymphatic surface of the epithelium, as well as
diffusion of Ca21-mobilizing second messengers
across GJ channels (43) (Figure 1). IP3 is a key inter-
mediate that mediates ATP-dependent Ca21
responses of cochlear sensory (50) and non-sensory
(35,46,51,52) cells.
What could be the significance of ICS
exchange, and of the strictly related [Ca21]i
oscillations?
Although precise answers are not yet available, it is
tempting to propose a developmental role. Thus, in
non-excitable cells, the dynamics of gene expression
have been shown to depend on [Ca2]i changes
through NFκB signalling (53–55). Ortolano et al.
(56) showed that impaired propagation of ICS,
evoked by focal UV photorelease of IP3 in this imma-
ture OoC is associated with Cx26 down-regulation
at both protein and mRNA levels in Cx30(–/–) cul-
tures. Conversely, ablation of Cx26 caused concur-
rent down-regulation of Cx30 and impaired ICS
propagation. Molecular characterization of the
coregulation mechanisms implicates IP3, intracellular
Ca2 and NFκB, a Ca2-dependent transcription
factor, as important players. Interestingly, a binding
site for NFκB has been identified in the promoter
region of GJB2 (57,58), and NFκB is reported to
 4 F. Mammano
mono for print colour online

Figure 1. Schematic representation of ICS propagation across the network of GJ coupled cochlear supporting and epithelial cells. CBX,
carbenoxolone, is a non-specific inhibitor connexin hemichannels and GJ channels (74); ARL67156, 6-N,N-diethyl-D-β-y-dibromomethylene
adenosine triphosphate, originally named FPL 67156, is a commercially available inhibitor of ecto-ATPases (75). Apyrase is an enzyme
that catalyses the hydrolysis of ATP to yield AMP and inorganic phosphate (76,77). La31 is a blocker of connexin hemichannels (40) that
does not affect GJ channels when applied extracellularly to cochlear cultures (43). P2YR, G-protein coupled P2Y receptor; IP3R, receptor
for inositol 1,4,5-trisphosphate (also commonly known as triphosphoinositol, abbreviated InsP3 or IP3).

regulate the expression of at least one other connexin
(connexin 43, reference (59)). These data (56) also
offer crucial insight into the observations that some
deafness associated DFNB1 alleles are characterized
by hereditable significant reduction of both GJB2
and GJB6 (60) and that GJB6 deletions in trans with
recessive mutations of GJB2 cause deafness in
humans (61–63) (see also reference (64)).
Furthermore, it is known that the acquisition
and maturation of mechanoelectrical transduction
in hair cells occurs in a gradient from the base to
the apex of the cochlea, and that IHCs differentiate
prior to OHCs (65). P2X2 and transiently expressed
P2X3 ionotropic channels on the endolymphatic
surface of IHCs activate in response to extracellular
ATP (66,67), which is released from supporting
cells into the endolymph through hemichannels
formed by Cx26 and Cx30 (43), causing nearby
IHCs to fire bursts of Ca2 action potentials in the
developing OoC (68). This firing activity promotes
the release of the afferent neurotransmitter gluta-
mate at the synaptic pole of the IHC (69,70), which
in turn triggers discrete bursts of Ca2 action poten-
tials in primary auditory neurons (71). Further-
more, although immature OHCs are not known to
fire spontaneous action potentials (66) and the role
of OHC afferent signalling remains obscure (72),
the endolymphatic surface of the OHC is a primary
region for purinergic influence, where ATP activates
Ca2 signalling through the combined action of P2X
and P2Y receptors (50,66).
ICS and [Ca21]i oscillations of are of great inter-
est also in relation to the responses evoked by damag-
ing stimuli delivered to hair cells (45,73). Although
the picture is far from complete, what is beginning
to emerge is an intriguing set of interactions between
cochlear sensory hair cells and their supporting cells,
with ATP playing a key signalling role.
Acknowledgements
This work has been funded by grants to FM from
Fondazione Cariparo (Progetti di Eccellenza 2006),
Telethon Italy, Italian Ministry of Research and the
European commission FP6 Integrated Project Euro-
Hear under the Sixth Research Frame Program of
The European Union.
Declaration of interest: The authors report no
conflicts of interest. The authors alone are respon-
sible for the content and writing of the paper.

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