Cell Calcium (2000) 28 (3), 195–203

Research © 2000 Harcourt Publishers Ltd

doi: 10.1054/ceca.2000.0145, available online at http://www.idealibrary.com on

Cholinergic control of
membrane conductance and
intracellular free Ca2+ in outer hair
cells of the guinea pig cochlea
M. G. Evans,1 L. Lagostena,2 P. Darbon,1 F. Mammano2
1
MacKay Institute of Communication and Neuroscience, School of Life Sciences, Keele University, STAFFS ST5 5BG, UK 2Biophysics Sector and Istituto
Nazionale di Fisica della Materia, International School for Advanced Studies, 34014 Trieste, Italy

Summary We have studied the action of cholinergic agonists on outer hair cells, both in situ and isolated from the
cochlea of the guinea pig, combining new fast CCD technology for Ca2+ imaging and conventional patch-clamp
methods. Carbachol (1 mM) activated a current with a reversal potential near –70 mV and a bell-shaped I-V curve,
suggesting that it was a Ca2+-activated K+ current. In a few cells, this current was preceded by a transient inward
current, probably owing to an influx of Ca2+ and other cations through the acetylcholine (ACh) receptors. The amplitude
of the Ca2+ signal was maximal in a circumscribed region at the basal pole of the cell and decreased steeply towards
the apical pole, compatible with Ca2+-influx and/or Ca2+-induced Ca2+-release at the cells base. The time course of the
Ca2+-rise was fastest at the base, but it was still slightly slower, and more rounded, than that of the K+ current. In some
recordings the K+ current was observed without any measurable change of intracellular Ca2+. The K+ current was poten-
tiated (18%) by caffeine (5 mM), and decreased (19%) by ryanodine (0.1 mM) in the majority of cells tested. The results
are discussed in terms of a labile intracellular Ca2+ store located at the base of the cell, close to the Ca2+-permeable
ACh receptor channels and Ca2+-activated K+ channels, whose contribution to the Ca2+ rise occurring in the region of
the channels is variable, and probably dependent on its ability to refill with Ca2+. © 2000 Harcourt Publishers Ltd

INTRODUCTION explanation for the unusual cholinergic receptor pharma-

Outer hair cells (OHCs) are the target of an efferent
innervation originating in the brainstem [1, 2]. The prin-
cipal neurotransmitter of this efferent system is acetyl-
choline (ACh) [3]. Whole-cell recordings from isolated
OHCs have provided evidence for cholinergic receptors
localized around the base of the cell [4], where the effer-
ent synapses are located [5]. The action of ACh on OHCs,
which results in the activation of an apamin-sensitive
Ca2+-dependent (activated) K+ current [6] requires extra-
cellular Ca2+ [7, 8] and is accompanied by changes of
intracellular Ca2+ concentration [8, 9]. The finding that
the α9 subunit of the nicotinic acetylcholine receptor
is expressed in cochlear OHCs [10] has provided an
Received 8 May 2000
Revised 28 July 2000
Accepted 29 July 2000
Correspondence to: Dr M.G. Evans. Tel: + 44 1782 583594; Fax: +44 1782
583055; E-mail: M.G.Evans@cns.keele.ac.uk
cology of the olivocochlear efferent innervation [11].
Recently, it has been shown that the recombinant
α9 receptor is highly permeable to Ca2+, providing a
pathway for Ca2+ influx leading to activation of the
Ca2+-dependent K+ current [12].
OHCs possess a unique form of motility, called electro-
motility, that depends on voltage-sensing protein aggre-
gates in the cells’ lateral plasma membrane (reviewed
in [13]). Recently, Dallos et al. [14] showed that ACh
increases the electromotile responses of OHCs and attrib-
uted this effect to a decreased axial stiffness of the cell.
The mechanical responses occur with a significantly
greater latency than the membrane current and potential
changes evoked by ACh. They are therefore thought to
be related to slow efferent effects, which follow the
classical fast effects of efferent nerve stimulation after a
similar delay (about 10 s) [15]. Both effects comprise a
suppression of the compound action potential of the
auditory nerve and an enhancement of the cochlear

195
196 MG Evans, L Lagostena, P Darbon, F Mammano

Fig. 1 Picture gallery of cochlear turn 3. (A) low magnification view from scala media, after the removal of the upper turn 4. The border of
the organ of Corti is marked by the Hensen’s cells, with their characteristic lipid inclusions. Bar: 500 µM. (B) outer hair cells in row 3, seen
through the layer of supporting Hensen’s and Deiters’ cells. Bar: 50 µM. (C) outer hair cells in row 3, after the removal of Hensen’s cells.
The base of each cell is cupped in its corresponding Deiters’ cell and therefore appears more blurred than the apex. Intact stereocilia stand
out beneath the border of the tectorial membrane. Bar: 25 µM. (D) showing the basal segment of OHCs in row 2, after the removal of the
entire row 3 and of row 2 Deiters’ cells. Notice thin nerve fibres and synaptic boutons reaching the cell synaptic poles. Bar: 10 µM.

microphonic potential, largely generated by the OHCs,
and they are both antagonized by the same blockers,
indicating a link to the same nicotinic ACh receptor [15].
The slow efferent effects are selectively enhanced by
antagonists of the sarcoplasmic/endoplasmic calcium
ATPase, suggesting a role for Ca2+ release from intra-
cellular stores in their initiation [16].
The leading candidate for an intracellular Ca2+ store
contributing to the hair cells’ synaptic response is the
subsynaptic cistern that lines the inner surface of the
OHC membrane opposite each efferent synapse [17, 18]
The subsynaptic cistern integrates with the extensive
network of subsurface cisterns that line the rest of the
basolateral cell membrane [19, 20]. Pores of about 8.5 nm
are present on both surfaces of subsynaptic cisterns, and
on the outer surface the pores appear to align with pillars
that cross the gap between the cistern and the cell mem-
brane [21]. The subsynaptic cisterns are within a few
nanometres of, and are probably connected to, the cell
membrane. They are, therefore, well placed to contribute
to the ACh-evoked Ca2+ rise in the postsynaptic region of
the hair cell, by providing Ca2+ release in response to Ca2+
influx, and also by acting as a barrier to the diffusion of
Ca2+ away from this region [18].
We have studied the action of the cholinergic agonists,
ACh and carbachol, on OHCs both in situ and isolated
from the cochlea of the guinea pig, combining new fast
CCD technology for Ca2+ imaging [22, 23] and conven-
tional patch-clamp methods. Our results indicate that
ACh (1) affects membrane conductances, both directly
via the nicotinic ACh receptor and probably also indi-
rectly via Ca2+ release from intracellular stores; and (2)
promotes the development of substantial gradients of
intracellular free Ca2+ concentration ([Ca2+]i). The ampli-
tude of the Ca2+ signal is maximal in a circumscribed
region at the basal pole of the OHC and decreases steeply
towards the apical pole. Preliminary accounts of this
work have been published [24, 25].
MATERIALS AND METHODS
Cell preparation
Adult guinea pigs (200–450 g) were killed by cervical dis-
location and decapitated. The temporal bones were
removed from the skull and placed in modified Leibowitz
cell culture medium (L-15) containing (in mM): NaCl,
137; KCl, 5.36; CaCl2, 1.25; MgCl2, 1.0; Na2HPO4, 1.0;
KH2PO4, 0.44; MgSO4, 0.81. pH was adjusted to 7.35 with
NaOH and osmolarity adjusted to 320±2 mOsm/L with
D-glucose.
Most Ca2+ imaging experiments were conducted on
cells in the whole organ of Corti, using an isolated
cochlea preparation. The preparation and methods of
recording and Ca2+ imaging in the organ of Corti have
been described previously [22, 23, 26]. The majority of
recordings were performed from cells in the third
cochlear turn (Figs 1A–B), with the patch pipette posi-
tioned close to the cells’ base. To gain access to the cells,
a cut was made was through the external supporting
cells with a suction pipette after first locally applying
collagenase type I (2.5 mg/ml) under pressure from a
pipette (Figs 1C–D).
Some experiments were performed on isolated OHCs
from turns 2 and 3. To isolate the cells, the bulla was
opened, the cochlea exposed and the otic capsule was

Cell Calcium (2000) 28(3), 195–203 © Harcourt Publishers Ltd 2000


chipped away with a surgical blade, starting from the apex.
Strips of the organ of Corti were dissected from the modi-
olus with a fine needle and transferred to a 100-µl drop of
external solution. In some experiments, collagenase was
added (final concentration 0.5 mg/ml). Cells were dissoci-
ated by reflux through the needle of a Hamilton syringe
(N. 705, 22 gauge) or through a pipette tip (Gilson, 200 µl)
and allowed to settle on the slide for 5 to 10 min.
Thereafter they were placed in a laminar flow bath (100
µl), where solution exchange (>150 µl/min) was achieved
by gravity feed. For Ca2+ imaging, physiological solutions
were made as already described. For the experiments
involving exposure to caffeine or ryanodine, the external
solution comprised (in mM): NaCl 128; KCl, 5.4; MgCl2,
0.5; Na2HPO4, 0.34; KH2PO4, 0.44; MgSO4, 0.4; CaCl2,
1.26; HEPES 25; D-Glucose, 6; pH 7.4. Drugs were pur-
chased from Sigma or RBI.
Patch-clamp recordings and drug delivery
Conventional whole-cell patch-clamp recordings were
made under visual control after mounting the recording
chamber on a microscope stage. OHCs were visualized and
the following morphological features were used to deter-
mine viability: uniform cylindrical shape, basal location of
the nucleus, membrane birefringence and intact stereocilia
(Fig. 1C). Cells used in this study ranged in length from 40
to 70 µm and were maintained at room temperature
(22–25°C) throughout the experiment. A List EPC-7 patch-
clamp amplifier (Heka, Lambrecht, Germany) was used to
drive pipettes that had been pulled on a two-stage vertical
puller (PP-83, Narishige, Tokyo, Japan) from 2.0 mm o.d.
borosilicate glass (Clark Electromedical, Pangbourne, UK).
Current and voltage were sampled at rates between 1 and
50 kHz using a standard laboratory interface (1401 Plus,
Cambridge Electronic Design, Cambridge, UK) controlled
by customized software operating under Windows NT.
For experiments involving Ca2+ imaging, pipettes were
filled with an intracellular solution containing (in mM):
KCl, 150; MgCl2, 2.0; Na2HPO4, 8.0; NaH2PO4 1.0, Na2ATP,
1.0; Na2GTP, 0.3; EGTA, 0.5; adjusted to pH 7.2 with
KOH and brought to 320 mOsm/L with D-glucose. The
cell-impermeable form of the Ca2+-selective fluorescent
dye Oregon Green 488 BAPTA-1 (100 µM; O-6806,
Molecular Probes, Eugene, OR, USA) was added to this
solution. Following establishment of the whole-cell
recording configuration, we waited for about 5–10 min
for the dye to diffuse into the cell. At the end of this time
a few frames were captured, allowing us to centre the
image of the OHC onto the CCD camera and check the
overall level of fluorescence following illumination from
a xenon arc lamp [22]. A puff pipette, prepared similarly
to the patch pipette and filled with carbachol (0.25–1.0
mM, dissolved in the extracellular solution), was placed
© Harcourt Publishers Ltd 2000
Cholinergic response of outer hair cells 197
near the cell’s synaptic pole and pressure was applied at
its back by a Pneumatic PicoPump (PV800, World
Precision Instruments, Stevenage, UK) gated by a transis-
tor-transistor logic (TTL) pulse under software control.
During the positioning of the puff pipette, a small hold-
ing pressure was applied to prevent uptake of fluorescent
dye (that may have leaked into the bath during position-
ing of the recording pipette). In most experiments this
holding pressure was removed before the pipette was
brought close to the cell. Failure to do this may have pre-
vented us from observing fluorescence changes in some
cells (e.g. Fig. 2C).
For non-imaging experiments the intracellular solution
contained (in mM): KCl, 130; EGTA, 5; Na2ATP, 2; MgCl2,
4; HEPES, 5; adjusted to pH 7.3 with KOH. The concen-
tration of the Ca2+ buffer EGTA was different in the two
intracellular solutions. The former (for Ca2+ imaging) had
only 0.5 mM EGTA, and was designed to maximize the
Ca2+ rise, while still containing sufficient Ca2+ buffer to
maintain cell viability. The latter (for recording the ACh-
evoked conductance alone) had the usual 5 mM EGTA,
designed to stabilize Ca2+ sufficiently to minimize run-
down of this conductance [7]. In both cases potentials
were corrected for liquid-junction potentials, estimated
to be –4 mV [27]. The pipette resistance was typically
5 MΩ when measured in the bath. Results are given as
mean ± SEM (n = number of cells).
RESULTS
Potassium currents activated by carbachol in OHCs in
situ
In order to gain access to OHCs for the recording and
puffer (drug application) pipettes, a cut was made
through the external supporting cells with a suction
pipette. Recordings were performed from cells in rows 3
(outermost) and 2 in the third cochlear turn with the
patch pipette positioned close to the cells’ base, where
synaptic boutons were clearly visible (Fig. 2A). Carbachol
(1 mM) activated a current with a reversal potential near
–70 mV and a bell-shaped 1-V relation (Figs 2B & C),
suggesting that it was primarily due to a Ca2+-activated
K+ current. In a few cells, this current was preceded by a
transient inward current (Fig. 2D), which is likely to be
due to influx of Ca2+ and other cations through the ACh
receptors, thereby producing a rise in [Ca2+]i and opening
the Ca2+-activated K+ channels through which the
outward current flows [7, 8, 28].
Simultaneous Ca2+ imaging and whole cell recording
During the recordings described above, we also moni-
tored the fluorescence of Oregon Green 488 BAPTA-1, a
single wavelength probe highly selective for Ca2+, that
Cell Calcium (2000) 28(3), 195–203
198 MG Evans, L Lagostena, P Darbon, F Mammano

Fig. 2 Current responses of OHCs in cochlear turn 3 to carbachol, a cholinergic agonist. (A) four images of row 2 OHCs taken at different
focal planes. A patch pipette (entering from the left) contacted one OHC at its base. A puff pipette (entering from the right) was loaded with
carbachol and positioned in the proximity of the synaptic pole. Notice synaptic boutons in the rightmost frame. OHC diameter 10 µM.
(B) current-voltage (I–V) relationships derived from the responses to a voltage ramp before (control, dashed line) and during the application
of Carbachol (1 mM, dash-dot line). Solid line (difference) is the carbachol-sensitive fraction of the current, which reversed near –70 mV and
peaked around –20 mV. Membrane voltages were corrected for the error due to the access resistance (9.2 MΩ). (C) current responses to
repeated applications of carbachol (1 mM, horizontal line) at the holding potential indicated for each trace. Notice current reversal between
–71 mV and –69 mV. (D) another set of responses from a different cell, in row 3, at the indicated holding potentials. Notice biphasic
response at –45 mV and –38 mV, due to the activation of an early inward current.

had been loaded into the OHCs through the patch
pipette. Despite the activation of both inward and out-
ward currents, we detected no change in fluorescence.
We took this as evidence for the labile nature of the
cholinergic response in OHCs, perhaps resulting from
depleted intracellular Ca2+ stores, and thereby tried to
improve the physiological state of the cells in the prepa-
ration. By limiting the exposure of the cells to carbachol
(see Methods), we were able to demonstrate the simulta-
neous rise of Ca2+ fluorescence and outward current
(Fig. 3). The largest Ca2+ signal was at the synaptic pole.
The magnitude of the fluorescence change was reduced
towards the cells’ apex, where it rose more slowly.
These findings were confirmed by experiments on iso-
lated cells (Fig. 4A), that also showed a rapid elevation
of [Ca2+]i in a confined region at the cells’ synaptic pole
(Figs 4B & C). Towards the apex of the cell, Ca2+ rose
more slowly, and had a distinctive sigmoidal time-course
(Fig. 4B). The K+ current rose rapidly after a delay of about
100 ms and then decreased slightly during the carbachol
application, presumably because of desensitization.
Comparing the time course of the simultaneous rise in
Ca2+ at different regions of interest (denoted by the boxes
in Fig. 4A), and from the entire cell (Fig. 4B, lower panel),
it is clear that the Ca2+ rise in box 1 most closely resem-
bles the activation of the K+ current. This is probably
because, like the ACh receptors [4], the Ca2+-activated K+
channels activated by carbachol are concentrated at the
cells’ base. The time course of the K+ current and of the
Ca2+ rise in box 1 were not identical, with the Ca2+ rise
developing after a longer delay (about 150 ms) and dis-
playing a more rounded time course (the K+ current
reached a peak at a time when the Ca2+ was still rising).
In addition, we noticed that with repeated applications of
carbachol, the magnitude of the Ca2+ signal became suc-
cessively smaller, until it disappeared altogether after
about 4 applications. This occurred in both the prepara-
tions used. The K+ currents also tended to rundown in
parallel, although small K+ currents usually remained
after the Ca2+ signals had disappeared completely. For the
cell shown in Figure 4, a subsequent application of carba-
chol at a holding potential of –50 mV produced a Ca2+

Cell Calcium (2000) 28(3), 195–203 © Harcourt Publishers Ltd 2000


Fig. 3 Simultaneous recording of Ca2+ and current responses. (A)
bright field (top) and fluorescence image (bottom) of an OHC in row
2 of turn 3, loaded with 100 µM Oregon-Green 488 BAPTA-1
through the patch pipette (left). A puff pipette (right) was loaded
with carbachol and positioned near the synaptic pole. Bar: 10 µm.
(B) top: whole-cell current evoked by the application of carbachol
(1 mM, horizontal line); bottom, corresponding fluorescence
change. Traces were generated by averaging pixel signals within
the three number-coded boxes superimposed on the cell image in
A. The largest signal was at the synaptic pole.
signal that was 60% of that seen at –20 mV, in spite of
the larger driving force for Ca2+ entry at the more nega-
tive voltage (not shown). This Ca2+ rise, however, had
similar kinetics and spatial properties (in each of the
regions of interest denoted by the boxes in Fig. 4A) to
that shown at –20 mV.
In experiments where we observed simultaneous Ca2+
signals and K+ currents, it was clearly the Ca2+ rise at the
basal pole of the cell that most closely resembled the
kinetics of the K+ current. In general, however, the ampli-
tude of the current and the speed of its recovery showed
no strict correlation with the amplitude and dynamics of
the Ca2+ signals, and in some instances the carbachol-
evoked current occurred without any measurable change
in intracellular Ca2+. One possible explanation for these
results would be to suppose that the Ca2+ required to
activate the K+ channels comes both from Ca2+ influx
through ACh receptors and from Ca2+-release from intra-
cellular stores, and that the latter is physiologically labile
and provides all of the measured Ca2+ signal.
© Harcourt Publishers Ltd 2000
Cholinergic response of outer hair cells 199
Effects of caffeine and ryanodine on the ACh-evoked K+
current
To obtain evidence for the involvement of Ca2+-release
from intracellular stores, we investigated the effects of
caffeine and ryanodine, agents that modify Ca2+ release
from intracellular stores via ryanodine receptors. Caffeine
induces Ca2+ release by shifting the Ca2+-sensitivity of
Ca2+-induced Ca2+-release (CICR) to lower Ca2+ concen-
trations [29]. Ryanodine acts as a blocker of the ryan-
odine receptor when used in the micromolar range [30].
For these experiments we simply recorded the Ca2+-acti-
vated K+ current evoked by ACh in isolated OHCs, thus
avoiding the problems associated with Ca2+-imaging
described above. Thus this current might be expected to
be increased by caffeine and decreased by ryanodine.
The amplitude of the outward current (Ca2+-activated
K+ current) in response to ACh (70 µM) was continuously
monitored before, during, and after, the introduction of
caffeine (5 mM) or ryanodine (0.1 mM) in the superfusate
for 3 min. At these concentrations, caffeine induced a
slow rise of intracellular Ca2+ in isolated OHCs [31], and
ryanodine antagonized the effects of ACh on basilar
membrane mechanics [32]. In most OHCs, these agents
modified the amplitude (and sometimes the offset kinet-
ics) of the ACh-evoked outward current. Caffeine
increased the amplitude of this current in 8/12 cells. The
mean increase was 18% (from 197 ± 36 pA to 233 ± 36
pA; n = 8); P < 0.001 (Students t-test for paired data).
Following this potentiated response, the amplitude of the
K+ current either recovered back to the baseline after caf-
feine removal (not shown), or decreased rapidly without
recovery (Figs 5A & B). Although the ACh-evoked current
tends to get smaller with repeated applications of ACh
under normal conditions [7] (see also Fig. 5C), after caf-
feine the rate of decline was much faster. Ryanodine
decreased the amplitude of the current in 3/4 cells, one
of which is shown in Figures 5C & D. The effect of ryan-
odine was more pronounced in cells with large ACh-
evoked currents, and cells with responses below 100pA
were excluded from the analysis. The reduction in cur-
rent amplitude induced by ryanodine was 19% (from 348
± 166 pA to 281 ± 170 pA (n = 3)). In this calculation, the
control amplitude was taken as the mean of several mea-
surements before and after ryanodine. One other effect of
ryanodine was noted in these three cells, although it was
particularly pronounced in this example: it slowed the
recovery of the current back to baseline (Fig. 5D). This
observation is consistent with the idea that ryanodine
impairs the ability of the stores to refill with Ca2+ [33].
DISCUSSION
Electrical stimulation of the efferent system innervat-
ing OHCs is known to reduce the amplitude of the
Cell Calcium (2000) 28(3), 195–203
200 MG Evans, L Lagostena, P Darbon, F Mammano

Carbachol 1mM
B V = –20 mV

150 pA
A
6 1
5
2
4

3% ∆F/F°
3
3
4
2 5
6
1
3% ∆F/F°

1s

173 ms 400 ms 2108 ms

C 7.5% 1116 ms

∆F/F
°

Fig. 4 Development of intracellular [Ca2+]i gradients. (A) fluorescence image of an OHC isolated from turn 3 and loaded with 100 µM
Oregon-Green 488 BAPTA-1 through the patch pipette (entering from the right). A puff pipette (not shown) was loaded with carbachol and
positioned near the synaptic pole. Six number-coded boxes are superimposed on the image of the cell body. Bar: 10 µm. (B) top: whole-cell
current elicited by the application of carbachol (1 mM, horizontal line); the vertical arrow indicates the time of drug delivery. Middle section:
corresponding fluorescence change traces, generated by averaging pixel signals within the number-coded boxes superimposed on the cell
image in A. Note steep decrease of signal amplitude from basal to apical cell pole. Bottom: fluorescence change generated by averaging
pixel signals over the entire cell. Holding potential: –20 mV. (C) four false-colour fluorescence images, coded according to the colour-scale
bar (first frame), captured at the times shown (in ms, from the start of carbachol application). Note lag in the generation of the response and
formation of a localized maximum at the base of the cell.

sound-evoked activity of the afferent nerve fibers within
100 ms [34]. This fast effect is compatible with the rela-
tively short latency of the cholinergic current response
measured in the present and previous experiments (e.g.
[7, 8, 35]). It has also been suggested that there is an addi-
tional slower suppression, occurring on a time scale of
seconds, mediated by an increase of [Ca2+]i due to CICR
initiated at the subsynaptic cistern and spreading to sub-
surface cisterns, promoting further Ca2+ release [16]. Our
Ca2+ imaging experiments show a spatially defined
region at the OHC synaptic pole where [Ca2+]i increases
rapidly (time to peak ≤ 1 s) and thereby spreads to the
rest of the cytoplasm with no evidence of secondary
release. However, we cannot exclude the possibility that
the lack of secondary CICR along the cell lateral wall is a
consequence of the poor metabolic state of the OHCs in
these testing experimental conditions.
A feature of these recordings was the lack of correla-
tion between the amplitude of the K+ current and the
Ca2+ responses. In some experiments, we did not observe
any increase in [Ca2+]i following the application of carba-
chol, despite the activation of Ca2+ dependent K+ currents
(Fig. 2). Furthermore, in cells where we did observe a
simultaneous increase in both [Ca2+]i and Ca2+-dependent
K+ current, this response was short-lived, and following a
few applications of carbachol only small K+ currents
remained. This was true for both preparations used in the
Ca2+-imaging experiments. In the experiments on iso-
lated OHCs were whole-cell recording was performed
alone, the responses to ACh were generally observed
over a longer time period, although exposure to caffeine
was shown to lead to a rapid and irreversible diminution
of the response in some instances. The more long-lasting
response under these recording conditions may have

Cell Calcium (2000) 28(3), 195–203 © Harcourt Publishers Ltd 2000

 Cholinergic response of outer hair cells 201

Fig. 5 Modulation of the current induced by ACh by caffeine and ryanodine. (A&C) Amplitude of the ACh-evoked current in different
isolated OHCs in response to caffeine (5 mM, A) and ryanodine (0.1 mM, C), applied as indicated by the filled horizontal bars. The numbers
indicate when the corresponding records shown in B (for the experiment shown in A) and D (for the experiment shown in C) were acquired.
(B&D) Examples of the current evoked by ACh (70 µM; horizontal bars) taken before (1), during (2) and after (3) the applications of caffeine
and ryanodine respectively. Holding potential –60 mV.

had something to do with the higher concentration of
Ca2+ buffer (5 mM EGTA) in the intracellular solution.
Thus, a general build-up of intracellular Ca2+ in cells dial-
ysed with a low concentration of EGTA (0.5 mM) with
each application of cholinergic agonist may have con-
tributed to store depletion, presumably as a result of
inadequate store refilling in the face of CICR. In addition,
the use of a lower concentration of ACh (compared to
1 mM carbachol) may have helped reduce the rate of
decline in the K+ current. Although carbachol is less
potent than ACh as an agonist for the hair cell ACh
receptor [36], the high concentration of carbachol used
would more than compensate for this, and might also
have contributed to store depletion.
The picture that emerges is of a labile Ca2+-store,
releasing Ca2+ in response to Ca2+-influx through the
ACh receptors, and prone to depletion over time or after
exposure to caffeine. One way of explaining the lack of
correlation between the Ca2+-activated K+ channels and
the rise of [Ca2+]i is to suggest that in OHCs, as well as in
other vertebrate hair cells [37], Ca2+-activated K+ chan-
nels co-localize with Ca2+-permeable channels (in this
case the hair cells’ ACh receptor). Therefore, Ca2+ need
not diffuse more than a few tens of nanometres from the
site of entry to activate the K+ current [38, 39]. In OHCs,
confinement of Ca2+ may be partly due to the close appo-
sition, within a few nanometres, of the synpatic cistern to
the plasma membrane [18]. In the absence of Ca2+
release, due to store depletion, such a confined entry of
Ca2+, being well below the ~1 µm spatial resolution of
our system [22], is likely to have remained undetected as
a fluorescence change.

© Harcourt Publishers Ltd 2000 Cell Calcium (2000) 28(3), 195–203

202 MG Evans, L Lagostena, P Darbon, F Mammano
In conclusion, our results show that in response to
application of carbachol, Ca2+ rises rapidly at the synaptic
pole of the OHCs and much more slowly closer to the
apex. This is indicative of diffusion of Ca2+ from base to
apex. We have not managed to determine the importance
of the Ca2+ stores in mediating the response, although
the fact that the ACh-evoked current was modulated by
both caffeine and ryanodine does imply that the Ca2+
stores release Ca2+ in response to cholinergic agonists.
Thus, to explain our results we have drawn on the exist-
ing notion of basally located Ca2+ store, possibly the sub-
synaptic cisternae [16], and have added to it a tendency
for the store to empty. Whatever the precise role of the
Ca2+ store in initiating the K+-based hyperpolarization
produced by ACh in OHCs, the major question now con-
cerns how the changes the membrane potential and
[Ca2+]i control the mechanical properties of OHCs [40,
41], leading to the changes in cochlear mechanics that
are thought to be responsible for efferent inhibition [42].
ACKNOWLEDGEMENTS
This work was supported by Istituto Nazionale di Fisica
della Materia, Ministero della Ricerca Scientifica, The
Wellcome Trust (grant 046090 to MGE), The Physio-
logical Society and Defeating Deafness. We thank Ivo
Priogioni, Paul Fuchs and David Furness for their com-
ments on this paper.
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