Brain Research 922 (2001) 65–70

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Research report

Intracellular calcium and outer hair cell electromotility

¨ a,b , David Z.Z. He a , Otto´ Ribari
Magdolna Szonyi ´ b , Istvan
´ Sziklai c , Peter Dallos a , *
a
Auditory Physiology Laboratory, Departments of Communication Sciences and Disorders and Neurobiology and Physiology,
Frances Searle Building, Northwestern University, 2299 North Campus Drive, Evanston, IL 60208, USA
b
Department of Oto-Rhino-Laryngology, Head and Neck Surgery, Semmelweis University Medical School, Budapest, Hungary
c
Clinic of Oto-Rhino-Laryngology, Debrecen University Medical School, Debrecen, Hungary

Accepted 25 September 2001

Abstract

The influence of increased intracellular calcium level on outer hair cell (OHC) electromotility was examined by means of transcellular
electrical stimulation in a partitioning microchamber. Electromotile activity was measured before and after application of the calcium
ionophore ionomycin, which promotes the inflow of extracellular calcium, as well as its release from intracellular calcium stores. The
ionomycin solvent, dimethyl sulphoxide (DMSO), by itself elicited a significant decrease in the magnitude of OHC electromotility. The
DMSO effect was counteracted by 10 mM ionomycin and was reversed by 50 mM ionomycin. The increase in electromotility is partially
mediated by a calmodulin-dependent mechanism, since W7, a calmodulin antagonist, attenuated the 50 mM ionomycin-induced motility
increase. Our results suggest that the electromotility magnitude increase in isolated OHCs due to ionomycin is a calcium / calmodulin-
dependent phenomenon.  2001 Elsevier Science B.V. All rights reserved.

Keywords: Electromotility; Outer hair cell; Calcium; Ionomycin; Signal transduction

1. Introduction tudinal elongations are produced [5]. The longitudinal

Outer hair cells (OHCs) of the organ of Corti generate
motile responses at acoustic frequencies and presumably
amplify the movements of the cochlear partition. OHC
motility is driven by the cell’s receptor potential [14] and
is considered to be responsible for sharp mechanical tuning
and high sensitivity of the inner ear [2]. Electromotility is
independent of energy consumption, and is detectable in
Ca 21 and ATP depleted cells [10]. The electromotile
performance of OHCs is known to be modified by the
efferent neurotransmitter acetylcholine (ACh) [19]. This
influence of ACh is mediated by the entrance of calcium
into the cells through nicotinic ACh receptors [3,4]. The
intracellular signaling pathway between calcium influx and
electromotility magnitude increase is not fully understood.
Intracellular calcium stores are demonstrated to exist in
OHCs; both inositol trisphosphate (IP3) [11] and
ryanodine-sensitive receptor [17,18] operated pools are
present. Rising [Ca 21 ] i also induces a slow shape change
in isolated OHCs: circumferential shortening and longi-
component of the shape change is probably due to activa-
tion of phosphorylating enzymes: myosine light chain
kinase and the Ca 21 / calmodulin-dependent kinase II [13].
Both motor and cytoskeletal proteins may be targets of
phosphorylation which can change the magnitude of
electromotility [21]. Protein kinase A (PKA) or protein
kinase C (PKC) have no modulatory effect, however
cGMP increases motility magnitude [22].
We report the influence of [Ca 21 ] i on the electromotility
of OHCs by means of the application of ionomycin to the
lateral membrane of isolated OHCs inserted into a parti-
tioning microchamber.
Detailed descriptions of Ca 21 imaging experiments are
available in several reports, showing the results of apply-
ing 10 mM ionomycin solved in 0.1% DMSO to isolated
OHCs [1,5,7]. Therefore, in this study, calcium imaging
was not performed.
2. Materials and methods

*Corresponding author. Tel.: 11-947-491-3175; fax: 11-847-467- OHC electromotility was measured before and after 4
4327. min drug application. The following drugs were used: L15
E-mail address: p-dallos@nwu.edu (P. Dallos). medium and 0.1% DMSO as controls. Ionomycin (10 and

0006-8993 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved.
PII: S0006-8993( 01 )03150-X
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M. Szonyi et al. / Brain Research 922 (2001) 65 – 70
50 mM) was applied alone or coapplied with the cal-
modulin antagonist W7. Ionomycin was dissolved in 0.1%
DMSO. Twelve cells each were measured in the control as
well as in the experimental groups.
2.1. Experimental techniques
Three- to 5-weeks old guinea pigs were euthanized with
intraperitoneal injection of 1 ml pentobarbital. The organ
of Corti was dissected from all turns of the cochlea and
transferred into collagenase (Type IV, Sigma) solution (2
mg / ml in L15). After 15 min digestion, the organ of Corti
pieces were placed in an experimental chamber and the
OHCs were isolated by gentle trituration with a 100 ml
pipette. All experimental methods were approved by the
Northwestern University Animal Care and Use Committee.
Descriptions of the apparatus and method for motility
measurements are published elsewhere [6,9,22]. Briefly,
isolated OHCs were partially inserted with their synaptic
pole first into a microchamber with aperture diameter 8–9
mm (Fig. 1). The experimental bath was filled with L15
medium. OHCs were observed with an inverted micro-
scope (Zeiss LM201). The inserted cell formed a me-
chanically stable seal with the microchamber, but it could
be moved in and out of the chamber with a micrometer-
driven syringe which produced negative or positive pres-
Fig. 1. An OHC inserted into the microchamber with its synaptic pole inside. All the measurements were performed in this configuration. Command
voltage was delivered between electrolytes inside and surrounding the microchamber. Drugs were applied via a puffer pipette to the excluded cell segment.
sure. Cells of different length (40–75 mm) were selected
without obvious sign of damage, swelling, nucleus disloca-
tion or granulation. Drugs were delivered to the cells via a
2 mm tip diameter puffer pipette, located approximately 50
mm distance from the cell.
Our apparatus for electromotility measurement can
detect responses up to 2.5 kHz (3 dB cut-off frequency)
and 10 nm (responses to five presentations were averaged).
Cell motility was quantified by the change in the current of
a photodiode when the magnified image of the ciliated pole
of the cell was projected onto the photodiode through a
rectangular slit. The photocurrent response was calibrated
to length-change units by moving the slit a known distance
(2 mm at the image plane) for each experimental run.
The electrical stimulus was a sequence of 10 ms
duration rectangular pulses of alternating polarity separated
by 40 ms, increasing in magnitude from 640 to 6240 mV
in 40 mV steps (Fig. 2). Cells were inserted approximately
50% into the microchamber using negative pressure. The
motility of the excluded segment was measured. The
membrane potential change that drives the motile response
is unknown; it is determined by the electrical voltage
divider formed by included and excluded cell-membrane
segments [6]. The actual driving voltage is significantly
less than the applied voltage.
After inserting the cell into the microchamber, the first
measurement was performed. The motility of mechanically
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M. Szonyi et al. / Brain Research 922 (2001) 65 – 70
Fig. 2. Upper traces: Waveforms of the electrical stimuli. The voltage is
stepped between 2240 and 1240 mV in 40 mV steps. Representative
motile response waveforms from one cell before (A) and after (B) 50 mM
ionomycin application. In this and all subsequent figures, cell contraction
is plotted downward. (C) Voltage-to-length change curves. Abscissa is
command voltage (mV), the ordinate is motile response (nm). The
abscissa shows the polarity of the stimulus, thus negative voltages applied
to the bath induce depolarization of the excluded segment. Squares
represent control measurements before drug administration; diamonds
give the data with the experimental drug. It is noted that the actual
voltage applied to the cell is reduced from the command voltage by the
voltage divider composed of the excluded and included cell membrane
segments. Cell motility was measured as in Fig. 1.
stable cells was measured after 4 min drug application only
if they did not bend out of the focal plane or moved from
the slit in front of the photodiode. Mechanically unstable
cells were rejected.
2.2. Drugs
All drugs were dissolved in an L15 culture medium
(Gibco), pH 7.4, 300 mOsm, balanced with 15 mM N-2-
hydroxyethyl piperazine-N9-2-ethansulfonic acid (HEPES).
Ionomycin and W7 were purchased from Calbiochem. One
millimolar stock solutions were made and were stored at
225C8. Ionomycin was dissolved in DMSO; the stock
solution was freshly diluted before experiments. The final
concentration of DMSO was 0.1%.
67
2.3. Statistics
The length change vs. applied voltage curves of the two
measurements, before and after drug application, were
plotted for each cell. For each curve, 12 motility data
points were measured for command voltages ranging from
2240 to 1240 mV in 40 mV steps. The statistical treatment
applied to the data examined the differences between pre-
and post-treatment curves. Portions of the curves were
compared separately for hyperpolarizing and depolarizing
stimuli. For each half-curve, a 1 or 2 sign was assigned,
depending on the direction of change from pre- to post-
treatment. Thus, if for hyperpolarization the response
became larger when the drug was applied, a 1 sign was
assigned. If there was no change, a zero was assigned. In
virtually all cases, an unambiguous assignment of sign
could be made. The various treatments were then examined
as to their significance. The sign-test for matched pairs of
observations [15] was used. This is a non-parametric
statistical test that takes only the direction of change into
account, irrespective of the magnitude of the change. No
change is considered a tie and those data do not enter into
the computation. Treatment is based on the simple bino-
mial distribution and the test computes the likelihood of a
given occurrence, given the number of trials. After sum-
ming the occurrences having positive and negative signs
separately, the probability applicable to the smaller sum is
computed. If the probability is smaller than 0.1 in a
two-tailed test, the null hypothesis is rejected, and the drug
treatment is concluded to have significantly altered the
motile response. Only those pairs of data files were
included in the treatment whose control magnitude reached
at least 150 nm.
3. Results
L15 culture medium treatment of 12 cells in the control
experiment resulted in no significant change of motility
magnitude (P50.81) (Table 1). The control measurement
in the computation of the statistical test and of the
electromotility magnitude percentage change is the pre-
drug measurement in L15 medium. In our statistical test,
voltage vs. length change depolarizing and hyperpolarizing
half-curves were used, but, for clearer presentation, the
average percentage change for each drug was also com-
puted and is shown in Fig. 3. In Fig. 2C and 3, stimulus
polarity is used. When hyperpolarization or depolarization
is mentioned in the article, reference is made to command
voltages. The effective polarity is opposite: a negative
command voltage produces depolarization of the excluded
(measured) cell segment.
DMSO application (0.1%) elicited a significant decrease
in electromotility magnitude for all voltage steps (P,
0.002) (Fig. 3A). Out of 24 hyper- and depolarization
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M. Szonyi et al. / Brain Research 922 (2001) 65 – 70

Table 1
Results
L15 DMSO Iono 10 Iono 50 W7
Increase 11 2 9 13 9
Decrease 9 19 8 8 14
No change 4 3 7 3 1
Sign test P50.81 P,0.002 P51.00 P50.38 P50.4
Increases, decreases and no changes for each drug, as well as the results of the sign test, are shown. DMSO induced a statistically significant electromotility
magnitude decrease. The higher concentration ionomycin (50 mM) induced an electromotility magnitude increase for 13 data sets. Greater increases were
found for the 50 mM than for the 10 mM group. The administration of W7 attenuated the ionomycin effect, decreases again becoming dominant.

half-curves, 19 showed a decrease, two an increase and
three no change.
The Ca 21 ionophore ionomycin dissolved in DMSO was
used in two different concentrations. The lower concen-
tration (10 mM) counteracted the DMSO effect observed
for all the responses to depolarizing stimuli, but for four of
six hyperpolarizing stimuli, a motility magnitude decrease
was still seen due to the DMSO solvent (Fig. 3B).
Electromotility increased in nine, and decreased in eight
curves. The remaining seven response curves did not
change after treatment. (Table 1). A higher concentration
(50 mM) of the drug evoked an electromotility magnitude
increase in 13 cases of the 24 half-curves and significantly
increased the magnitude of the electromotility in com-
parison to the decrease evoked by the DMSO solvent (Fig.
3C).
A majority of the curves showed a decreased magnitude
after coapplication of 50 mM ionomycin and the water-
soluble calmodulin antagonist W7. In this group, we
observed 14 decreases, nine increases and one no change.
The average percentage changes are shown in Fig. 3D in
comparison to the 50 mM ionomycin condition. The effect
due to 50 mM ionomycin, while significantly reduced, was
not completely abolished by W7. For hyperpolarizing
stimuli, an electromotility magnitude decrease due to
DMSO was observed, but for depolarizing stimulation the

Fig. 3. The abscissa is the command voltage, the ordinate is the percentage length change. L15 medium caused a random increase and decrease within a
10% range. DMSO induced a statistically significant electromotility-magnitude decrease at all voltage step stimuli. In panels (B) and (C) the DMSO
response patterns are shown for comparison with the different drug treatments. Panel (B) shows the percentage length change induced by 10 mM
ionomycin. This drug was dissolved in DMSO, thus ionomycin counteracted the DMSO effect: the significant electromotility decrease disappeared. For
depolarizing stimuli, the ionomycin effect is more pronounced than for the hyperpolarizing stimuli. In panel (C), 50 mM ionomycin is shown to induce a
motility magnitude increase despite being dissolved in DMSO; it reversed the DMSO response at all voltage steps. Panel (D) demonstrates the
coapplication of 50 mM ionomycin and W7, a calmodulin antagonist. The electromotility magnitude decrease (due to DMSO) again became dominant.
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M. Szonyi et al. / Brain Research 922 (2001) 65 – 70
ionomycin effect is not completely eliminated by W7. The
statistics performed on the W7-treated cells, with L15
medium as control, yielded P50.4, a non-significant
change.
4. Discussion
The main finding of this study is that the ionomycin-
induced [Ca 21 ] i increase in isolated OHCs is an effective
cofactor in the enhancement of the electromotile response
of these cells.
Ca 21 ions are maintained at low levels in resting cells.
Na 1 / Ca 21 exchangers and Ca 21 pumps remove Ca 21 ions
from the cell. Ca 21 binding proteins and Ca 21 stores
(endoplasmic reticulum and the mitochondria) also control
the Ca 21 level in the cytoplasm and restrict Ca 21 diffu-
sion. Under physiological conditions, Ca 21 spikes and
fast-spread waves signify Ca 21 signals.
Ionomycin evokes calcium inflow from the extracellular
fluid, as well as calcium release from intracellular stores. A
transient, generalized increase of intracellular calcium is
induced by this drug with an effect along the whole cell
body. This phenomenon is well documented in several
studies [5,8,12,16]. In our experiments, ionomycin first
reached the apical part of the cell, since the cell was
inserted half-way into the microchamber with its synaptic
pole inside. IP3-sensitive stores are localized to the lateral
cell membrane of OHCs [7], thus ionomycin may release
Ca 21 from these stores as a first step, then the Ca 21 signal
may spread along the lateral cell membrane.
Previously, a relation between increasing intracellular
Ca 21 level and slow shape changes of isolated OHCs was
demonstrated. The effect of the Ca 21 ionophore could be
completely blocked by the calmodulin antagonist tri-
fluoperazine [5] and by ML9, a Ca 21 / calmodulin-depen-
dent kinase inhibitor [1]. The most likely candidate
enzymes mediating the slow shape change are myosin light
chain kinase and Ca 21 / calmodulin-dependent protein ki-
nase II [13].
ACh was also applied in our previous experiments and
an electromotility magnitude increase was detected that
could be blocked by coapplication of KN-62, a calcium-
calmodulin protein kinase II inhibitor. These data are
published elsewhere [19,20,22]. As shown in the Results
section, the calmodulin antagonist W7 abolished the
ionomycin-evoked electromotility magnitude increase in
the case of hyperpolarizing stimuli, but, for highly de-
polarizing voltages, the ionomycin effect was not coun-
teracted. W7 shifted the voltage sensitivity of the nonlinear
membrane capacitance (an electrical signature of motility)
of isolated OHCs in the depolarizing direction [8]. This
phenomenon may explain the different W7 effects for
hyper- and depolarizing stimuli.
Caffeine-evoked calcium release from intracellular
stores also induces an electromotility magnitude increase
69
when the drug is applied to the synaptic pole of OHCs
¨
(Szonyi et al., unpublished data). The ionomycin effect is
similar to the ACh-induced [Ca 21 ] i increase; both elevate
the electromotility magnitude and both can be blocked by
calmodulin inhibitors. Acetylcholine and ionomycin may
act via a calcium / calmodulin-dependent protein kinase.
These kinases probably phosphorylate certain cytoskeletal
proteins [21] and thereby change the axial stiffness of the
hair cell [3]. Such an internal stiffness change alters the
load on the motility motors and thereby modifies the
magnitude of the electromotile response.
Acknowledgements
This work was supported by OTKA (Hungarian Nation-
al Research Fund) grants T025050 and T 25231.
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