Cell Calcium 33 (2003) 185–195

Regulation of outer hair cell cytoskeletal stiffness by intracellular Ca2+:

underlying mechanism and implications for cochlear mechanics
Gregory I. Frolenkov a,∗ , Fabio Mammano b , Bechara Kachar a
a Section on Structural Cell Biology, National Institute on Deafness and Other Communication Disorders (NIDCD),
National Institutes of Health (NIH), Bldg. 50, Room 4346, Bethesda, MD 20892-8027, USA
b Istituto Nazionale di Fisica della Materia (INFM) and Venetian Institute of Molecular Medicine (VIMM), Padova, Italy
Received 14 August 2002; received in revised form 25 November 2002; accepted 25 November 2002

Abstract

Two Ca2+ -dependent mechanisms have been proposed to regulate the mechanical properties of outer hair cells (OHCs), the sensory–motor
receptors of the mammalian cochlea. One involves the efferent neurotransmitter, acetylcholine, decreasing OHC axial stiffness. The other
depends on elevation of intracellular free Ca2+ concentration ([Ca2+ ]i ) resulting in OHC elongation, a process known as Ca2+ -dependent
slow motility. Here we provide evidence that both these phenomena share a common mechanism. In whole-cell patch-clamp conditions, a
fast increase of [Ca2+ ]i by UV-photolysis of caged Ca2+ or by extracellular application of Ca2+ -ionophore, ionomycin, produced relatively
slow (time constant ∼20 s) cell elongation. When OHCs were partially collapsed by applying minimal negative pressure through the patch
pipette, elevation of the [Ca2+ ]i up to millimole levels (estimated by Fura-2) was unable to restore the cylindrical shape of the OHC.
Stiffness measurements with vibrating elastic probes showed that the increase of [Ca2+ ]i causes a decrease of OHC axial stiffness, with
time course similar to that of the Ca2+ -dependent elongation, without developing any measurable force. We concluded that, contrary to a
previous proposal, Ca2+ -induced OHC elongation is unlikely to be driven by circumferential contraction of the lateral wall, but is more
likely a passive mechanical reaction of the turgid OHC to Ca2+ -induced decrease of axial stiffness. This may be the key phenomenon for
controlling gain and operating point of the cochlear amplifier.
© 2003 Elsevier Science Ltd. All rights reserved.

Keywords: Cochlear amplifier; Prestin; Olivocohlear bundle; Acetylcholine; Intracellular calcium stores; Slow motility

1. Introduction cochlear partition, which is achieved by keeping the basilar

Cochlear OHCs are responsible for amplification of
sound-induced vibrations within the organ of Corti (re-
viewed in [1,2]) due to their ability to elongate and shorten
at acoustic frequencies following the changes of intracellular
potential (for review, see [3,4]). A unique, ATP-independent
[5], membrane-based [6] form of cellular motility is re-
sponsible for these fast changes of OHC shape, known
as electromotility. A recently cloned membrane protein,
prestin [7], populates the lateral plasma membrane of OHCs
[8] and serves as a direct electro-mechanical transducer
[4], presumably providing the force required for cochlear
amplification.
The main function of the cochlear amplifier is to coun-
teract intrinsic viscous losses of vibrating energy of the
∗ Corresponding author. Tel.: +1-301-402-4211; fax: +1-301-402-1765.
E-mail address: gregory frolenkov@nih.gov (G.I. Frolenkov).
membrane at the threshold of spontaneous oscillations (for
review, see [9]). Enormous range of input sound pressures
of about six orders of magnitude requires especially dedi-
cated mechanisms capable of adjusting the gain of cochlear
amplifier. Due to the strategic location of OHCs in the organ
of Corti, any change of their mechanical properties, par-
ticularly somatic length and cytoskeletal stiffness, is bound
to profoundly affect cochlear mechanics, for instance by
shifting the operating point of the mechano-sensory appara-
tus hosted in the cell stereocilia and/or the local resonance
frequency of the cochlear partition.
In fact, OHCs are the target of an abundant efferent
innervation originating in the brainstem, whose activation
results in elevation of hearing threshold, i.e. a decrease of
system’s sensitivity (reviewed in [10]). The principal neu-
rotransmitter of this efferent system is acetylcholine (ACh)
(for review see [11]). Whole-cell recordings from isolated
OHCs have provided functional evidence for cholinergic
receptors localized around the base of the cell, where the

0143-4160/03/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0143-4160(02)00228-2
186 G.I. Frolenkov et al. / Cell Calcium 33 (2003) 185–195
efferent synapses are located [12]. The action of ACh on
OHC currents is relatively fast (time scale of tens or hun-
dreds of milliseconds [12,13]), requires extracellular Ca2+
[13,14], is accompanied by changes of [Ca2+ ]i [15,16],
and is probably mediated by a novel heteromeric receptor,
assembled from ␣9 and ␣10 subunits [17].
In addition to fast Ca2+ -dependent ionic currents stim-
ulated by ACh, two slower Ca2+ -dependent mechanisms,
operating in a time scale of tens of seconds and regulating
the mechanical properties of cochlear OHCs, have been
proposed. On the one hand, ACh would decrease OHC
axial stiffness [18] presumably through Ca2+ -dependent
phosphorylation of unspecified cytoskeletal proteins [19]
without appreciable effects on OHC electromotile mecha-
nism [20]. On the other hand, elevation of [Ca2+ ]i would
trigger a circumferential contraction of the OHC cortical
cytoskeleton resulting in axial elongation of the OHC [21].
This elongation, known as “Ca2+ -dependent slow motility”,
involves Ca2+ /calmodulin-dependent protein phosphoryla-
tion [22,23].
Here we used patch-clamp recordings, UV photolysis,
Ca2+ -imaging, and stiffness measurement techniques to
study the properties Ca2+ -induced slow motility and its rela-
tionship with cytoskeletal stiffness.
2. Methods
2.1. Cell preparation
Adult guinea pigs (200–400 g) were sacrificed by suffoca-
tion with carbon dioxide and decapitated according to NIH
Guidelines for Animal Use. The temporal bones were re-
moved from the skull and placed in a modified Leibowitz cell
culture medium (L-15) containing (in mM): NaCl (137), KCl
(5.4), CaCl2 (1.3), MgCl2 (1.0), Na2 HPO4 (1.0), KH2 PO4
(0.44), MgSO4 (0.81). Osmolarity and pH were adjusted to
325 ± 2 mOsm with d-glucose and 7.35 mOsm with NaOH,
respectively. To isolate OHCs, the bulla was opened to ex-
pose the cochlea, and the otic capsula was chipped away
with a surgical blade starting from the base. Strips of the
organ of Corti were dissected from the modiolus with a
fine needle, transferred with a glass pipette to a 100 ␮l
drop of medium containing 1 mg/ml of collagenase type
IV (Life Technologies, Rockville, MD), and incubated for
15–20 min. When experiments required preincubation with
the AM-ester form of Ca2+ -sensitive indicators and/or pho-
tolabile Ca2+ -chelators (see the description later), collage-
nase was added 15–20 min before the end of the incubation
period. After incubation, cells were dissociated by gentle
reflux of the tissue through the needle of a Hamilton sy-
ringe (N. 705, 22 gauge) and allowed to settle on the slide
for 5–10 min. OHCs were placed in a laminar flow bath
(100 ␮l), with exchange of L-15 medium (about 5 ml/h) by a
pressurized perfusion system (BPS-4; ALA Scientific Instru-
ments, Westbury, NY), and maintained at room temperature
(22–24 ◦ C) throughout the experiments. In several control
experiments, OHCs were placed in Ca2+ -free L-15 medium,
prepared as described above with CaCl2 replaced by 2 mM
EGTA.
2.2. Patch-clamp recordings
OHCs were visualized with an inverted microscope (Ax-
iovert 200, Carl Zeiss, Gottingen, Germany) and the follow-
ing morphological features were used to determine viability:
uniform cylindrical shape, basal location of the nucleus,
membrane birefringence and intact stereocilia. Patch-clamp
pipettes were filled with an intracellular solution containing
(in mM): KCl (144), MgCl2 (2.0), EGTA (0.5), Na2 HPO4
(8.0), NaH2 PO4 (2.0), Mg-ATP (2.0), Na-GTP (0.2), ad-
justed to pH 7.2 with KOH and brought to 325 mOsm
with d-glucose. Patch-clamp recordings were performed
using an Axopatch 1D amplifier (Axon Instruments, Foster
City, CA). Pipettes for conventional whole-cell recordings
were formed on a programmable puller (P87, Sutter Instru-
ments, Novato, CA) from 1.0 mm O.D. borosilicate glass
(#30-30-0, FHC, Bowdoinham, ME). Current and voltage
were sampled at 100 kHz using a standard laboratory in-
terface (Digidata 1200A, Axon Instruments) controlled by
pClamp 7.0 software (Axon Instruments). The uncompen-
sated pipette resistance was typically 3–5 M when mea-
sured in the bath and the access resistance did not exceed
15 M under whole-cell patch-clamp conditions. Potentials
were corrected off-line for the error due to the access resis-
tance. Junction potential was −4.2 mV, as computed by the
pClamp 7.0 software based on the given solution compo-
sition. This value was rather small and no correction was
applied to the data for liquid-junction potential.
2.3. Cell loading
To monitor changes of [Ca2+ ]i , we used two Ca2+ -sensi-
tive fluorescent indicators (Molecular Probes, Eugene, OR):
Calcium Green-1 for experiments requiring photolysis of
caged compounds, and Fura-2, otherwise. These dyes were
loaded into the cell either through the patch pipette or by in-
cubation with their cell-permeable AM-ester derivatives. In
the patch-clamp experiments, the intra-pipette solution was
prepared as described earlier, omitting EGTA, and supple-
menting it with 50 ␮M of the cell-impermeable form of one
of these dyes. In the experiments with free isolated OHCs,
the acetoxymethyl ester form of the dye (10 mM stock so-
lution in DMSO) was dispersed with the equal amount of
20% DMSO-based solution of Pluronic F-127 (Molecular
Probes), diluted to the final concentration of 10 ␮M with
L-15, and used to pre-incubate OHCs for 30 min at room
temperature.
The increase of [Ca2+ ]i was induced either by ex-
tracellular application of the Ca2+ -ionophore ionomycin
(Sigma-Aldrich, St. Louis, MO) or by photolysis of a pho-
tolabile Ca2+ chelator, pre-complexed with Ca2+ . In the
 G.I. Frolenkov et al. / Cell Calcium 33 (2003) 185–195
patch-clamp experiments, NP-EGTA (Molecular Probes)
at concentration of 2 mM was mixed with Ca2+ (1.5 mM),
added to the intra-pipette solution without EGTA, and sup-
plemented with 50 ␮M of Calcium Green-1. In other experi-
ments, OHCs were incubated with the cell-permeable caged
Ca2+ compound Nitr-5/AM (Calbiochem, San Diego, CA)
at 25 ␮M concentration for 30 min (at room temperature).
In the latter case, the chelator bound only the free Ca2+
inside the cell and the exact amount of Ca2+ complexed to
the photoactive substance remained undetermined.
2.4. Drug delivery
Stock solution of ionomycin in DMSO (10 mM) was di-
luted in the extracellular solution to a final concentration of
5 or 10 ␮M and was used to fill a puff pipette, prepared sim-
ilarly to the patch pipette. This pipette was placed near the
basolateral wall of the OHC and pressure (10–15 kPa) was
applied to its back by a pneumatic injection system (PLI-100,
Medical Systems Corp., Greenvale, NY) gated under soft-
ware control. Typical drug delivery time was ≤30 ms, as de-
termined by positioning the patch pipette in front of the puff
pipette and monitoring the change in junction potential.
2.5. Ca2+ imaging and photolysis of caged compounds
Light from a 175 W stabilized Xenon arc source (Lambda
DG-4, Sutter instruments) was coupled via a liquid light
guide to the epifluorescence port of our Axiovert 200
microscope, equipped with an oil-immersion UV-passing
objective (Carl Zeiss, F-Fluar, 40×, 1.3 N.A.). The follow-
ing excitation filters were mounted into the light source:
(1) a band-pass filter used to excite Calcium Green-1
(HQ480/40×, Chroma Technology, Brattleboro, VT); (2) a
broad-band UV filter (330WB80, Omega Optical, Brattle-
boro, VT) for uncaging photolabile Ca2+ chelators; (3) a
band-pass filters to excite Fura-2 (340HT15 and 380HT15,
Omega Optical). The light source was pre-programmed to
deliver 0, 25, 50, and 75% light attenuation at each filter
position. Additional attenuation was provided to the 480 nm
(Calcium Green-1) excitation channel by a neutral density
filter. Switching of the filter positions and attenuation steps
was controlled by the acquisition software (MetaMorph,
Universal Imaging Co., Downingtown, PA). The illumi-
nation intensity was adjusted to avoid phototoxicity by
reducing dye photo-bleaching rates to ≤0.1%/s. Decompo-
sition of the caged compounds was minimized by inserting
a green interference filter into microscope condenser to cut
off the wavelengths in the near-UV range from bright-field
illumination.
The reflector turret of the microscope was equipped
with a special dichroic mirror for simultaneous excitation
in the UV and 480 nm spectral regions (#74100, Chroma
Technology) and a band-pass observation filter optimized
for the Ca2+ -selective dyes Calcium Green-1 and Fura-2
(HQ535/50m, Chroma Technology). Fluorescence images
187
were formed on a scientific grade cooled CCD sensor (Mi-
cromax 1300Y, Roper Scientific, Trenton, NJ) using 1.6×
secondary magnification. Sensor’s output was binned 2 × 2,
digitized at 12 bit/pixel, recorded to a host PC controlled by
the MetaMorph software, and analyzed off-line.
Before subjecting OHCs to any sort of stimulation, 30
control images were acquired to determine baseline. (Here
and thereafter, we shall designate “image” as a single
non-ratiometric fluorescent image of the cell or the pairs
of images taken at different wavelengths when the cell was
loaded with Fura-2.) During the experiment, either the injec-
tion system was activated to apply ionomycin, or broadband
UV illumination of maximal intensity was delivered for
1–3 s to initiate photolysis of caged compounds. To track the
effect of stimulation, 120 images were acquired. Exposure
times depended on the intensity of fluorescence and were
typically in the range of 200–400 ms for non-ratiometric
imaging and 200–800 ms for ratiometric imaging. Time de-
lay for switching filters/attenuations was negligible (∼1 ms)
compared to typical exposure times. Overall, sampling rate
was in the range from 1 to 2.5 images per second.
When the cell was loaded with Fura-2, fluorescence
was excited at the standard wavelength pair of 340 and
380 nm to estimate [Ca2+ ]i from consecutive pair ratios
[24]. Background-subtracted fluorescence ratio images were
calibrated using a calcium calibration buffer kit (C-3722,
Molecular Probes). Background fluorescence was calcu-
lated for each image individually as the average intensity of
fluorescence in a manually selected reference region, com-
mon to the whole sequence, devoid of any obvious cellular
structures.
To estimate the overall changes of [Ca2+ ]i in the OHC,
we constructed a region of interest covering the whole
cell body for each image in the sequence. For ratiometric
measurements, the average ratio of 340/380 nm fluorescent
signals from this region was used to estimate changes of
average [Ca2+ ]i in OHCs. When the single-wavelength
indicator was used, average fluorescence signals from
this region of interest was computed as ratio F /F =
[F (t) − F (0)]/F (0), where t is time, F(t) is fluorescence
following a stimulus that causes Ca2+ elevation within the
cell, and F(0) is pre-stimulus (baseline) fluorescence. This
procedure monitors changes of [Ca2+ ]i correctly only if the
cell does not change the shape. In our experiments, OHCs
elongated, the area of the corresponding region of interest
increased, and changes of the [Ca2+ ]i were thus generally
underestimated. Aware of this potential pitfall we refrained
from drawing quantitative conclusions from non-ratiometric
[Ca2+ ]i measurements.
2.6. Stiffness measurement
Elastic probes were pulled from the same 1.0 mm O.D.
borosilicate glass used for patch pipette manufacturing. The
tapered fiber of each probe was 10–15 mm in length and
∼0.5 ␮m in tip diameter. We did not measure the actual
188 G.I. Frolenkov et al. / Cell Calcium 33 (2003) 185–195

Fig. 1. Measurement of OHC axial stiffness. (A) Positioning of the elastic probe (P) on the cuticular plate (CP) of the OHC; St, stereocilia. (B) Apparent
ratio of cell/probe stiffness (see Section 2) estimated at four different positions of the probe, starting from a point above the cuticular plate and moving
towards the cell (arrows). Shaded region indicates the position of the cell edge, where the probe contacted the cuticular plate. Inset—movements of the
elastic probe unattached to the cell (unloaded) and maximally pushed against the cuticular plate (loaded).

stiffness of the probe, but our manufacturing protocol was
optimized to ensure that it was comparable to the OHC ax-
ial stiffness. For all OHCs included in the analysis, this was
ascertained by pushing the probe against the cell’s cuticular
plate and confirming that cell compression and probe de-
flection were comparable. To measure relative stiffness, the
probe was attached to a vibrating piezo actuator mounted on
a 3-axis micromanipulator (Narishige, Tokyo, Japan). The
amplitude of vibration was <1.5 ␮m to minimize poten-
tial traumatic effects of external mechanical stress on the
cell.
The isolated OHCs were held by the patch pipette, located
just above the nucleus, by applying gentle suction without
breaking the patch. The probe was oriented parallel to the
OHC cuticular plate and approximately perpendicular to the
long axis of the cell (Fig. 1A). OHCs with tilted cuticular
plates were not included in this study. Care was taken to
keep the probe away from the hair bundle, as far as possible,
without compromising its ability to produce axial compres-
sion of the cell and without tilting the cuticular plate. This
arrangement provides a predominant compression of the cell
body in axial direction even for curved OHCs. Despite some
possible radial component, we will designate stiffness esti-
mated in this arrangement as “axial stiffness”.
Sinusoidal command voltages with a frequency of 1–3 Hz
drove the vibrating motion of the probe along the OHC axis
in such a way that it would alternatively compress and relax
the cell. For each cell, the motion of the freestanding probe
(unloaded vibration) was recorded first (Fig. 1B, inset). The
probe was then carefully advanced to compress the cell, until
its vibration amplitude stopped decreasing, indicating full
contact. Under these conditions, the apparent cell stiffness
reached a plateau value (Fig. 1B). The effects of [Ca2+ ]i
changes on the OHC stiffness were monitored by measuring
vibration amplitude around this position. Before stimulating
the cell, a baseline record of 15–40 s duration was obtained,
ensuring that recording conditions or cell stiffness did not
change spontaneously.
Assuming the probe and the cell as two sequentially con-
nected elastic elements with stiffness Kp and Kc , respec-
tively, the stiffness ratio could be estimated as: Kc /Kp =
Au /Al − 1, where Au and Al are unloaded and loaded vi-
bration amplitude, respectively. Since only Kc is expected
to change after OHC stimulation, percent changes of Kc /Kp
simply reflect underlying percent changes of Kc .
2.7. Measurements of cell and probe movement
Measurements of cell and/or elastic probe motion were
performed as described in [25]. Briefly, motion was recorded
with a video camera interfacing with an inverted microscope
equipped with Differential Interference Contrast (DIC) op-
tics to an optical disk recorder (Panasonic TQ-3031F). Dig-
itized images were analyzed off-line with the MetaMorph
image-processing system. For motion quantification, a mea-
suring rectangle ranging in length from 5 to 20 ␮m and
composed of 3–15 rows of pixels was positioned across the
moving edge of the cell or the probe. The average intensity
profile across the edge of the object was calculated and the
number of points in the profile was increased 10 times by cu-
bic spline interpolation. Motion of the object was calculated
from the frame-by-frame shift (computed by a least-square
procedure) in the interpolated intensity profiles. The sensi-
tivity of the measurement was ∼0.02 ␮m, as previously de-
termined [25].
2.8. Statistical analysis
All values are given as means ± S.E.M. unless otherwise
stated. Statistical significance was estimated using Student’s
t-test at P = 0.05 level. Curves were fitted to data by a
Levenberg–Marquardt algorithm using Origin 6.0 software
 G.I. Frolenkov et al. / Cell Calcium 33 (2003) 185–195 189

(Microcal Software, Northampton, MA). This software es-
timated also the standard errors of fitting parameters.
3. Results
3.1. Measurement of Ca2+ -induced elongation of OHCs
in whole-cell patch-clamp conditions
Fig. 2A shows a typical response of a relatively long
OHC to the pressure application of the Ca2+ -ionophore ion-
omycin. When applied at 5 ␮M concentration, ionomycin
produced a dramatic increase of the [Ca2+ ]i from base-
line values of 145–220 nM (typical of isolated OHCs) to
0.8–4.5 ␮M (n = 10 cells). In 12 out of 15 cells maintained
under whole-cell patch-clamp conditions, ionomycin also
elicited an outward current of 40–280 pA when measured
at holding potentials in the range from −70 to −55 mV
(Fig. 2C). This current, which has been previously ob-
served in OHCs [12,20,26], reflected likely the activation
of Ca2+ -dependent K+ -channels. Although the intracellu-
lar potential was fixed, the ionomycin-induced increase of
[Ca2+ ]i was followed by a relatively slow elongation of the
OHC. In a sample experiment shown in Fig. 2C, the latency
of this elongation was about 10 s and the time constant was
15.8 ± 0.9 s. Cell elongation and [Ca2+ ]i increase were sup-
pressed after more than 15 min of incubation in Ca2+ -free
extracellular medium (data not shown). Therefore, we shall

Fig. 2. Ca2+ -induced OHC currents and motile responses in whole-cell patch-clamp conditions. (A, C) Responses of an OHC to the application of
ionomycin (10 ␮M). (B, D) Responses of another OHC to increase of the [Ca2+ ]i produced by UV-photolysis of the photolabile Ca2+ -chelator NP-EGTA
(2 mM) pre-loaded with Ca2+ (1.5 mM) and introduced into the cell through the patch pipette. (A, B) Pairs of OHC images showing the distribution
of the [Ca2+ ]i before (left images in the pair) and after (right images) stimulation. [Ca2+ ]i changes were estimated with the ratiometric fluorescent
Ca2+ -indicator Fura-2 in A, and the single-wavelength indicator Calcium Green-1 in B. (C, D) From top to bottom: changes of the total Ca2+ -dependent
fluorescent signal emitted from OHCs; relative changes of OHC length; whole-cell current. The timing of ionomycin application is indicated by a
horizontal bar (C), the timing of UV-illumination is indicated by two arrows (begin–end, D). The measurement procedures are described in Section 2.
OHC in A and C was held at −48 mV, OHC in B and D was held at −53 mV.
190 G.I. Frolenkov et al. / Cell Calcium 33 (2003) 185–195

hereafter attribute the ionomycin-induced OHC elongation
to the “Ca2+ -induced slow motility” observed previously
in free isolated unpatched OHCs [21].
The use of UV-photolysis affords an alternative method
to gate the increase of the [Ca2+ ]i with better temporal
resolution. Photolysis of the UV-sensitive Ca2+ chelator,
NP-EGTA, pre-complexed with Ca2+ and introduced into
the cell through the patch pipette, produced fast and dis-
tinct increase of [Ca2+ ]i in OHCs (Fig. 2B). However,
we were unable to estimate the absolute values of [Ca2+ ]i
changes because of the difficulties involved in the simulta-
neous usage of the ratiometric Ca2+ -indicator Fura-2 with
photolabile compounds [27]. The fluorescence of OHCs
loaded with Calcium Green-1, a non-ratiometric indicator
excited in the visible region of the spectrum, increased
only by 5.4 ± 0.3% after photolysis (n = 4). The amount
of Ca2+ released by UV illumination depends on the in-
tracellular concentration of the chelator. After establish-
ing whole-cell configuration, the chelator slowly diffused
into the cell and repeated application of UV illumina-
tion produced progressively larger changes of fluorescent
signal, reaching a plateau after 15–30 min of whole-cell
recording. The Ca2+ -induced elongation of OHCs could
be observed during the first 10–15 min of whole-cell
patch-clamp recording, suggesting that it requires some
soluble intracellular components, which were inevitably
washing out in the whole-cell recording. For these reasons,
data collection was terminated after 15 min of whole-cell
recording.
The increase of the [Ca2+ ]i by UV-photolysis evoked a
noticeable outward current (50 pA for the cell illustrated in
Fig. 2D, 30–110 pA for four out of five OHCs tested). This
current was maximal at holding potentials around −20 mV,
as expected for a Ca2+ -activated K+ -current (data not
shown). This current was adapting even if [Ca2+ ]i remained
elevated (Fig. 2D). In contrast to the relatively fast changes
of [Ca2+ ]i and whole-cell current, changes of OHC length
occurred with slower time constant (∼10 s for the cell il-
lustrated on Fig. 2D), similar to the ionomycin-induced
elongation (Fig. 2A). In four out of five cells, the elonga-
tion of the OHC initiated by UV-photolysis was bi-phasic
(Fig. 2D) without obvious correlation with the time course
of the fluorescence change.
3.2. Comparing the time course of Ca2+ -induced OHC
elongation in free cells and under patch-clamp conditions
In a separate set of experiments, we studied Ca2+ -induced
elongation in free (unpatched) OHCs. To monitor [Ca2+ ]i
changes evoked by UV-photolysis, OHCs were loaded with
the cell permeant forms of Nitr-5 and Calcium Green-1 (see
Section 2). Fig. 3 presents average responses obtained in
these conditions and compares them with the correspond-
ing results of patch-clamp experiments. UV-activated Ca2+
photo-release was less effective in free unpatched OHCs than
in patch-clamped OHCs, as expected given that the mem-
brane permeant form of the photolabile Ca2+ chelator may
only complex with the limited amount of free intracellular
Ca2+ (less or about 200 nM, compared to 1.5 mM of the
patch pipette mixture; see Section 2). Nonetheless, measur-
able elongations of the OHC were still evoked under these
unfavorable conditions and were appreciably faster than in
the patch-clamp recordings. Furthermore, cell length in-
creased with short latency, typically shorter than the duration
of the UV-irradiation in these experiments (<3 s). Finally,
the time constant of OHC elongation (6.5 ± 0.7 s, Fig. 3A)
was significantly faster than that observed in patch-clamp
experiments (18 ± 1 s, Fig. 3B, P < 0.0001). Since this fast

Fig. 3. Ca2+ -dependent elongation is faster in isolated unpatched (A) vs. patched (B) OHCs. Rise of the [Ca2+ ]i in these cells was produced by
UV-photolysis of photolabile Ca2+ -chelators, introduced into the cell either by pre-incubation with the AM-ester derivative (Nitr-5 in A) or through the
patch pipette (NP-EGTA in B). Timing of UV illumination is indicated by two arrows (begin–end). Top records on each panel show the relative changes
of the Calcium Green-1 fluorescence signal averaged over the whole cell body. Bottom records illustrate relative changes of cell length. Each record is
an average of the responses observed in n = 8 (A) and n = 4 (B) cells, respectively. Error bars on the left side of each record indicate S.E.M. values.
 G.I. Frolenkov et al. / Cell Calcium 33 (2003) 185–195 191

phase of the Ca2+ -dependent OHC elongation was missing
when the intracellular potential was held constant, we con-
clude that it resulted from a voltage-driven elongation of
OHC activated by Ca2+ -induced hyperpolarization, whereas
the slow Ca2+ -induced component of the elongation was
probably too small to be detected under these experimental
conditions.
3.3. Ca2+ -induced changes of OHC turgor
The cylindrical shape of OHCs results from positive in-
tracellular pressure (cell turgor [28]). Thus, if cell elonga-
tion were actually driven by circumferential contraction of
the lateral wall, as proposed [21], the elevation of [Ca2+ ]i
should, in principle, promote restoration the shape of an
OHC partially collapsed by applying small negative pres-
sure via the patch pipette. This was never observed in any
of n = 4 cells tested (Fig. 4A). Instead, OHCs often tended
to lose their cylindrical shape after ionomycin (Fig. 4B).
Changes in cell turgor were most prominent when [Ca2+ ]i
increased above about 5 ␮M as determined by Fura-2 fluo-
rescence. Cell shape recovered only partially after washout
(Fig. 4B).
3.4. Ca2+ -activated decrease of OHC axial stiffness
OHC axial stiffness was monitored by pushing a vibrating
elastic probe against the cuticular plate (Fig. 5A). In a typi-
cal experiment, illustrated in Fig. 5B, the amplitude of probe
vibration decreased by about five times after applying me-
chanical force to the cell, suggesting that the cell/probe stiff-
ness ratio was close to 4. In the same experiment, application
of ionomycin increased the [Ca2+ ]i from a basal value of
230 nM to 4.5 ␮M. [Ca2+ ]i changes were followed by a slow
increase of probe vibration amplitude (Fig. 5C), indicating
a twofold decrease of OHC axial stiffness (or more), only
partially reversible after the end of application (Fig. 5D). In
all five cells tested, ionomycin produced a decrease of ax-
ial stiffness (range of changes: 16–80%). The time course
of these changes was similar to that of ionomycin-induced
OHC elongation (Fig. 2A). Ionomycin-induced changes of
OHC axial stiffness were inhibited either after more 15 min
of incubation in Ca2+ -free extracellular medium or dur-
ing the latest stages of patch-clamp recording (>15 min af-
ter the establishment of whole-cell configuration), when the
Ca2+ -induced elongation could not be evoked (data not
shown).
To address the question whether Ca2+ -induced elongation
of the OHC could produce any mechanical work against an
external load, we measured the resting length of the cell un-
der static compression by an elastic probe (Fig. 6). When
the stiffness of the elastic probe was comparable to the OHC
axial stiffness, ionomycin application invariably resulted in
further compression of the loaded cell, indicating that me-
chanical work was done against the cell (Fig. 6A). When
the cell was either not loaded, or loaded by a probe having
comparatively negligible stiffness, a clear ionomycin-evoked
elongation was observed (Fig. 6A). These responses were
dramatically different from electrically evoked motile re-
sponses observed in the same cells when the intracellular
potential was altered under voltage-clamp. In particular, the
electrically-evoked elongation evoked by hyperpolarization
easily deflected also those probes that possessed a stiffness
2–3 times larger than the cell stiffness, indicating that me-
chanical work was done against the elastic load of the (ex-
ternal) probe (Fig. 6B).

Fig. 4. Ionomycin-induced decrease of OHC turgor. (A) Fluorescent images of an OHC before (left) and 40 s after transient (20 s duration) application of
10 ␮M of ionomycin (right). The cell was slightly deflated by applying negative pressure to the patch pipette. Intensity of fluorescence monitors [Ca2+ ]i
in arbitrary units. OHC was in whole-cell patch-clamp at holding potential of −53 mV. (B) DIC images of another OHC with apparently normal turgor
before ionomycin application (left), 40 s following transient application of 10 mM of ionomycin, and 150 s thereafter (right). Arrows point to the locations
where OHC lateral wall curved inward, indicating that the cell started to lose their turgor. Holding potential was −20 mV.
192 G.I. Frolenkov et al. / Cell Calcium 33 (2003) 185–195

Fig. 5. Ca2+ reduces OHC axial stiffness. Panel A shows a differential interference contrast (DIC) image of an OHC (top) and the movement of the
elastic probe (bottom), which was vibrating in close proximity to the cuticular plate without touching the cell. Panel B shows DIC image (top, left)
and Fura-2 ratio image (top, right) of the same cell compressed longitudinally by the probe. Bottom record illustrates movement of the vibrating elastic
probe. Panel C is the same as B, 15 s following the application of ionomycin (10 ␮M). (D) Time course of the stiffness changes of this cell. Each point
represents an average of five stiffness measurements calculated for five consecutive vibration cycles. S.E.M. values are indicated. Horizontal bar marks
timing of ionomycin application.

Fig. 6. Ionomycin (A) and hyperpolarization (B) produce significantly different mechanical forces. Top records illustrate ionomycin and hyperpolarization
induced changes of OHC length without any mechanical load attached to the cell. Bottom records show the length responses of the cell compressed by
the elastic probe. Data on panel A were obtained in two different cells, data on panel B were recorded from the same cell. Horizontal bars mark either
timing of ionomycin application (A) or negative voltage command (B).

4. Discussion cochlear potentials were observed in vivo: a “fast” effect,

Efferent-mediated sound-induced changes of cochlear
function require 100–200 ms to develop in anesthetized cats
[29] and at least 13–15 ms to develop in awake humans and
guinea pigs [30,31]. Two distinct modulatory effects of the
electrical stimulation of the olivo-cochlear bundle on the
with a time constants of about 100 ms, and a “slow” one,
with characteristic time constant of 25–50 s [32]. Both ef-
fects are initiated by the entry of Ca2+ into OHCs through
acetylcholine receptor [33] and accompanied by changes
of the sound responses of the basilar membrane [34]. At
the level of OHCs, “fast” effects have been tentatively
 G.I. Frolenkov et al. / Cell Calcium 33 (2003) 185–195
attributed to ACh-induced ionic currents, while “slow” ef-
fects may be associated with slow Ca2+ -induced changes of
OHC shape and/or stiffness [18]. Here we show that “slow”
Ca2+ -dependent elongation of isolated OHCs is primarily
an effect of “slow” Ca2+ -induced decrease of axial stiffness.
4.1. OHC hyperpolarization has a minor effect in
Ca2+ -dependent slow motility
After the original experiments of Dulon et al. [21],
it was pointed out [22] that the generalized increase
of [Ca2+ ]i produced by Ca2+ -ionophores may induce
Ca2+ -dependent phosphorylation of numerous proteins, in-
cluding Ca2+ -dependent modulation of the various plasma
membrane ion channels. Changes of ionic conductance
likely affect intracellular potential, and in turn may result
in contraction/elongation of the cell because OHC length is
also a function of intracellular potential [35]. This can be
described as “activating fast OHC electromotility slowly”.
The involvement of a sub-class of K+ -conductances in
Ca2+ -induced OHC elongation has been excluded based on
the fact that length changes were not inhibited by tetraethy-
lammonium ions [21]. However, other conductances could,
in principle, effect these changes. Here we have shown
that Ca2+ -induced elongation of OHCs can be recorded
in patch-clamp conditions (Fig. 2), when the intracellular
potential is fixed. In other words, our data indicate that
the effect of Ca2+ -ionophores is not mediated by a slow
hyperpolarization of the cell. However, the fast component
of the fourfold smaller elongation observed in free un-
patched OHCs probably results from cell hyperpolarization
mediated by Ca2+ -activated K+ -channels (Fig. 3).
The results of our UV-photolysis experiments with photo-
labile Ca2+ -chelators, following relatively short (5–15 min)
loading of the cell via patch pipettes, indicate that
Ca2+ -induced OHC elongation occurs primarily in response
to changes of Ca2+ concentration in the cytoplasm. How-
ever, several intracellular membranes, including those of
sarcoplasmic reticulum [36] and mitochondria [37] become
permeable to Ca2+ after application of Ca2+ -ionophores.
Thus, the ionomycin experiments may include contributions
from different intracellular compartments. In OHCs, based
on our data with ACh [16,20], we expect Ca2+ -induced
Ca2+ -release from intracellular stores to play an essential
role in the process.
4.2. Axial stiffness of OHC is regulated by [Ca2+ ]i
Dulon et al. [21] also suggested that Ca2+ -induced elon-
gation of the OHC results from a circumferential contraction
of the cortical cytoskeleton, which, under constant volume
conditions, produced cell elongation due to incompressibil-
ity of the cytosol. This argument was based on the obser-
vation that the increase of [Ca2+ ]i decreased the volume
responses of the OHCs to the application of hypotonic extra-
cellular medium. This decrease of the volume responses was
193
interpreted as a result of increased cell rigidity [21]. How-
ever, this interpretation is challenged by others’, as well as
our own results. First, the OHCs show prominent regulatory
volume decrease (RVD) in response to hypo-osmotic stim-
ulation [38]. Any Ca2+ -dependent modulation of the RVD
mechanisms may affect steady-state volume measurements
like those reported by Dulon et al. [21]. Second, as shown
here, Ca2+ -induced elongation in free unpatched OHCs has
at most a minor component associated with cell hyperpo-
larization (Fig. 3). Hyperpolarization, in turn, increases the
axial stiffness of OHC [39,40], partially counteracting the
primary effect of Ca2+ . Finally, large changes of [Ca2+ ]i
may disturb OHC turgor, decreasing also the apparent vol-
ume changes in response to hypo-osmotic stimulation. In
the present experiments, we performed direct measurements
of the OHC axial stiffness with the elastic probe technique
and showed that it decreases in response to increase of
the [Ca2+ ]i .
Our stiffness measurement technique detects elastic defor-
mations of the cell, imposed by loading the cuticular plate,
which in turn act as a “piston”. Elastic forces contributing
to these deformations may include: (1) axial elasticity of the
mechanical skeleton of the cell; (2) some potential compo-
nent related to the bending elasticity of the cell skeleton;
(3) additional forces resulted from re-distribution of external
mechanical load by intracellular pressure. Of these compo-
nents, bending stiffness was probably irrelevant in our ex-
periments, because we observed Ca2+ -induced decrease of
cell stiffness even in perfectly straight OHCs. Axial stiffness
of OHC cytoskeleton is the most likely target for [Ca2+ ]i ac-
tion. However, externally driven cell compression certainly
increases intracellular pressure providing an additional elas-
tic force, which may depend also on the stiffness of non-axial
components. Therefore, the Ca2+ -induced decrease of OHC
stiffness observed in our experiments may include also con-
tributions from other non-radial mechanical elements of the
cell. In any case, such decrease is hardly compatible with
previously proposed circumferential contraction of the cor-
tical cytoskeleton [21].
A Ca2+ -dependent decrease of OHC axial stiffness was
also observed following the application of acetylcholine to
the basal pole of the OHC [18]. In isolated unloaded OHCs,
axial stiffness decrease is thought to be responsible for the
observed increase of electromotile responses [18]. This can
be easily explained if Ca2+ affects the cytoskeletal stiff-
ness more than the effective stiffness of the prestin-based
motors. We showed previously that ionomycin-induced
rise of [Ca2+ ]i mimics the effects of acetylcholine on the
electrically-evoked motile responses of OHCs and on the
OHC voltage-dependent capacitance [20], suggesting some
common mechanism. Here we round up those observations
reporting that the effects of the [Ca2+ ]i increase are, indeed,
associated with a decrease of OHC axial stiffness.
The biochemical pathway targeting OHC axial stiffness
either after acetylcholine application or following the in-
crease of [Ca2+ ]i , remains obscure. What is known is that
194 G.I. Frolenkov et al. / Cell Calcium 33 (2003) 185–195
several inhibitors of calmodulin and calmodulin-dependent
kinases affect Ca2+ -induced elongation of OHCs [21–23]
and acetylcholine-induced decrease of OHC stiffness [19].
The exact cellular mechanism, however, may be fairly com-
plicated including regulation of OHC cortical cytoskeleton
via Rho GTPases [41].
The apparent difference between the effects of ionomycin/
UV-photolysis and that of acetylcholine is the amount
of [Ca2+ ]i increase involved. Ca2+ -imaging experiments
rarely showed a significant increase of [Ca2+ ]i induced
by acetylcholine [15,16]. However, there is indirect evi-
dence that acetylcholine may activate spatially localized
Ca2+ -induced Ca2+ release in close vicinity to the plasma
membrane [16,33], which could not be detected due to lim-
ited resolving power of fluorescence microscopy. Therefore,
it is reasonable to conclude that Ca2+ -induced decrease of
axial stiffness is a characteristic response of OHCs, which
evolved as an essential component of the efferent-based
system controlling the mechanical properties of the organ
of Corti. Ca2+ -induced elongation of the OHC is nothing
but a by-product of this active regulation and represents
a passive reaction of the turgid cell to a decrease of axial
stiffness.
Acknowledgements
We thank David Robinson, Mark Ospeck, Claudia Racca,
and Mark Schneider for critical review of the manuscript.
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