HBlRlnG

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Hearing Research 106 (1997) 105-111

Oncomodulin is abundant in the organ of Corti
Michael T. Henzl a,*, Osamu Shibasaki b, Thomas R. Comegys h, Isolde Thalmann
Ruediger Thalmann b
b

b,

a Biochemistry Department, University of Missouri at Columbia, Columbia, MO 65211, USA
Department of Otolaryngology, Washington University School of Medicine, St. Louis, MO 63110, USA

Received 29 August 1996; revised 10 December 1996; accepted 23 December 1996

Abstract
A small, acidic Ca 2 +-binding protein (CBP-15) was recently detected in extracts of the mammalian auditory receptor organ, the
organ of Corti [Senarita et al. (1995) Hear. Res. 90, 169-175]. N-terminal sequence data for CBP-15 [Thalmann et al. (1995)
Biochem. Biophys. Res. Commun. 215, 142-147] implied membership in the parvalbumin family and possible identity with the
mammalian J}-parvalbumin oncomodulin. As shown herein, the latter conclusion is supported by strong cross-reactivity between
CBP-15 and isoform-specific antibodies to oncomodulin. Moreover, we have succeeded in amplifying the guinea pig CBP-15 coding
sequence from organ of Corti cDNA using degenerate oligonucleotide primers based on the rat oncomodulin sequence. The deduced
amino acid sequence of guinea pig CBP-15 displays 90%, 92%, and 98% identity with mouse, rat, and human oncomodulin isoforms.
Demonstration of the presence of oncomodulin in the organ of Corti is the first documentation of this substance in a postnatal
mammalian tissue.
Keywords: Oncomodulin; Parvalbumin; Organ of Corti; Guinea pig

1. Introduction

Parvalbumins are small, vertebrate-specific relatives
of calmodulin (Wnuk et a1., 1982; Gerday, 1988; Heizmann, 1984). The PV primary structure encodes two
functional 'EF-hand' Ca2+-binding sites and the nonfunctional remnant of a third (Kretsinger, 1980). Generally viewed as cytosolic Ca2+ buffers, PVs are abundant in select skeletal myofibrils and neurons where
they are believed to facilitate myofibrillar relaxation
(Gillis, 1985) and neuronal de-excitation (Celio, 1990).
The PV family contains two sub-lineages, ex and ~
(Goodman and Pechere, 1977; Maeda et a1., 1984).
The former are distinguished by the presence of an
additional C-terminal residue and relatively higher isoelectric points (pI;:::: 5).
The skeletal muscle of poikilotherms harbors multiple parvalburnin isoforms (Simonides and van Harde-

* Corresponding author. Tel.: + 1 (573) 882 7485;
fax: +1 (573) 8844812; e-mail: bchenzl@muccmai1.missouri.edu
0378-5955/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved
PI! S 0 3 7 8 - 5 9 5 5 ( 9 7 ) 0 0 0 0 5 - 1

veld, 1989; Gerday et a1., 1991; Schwartz and Kay,
1988). This polymorphism may serve as a mechanism
for tuning the relaxation properties of various types of
muscle fibers. By contrast, birds and mammals express
a single muscle isoform, belonging to the ex lineage. In
mammals, the identical isoform is detected in such diverse tissue settings as muscle, nerve, kidney, adipose
tissue, and skin (Heizmann, 1988). Although the mammalian genome also encodes a ~-PV isoform called oncomodulin (MacManus and Whitfield, 1983; MacManus et a1., 1987), normal expression of the protein is
believed to be restricted to fetal placenta (Brewer and
MacManus, 1985, 1987; MacManus et a1., 1985).
Previous studies have revealed the existence of an
apparently novel Ca2+-binding protein in the organ of
Corti (Ot,'), which was labeled CBP-15 (Senarita et a1.,
1995). The physical properties of CBP-15 (Mf 15,000;
pI 3.1) and preliminary N-terminal sequence data
(Thalmann et a1., 1995) suggested membership in the
parvalbumin (PV) family. We herein present irrefutable
evidence that CBP-15, expressed at high levels in the

106

M. T. Henzl et al. I Hearing Research 106 (1997) 105-111

OC, is in fact identical to oncomodulin. This finding
constitutes the first observation of the protein in a postnatal mammalian tissue setting.
2. Materials and methods
2.1. Tissue preparation

Young guinea pigs (250-350 g) were anesthetized to
a deep plane of surgical anesthesia with veterinary pentobarbital (33 mg/kg i.p.) and decapitated. Temporal
bones were rapidly removed from the skull, cleared of
soft tissues, immersed in Freon-12 chilled to its melting
point with liquid nitrogen, and freeze-dried at -40°C
for 5 days. The organ of Corti was dissected at room
temperature, at a relative humidity of 40% or less. Each
ear yielded approximately 15 ug (dry weight) of OC
tissue.
2.2. Preparation of antibodies

Recombinant rat OM was coupled to keyhole limpet
hemocyanin as described elsewhere (Serda and Henzl,
1991), emulsified with RIBI adjuvant (RIBI Immunochemical Co., Hamilton, MT), and injected at multiple
sites on the dorsal surface of a male NZW rabbit (Hum
and Chantler, 1980). This pre-immunization was repeated 5 weeks later. Thereafter, injections of OM alone
were made as necessary to boost the antiserum titer.
For the preparation of monoclonal antibodies, the
antigen was prepared similarly. Female BALB/c mice
received two 0.2 ml injections (i.p.) of the antigen-adjuvant emulsion 5 weeks apart. After resting the animals for 15 weeks, the immunization protocol of Stahli
et al. (1983) was initiated. The serum antibody titer i.e., the dilution factor required to reduce the maximal
ELISA signal by 50% - exceeded 105 at the time the
animal was killed.
The polyethylene glycol-induced fusion of splenocytes with murine myeloma cells employed a modification of standard protocols (Hum and Chantler, 1980;
Galfre and Milstein, 1981; Goding, 1983). Splenocytes
(2X 108 ) were combined with PAI-O myeloma cells at a
splenocyte/myeloma ratio of 10: 1, collected by centrifugation, and warmed to 37°C. Two milliliters of 50% (w/
w) PEG 1500 in RPMI-Hepes, pH 7.2, was then added
to the splenocyte-myeloma pellet over the course of 60
s. After an additional 60 s at 37°C, the PEG was diluted
with RPMI-Hepes - first with 2.0 ml at 1.0 ml/min,
then with an additional 8 ml added over the course of
1 min. After an additional 2 min at 37°C, the cells were
collected by centrifugation.
The resulting cell preparation was resuspended at
1.5 X 106 splenocytes/ml and dispensed into 96-well
plates. The RPMI-1640 plating medium was supple-

mented with NaHC0 3 (2 g/l), 10% heat-inactivated
FBS, 2 mM L-Gln, 80 ug/ml gentamycin sulfate, and
HAT components at standard concentrations. Cultures
were maintained on HAT medium until expansion to
24-well plates, then weaned on 50% HAT, followed by
25% HT.
Hybridomas producing anti-OM antibodies were
identified by ELISA and immunoblot assays on culture
supernatants. The lAlO antibody belongs to the IgG l
sub-class and harbors K light chains, as determined with
the Isotyper" kit from Boehringer-Mannheim.
2.3. Isoelectric focusing

Freeze-dried OC tissue was dissolved in deionized
water, followed by double-strength sample buffer containing 60% glycerol and 4% ampholytes, then centrifuged for 2 min at 10,000 Xg. Non-denaturing IEF was
performed at 15 W constant power in an SE600 vertical
slab unit (Hoefer Scientific Instruments, San Francisco,
CA), at 15°C. The 140x 160x 1.5 mm gels contained
5.5% acrylamide, 0.5% piperazine di-acrylamide, 10%
(v/v) glycerol, and ampholytes (3.75%: pI 3-10;
1.88%: pI 3-5; 0.37%: pI 2.5--4). Acetic acid (0.02 M)
and 0.02 M NaOH served as the anolyte and catholyte,
respectively. After pre-running for 15 min, samples were
loaded, and focusing was continued for 150 min. Proteins were visualized by silver staining (Oakley et al.,
1980).
2.4. Immunoblotting

Following IEF, proteins were transferred to nitrocellulose (Towbin et al., 1979) in a Hoefer Mighty Small
Transphor apparatus for 2 h at 90 V. After blocking for
1.5 h in 3% gelatin, the replica was probed sequentially
with the anti-OM primary antibody and an appropriate
alkaline-phosphatase-conjugated secondary antibody.
NBT and BCIP were used for visualization. The lAlO
hybridoma culture supernatant was used at a 1: 10 dilution, and the rabbit anti-OM antiserum at 1: 1000.
2.5.

rce amplification

An aliquot (107 cfu) of the Wilcox-Fex guinea pig
OC cDNA library (Wilcox and Fex, 1992) was grown
to stationary phase in 10 ml of LB broth containing
ampicillin (100 ug/ml), Total plasmid DNA served as
the template for amplification of the CBP-15 coding
sequence. The 50 III reactions contained 10 mM Tris,
pH 8.3, 50 mM KC1, 1.5 mM Mg 2+ , 0.2 mM dNTPs
(Amersham-USB), 5 mM 2-mercaptoethanol, 1 ug each
of the sense and anti-sense primers, 100 ng of template,
and 1 V of Taq polymerase (Boehringer-Mannheim).
Amplifications included 30 cycles of denaturation (1
min), annealing (1 min), and extension (1 min), fol-

M. T. Henzl et al. I Hearing Research 106 (1997) 105-111

lowed by a 10 min extension period. Annealing was
performed at 55°C, except when utilizing degenerate
oligonucleotide primers (47°C).
Aliquots (5.0 Ill) of the reactions were analyzed by
agarose gel electrophoresis. For cloning into pCRIl (Invitrogen, Inc.), the product of interest was recovered
with QIAEX II (Qiagen, Inc., Chatsworth, CA) following preparative electrophoresis in 2% agarose. Cloned
PCR products were sequenced with Sequenase v2.0 or
by automated fluorescence sequencing. For direct sequencing of PCR products, the amplified fragment
was purified electrophoretically in 6% acrylamide,
eluted in TE buffer, and recovered with QIAEX II.
The following oligonucleotide primers were synthesized by the University of Missouri DNA Core Facility:
CBP16S,
CARGARTGYCARGAYCCIGAYAC;
CBPl06AS,
ACCATYTCYTGRAAYTCRTCIGC;
CBP56S,
GGATACCTGGATGAAGAAGAG;
CBP53AS,
GTCATTGTCTATGAACCGGAA;
CBP5',
CCCACGCGTCCGCTCTTTCAACTG;
CBP3',
CAGGCCAATGACAAGAAGATGTCT;
M13F, CGTTGTAAAACGACGGCCAGT; M13R,
CAGGAAACAGCTATGACCATG. For the degenerate primers, CBP16S and CBPlO6AS, I is inosine, R is
a purine, and Y is a pyrimidine.
Procedures involving animals were performed according to protocols approved by the Animal Studies
Committees at the respective institutions, in accordance
with NIH Animal Care and Usage Guidelines.
3. Results
3.1. Western blot
Anti-OM antibodies react strongly with CBP-15, as
shown in Fig. 1. For these analyses, extracts from guinea pig and rat OC were resolved by l-D IEF, electrophoretically transferred to nitrocellulose, then probed
with the anti-OM primary antibody and an alkalinephosphatase-conjugated secondary antibody. A stained
portion of a representative gel is displayed in the left
panel. The proteins in the guinea pig and rat extracts
that display isoelectric points comparable to the recombinant OM standard correspond to the guinea pig
and rat CBP-15 isoforms. It is apparent from their intensities that CBP-15 is a major component in the OC
from both species.
As shown in the accompanying blots, the CBP-15
isoforms are recognized by anti-OM antibodies. It
should be noted that these antibody preparations are
highly isoform-specific. The lAlO monoclonal does
not recognize the a-PVs from pike, rat, or chicken,
nor does it recognize the two ~-PVs from chicken thymus tissue, ATH and CPV3. The rabbit polyclonal
preparation cross-reacts very weakly with the other iso-

107

GEL

BLOT
Polyclonal

Monoclonal

•

...OM

GP

Rat

OM

OC-GP OC-Rat

OM OC-GP

Fig. 1. Polyclonal and monoclonal antibodies to rat OM cross-react
strongly with CBP-15 from guinea pig Oc. Left: Silver-stained gel
with 0.24 ug oncomodulin standard (OM), 15 J..lg freeze-dried guinea
pig organ of Corti (GP) and 8 ug freeze-dried rat organ of Corti
(Rat). Center: Reaction of monoclonal antibody to rat OM with
0.24 J..lg OM standard, 15 J..lg guinea pig organ of Corti (OC-GP)
and 15 ug rat organ of Corti (OC-Rat). Right: Reaction of polyclonal OM antibody to 0.63 ug OM standard and 18 J..lg guinea pig organ of Corti.

forms, displaying titers 2-3 orders of magnitude lower
than that displayed for OM. Thus, the cross-reactivity
displayed in Fig. 1 suggests that the CBP-15 isoforms
share a very high degree of structural homology with
OM.
Note that the pI of rOM is not identical to those of
the CBP-15 isoforms. This difference may reflect heterogeneity at the N-terminus. Similar to placental OM,
the CBP-15 isoforms are known to be N'<acetylated
(22). By contrast, the N-terminal residue of recombinant OM is unmodified. This difference in post-translational processing could account for the slightly higher
isoelectric point of rOM.
3.2. Isolation of CBP-15 cDNA sequence
A 270 bp fragment of the CBP-15 coding sequence
was obtained in high yield by PCR, employing degenerate oligonucleotide primers based on residues 16-23
and residues 99-106 of rat OM (Fig. 2A, lane 1). Total
plasmid isolated from a guinea pig OC cDNA library
(Wilcox and Fex, 1992) served as the template for the
amplification. The 270 bp amplification product was
cloned into pCRIl to afford pCBP15-1. To obtain the
flanking cDNA sequences, we took advantage of the
fact that the Wilcox-Fex library had been directionally
cloned into the pSportl vector (see Fig. 2B). Thus, M13
reverse and primer CBP53AS were used to amplify the
5' end of the CBP-15 cDNA sequence. We had anticipated heterogeneity in the product, reflecting incomplete reverse transcription during construction of the

M. T. Henzl et al. I Hearing Research 106 (1997) 105-111

108

2

34M

®

-2176

-1033
653
517
394
298
234

®
5'

100

pSportl
I

I

200

I

300

ATG
CBP16S •

I

400

I

I
TAA

500

I

600

I

pSport1

3'

AAAA •••

• CBP106AS

M13R ...- - - - - - - . . . . CBP53AS
CBP56S ...- - - - - - - - - - _ . M13F
CBP5' ••- - - - - - - - - - - - - - -... CBP3'

Fig. 2. PCR amplification of CBP-15 cDNA. Reactions were carried out as described in Section 2. A: Amplification of core, 5', 3', and complete CBP-15 cDNA sequences. 5 III aliquots of the reactions were resolved on a 2% agarose gel, then stained with 0.0005% ethidium bromide
and photographed with UV illumination. Lane I: Amplification of residues 16-106, employing the oligonucleotide primers, CBP16S and
CBPI06AS. Lane 2: Amplification of S' end of CBP-15 cDNA employing the M13R and CBP53AS primers. Lane 3: amplification of 3' end
of CBP-15 cDNA using the CBP56S and M13F primers. Lane 4: Amplification of putative full-length cDNA with CBP5' and CBP3' primers.
Lane M: DNA standards (size, in bp, indicated at right). B: PCR amplification scheme.

cDNA library. In fact, the product displayed a narrow
size distribution, centered near 350 bp (Fig. 2A, lane 2).
Following gel-purification, the product was cloned into
the pCRIl cloning vector. Several clones harboring an
insert were identified, and the one containing the largest
insert, designated pCBP15-2, was purified and sequenced.
A similar strategy was employed to obtain the 3' end
of the cDNA. PCR was performed using the vectorspecific M13 forward primer and the CBP56S primer,
and the resulting product (z 600 bp, Fig. 2A, lane 3)
was cloned into the pCRIl vector, affording pCBP15-3.
The presence of a poly(A +) tail at the 3' terminus of the
product was confirmed by sequencing.
Sense and anti-sense primers corresponding to the
putative 5' and 3' termini of the CBP-15 cDNA sequence were designed, based on sequence information
from pCBP15-2 and pCBP15-3. These were used to
amplify the entire cDNA sequence from the WilcoxFex library. Both strands of the resulting PCR product
were sequenced directly. The sequence thus obtained
(Fig. 3) spans 670 nucleotides, including a 69-nucleotide
5'-UTR, 330 nt of coding information, and a 271-nucleotide 3'-UTR.
The translated sequence - 108 residues excluding the
initiating methionine - is unmistakably that of a parvalbumin. The primary structure includes two consensus EF-hand binding loops spanning residues 51-62 and
90-101, corresponding to the parvalbumin CD and EF

ion-binding sites. All 24 invariant PV residues (Kretsinger, 1980) are observed. It is further apparent that
CBP-15 is a ~-PV isoform. The C-terminal helix extends
just seven residues beyond the -Z ligand (E101), rather
than eight, consistent with membership in the ~-PV
lineage (Goodman and Pechere, 1977). Moreover, the
residues at positions 18 and 66 - cysteine and phenylalanine, respectively - are ~-lineage-specific markers.
Significantly, CBP-15 displays an aspartyl residue at
10

30

50

70

90

110

CCCACGCGTCCGCTCTTTCAACTGTTTTTCCCCCTGACTCTTCTTGGGGGAAAACGTAAA
AGGGTGAAGATGAGCATCACAGACGTGCTCAGTGCTGATGACATTGCCGCTGCCCTGCAG
M S I T D V L SAD D I A A A L Q

130
150
170
GAATGCCAAGATCCAGACACTTTTGAGCCCCAAAAATTCTTCCAGACATCAGGCCTCTCC
E C Q D PDT F E P Q K F F Q T S G L S
190
210
230
AAGATGTCAGCCAGTCAGGTGAAAGACGTTTTCCGGTTCATAGACAATGACCAGAGTGGA
K M S A S Q v K D V F R F I D N D Q S G
250
270
290
TACCTGGATGAAGAAGAGCTTAAGTTTTTCCTCCAGAAATTTGAGAGTGGTGCTAGAGAA
Y L D E E E L K F F L Q K F E S GAR E
310
330
350
CTCACCGAGTCAGAAACCAAATCTTTGATGGCCGCTGCAGATAATGATGGAGACGGAAAA
L T ESE T K S L M A A A D N D G D G K
370
390
410
ATTGGGGCTGATGAATTCCAGGAAATGGTGCATTCTTAAAACCCCAGTCAGTGGAGAACA
I GAD E F Q E M V H S *
430
450
470
GAGAGAAAGGGACACTCAGCTGGAAGGCCTCCAAAGTCTTGGGAATGGGAAACCCCACAT
490
510
530
CCTACCACAAACTAATTCACTAATTCCCCCCAAAATGCTTCTGAGATTTGCTCATTTCTC
550
570
590
AAGTGAGGAAATAAGACACACTCCAAGGCTTTTTTTGGTGATGGGTTTGCCCTGATGACT
610
630
650
CCTGTAAGGTCCCCTACAGGCAATGCCTTTAAAAATCACCCAATAAAGACATCTTCTTGT
670
CATTGGCCTG

Fig. 3. CBP-15 cDNA sequence and predicted amino acid sequence.
The putative polyadenylation signal is underlined.

M. T. Henzl et al. I Hearing Research 106 (1997) 105-111
1

10

20

CBP-15 SIT D V L SAD D I A A A L Q E C Q D PDT F E P Q
rOM
- - - - I - - - E - - - - - - - - - - - - ~ - - - - -

mOM

----I----------------------

CBP-15
rOM
mOM
hOM

K F F Q T S G L S K MS A S Q V K D V F R F I D N D Q
------------------1-------- - - - - - - - - - - - - - - L - - I - Q - - - - - -------------N-------------

CBP-15
rOM
mOM
hOM

z -y
-x
-z
65
70
80
S G Y L DEE ELK F F L Q K F E S GAR E L T ESE
- - - - - G D - - - Y - - - - - Q - D - - - - - - - ------D---Y---R-Q-D---------------------------------_

CBP-15
rOM
mOM
hOM

T
-

hOM

- - - - - - - - - - - - - - - - - - - - - - - - - - 30

K
-

S
-

50 x

40

85

L
-

MA
- D
- D
- -

A
-

A
-

x
D
-

N
-

y

D
-

G
-

z
D
-

G
-

-y

K
-

I
-

-x
-z
GAD E
- - - - - - - - E -

F
-

Q
-

E
-

Y

105

M
-

V
_
-

H
_
_
-

S
_
_
-

Fig. 4. Primary structures of CBP-15 and OM isoforms from rat
(rOM), mouse (mOM), and human (hOM).

position 59. The presence of aspartate at this position is
idiosyncratic for oncomodulin. In all other parvalbumin
isoforms sequenced to date, residue 59 is a glutamyl
residue. Whereas the latter is capable of directly coordinating the bound metal ion (Declercq et al., 1988;
Roquet et al., 1992; Swain et al., 1989; McPhalen et
al., 1994), the shorter side-chain of aspartate precludes
direct ligation. Instead, the aspartyl carboxylate coordinates indirectly via an intervening water molecule
(Ahmed et al., 1990, 1993). CBP-15 also displays leucine at position 58. The oncomodulin amino acid sequence is also distinguished by the presence of leucine-58. Virtually all other parvalbumin isoforms
employ isoleucine at this position.
The inferred amino acid sequence of CBP-15 is compared with the OM sequences from rat (Gillen et al.,
1987), mouse (Banville et al., 1992), and human (Fohr
et al., 1993) in Fig. 4. CBP-15 is identical to the rat
isoform at 99 of 108 positions (for 92% identity) and
identical to the mouse isoform at 98 positions (90%).
Interestingly, the human OM sequence coincides with
that of CBP-15 at 106 positions (98% identity).
4. Discussion
Discovered in extracts of a rat hepatoma nearly two
decades ago (MacManus, 1979), the function of oncomodulin remains conjectural. The parvalbumin CD and
EF sites are typically 'Ca2+/Mg2+ sites', displaying substantial affinity for both Ca 2+ and Mg2+ at physiological pH and ionic strength, with KCa = 1-10 nM and
K Mg =10-50 IlM (Serda and Henzl, 1991; Haiech et
al., 1979; Moeschler et al., 1980; Rinaldi et al., 1982;
Eberhard and Erne, 1994). Oncomodulin, however, displays uncharacteristically low affinity for the ions (Hapak et al., 1989; Cox et al., 1990). The calcium dissociation constants for the EF and CD sites are 45 and
800 nM, respectively. The corresponding Mg2+ constants are 250 IlM and > 1 mM. In fact, the oncomo-

109

dulin CD site meets the criteria for a 'Ca2+ -specific site',
fueling speculation that the protein may serve in a regulatory capacity.
To date, however, no biological effector for oncomodulin has been identified, and the relevant literature
pertaining to its regulatory capacity is controversial.
Claims that OM is capable of activating cyclic nucleotide phosphodiesterase (MacManus, 1981; Mutus et al.,
1985, 1988) have been refuted (Klee and Heppel, 1984;
Clayshulte et al., 1990), and there have been no confirmatory reports that oncomodulin is capable of activating a nuclear protamine kinase (MacManus and Whitfield, 1983) and inhibiting glutathione reductase
(Palmer et al., 1990). However, Blum and Berchtold
(1994) have observed calmodulin-like effects of OM
on the cell cycle in chemically transformed rat fibroblast
cell lines. Specifically, transcript and protein levels increase significantly at the G liS boundary, in analogy to
calmodulin (Chafouleas et al., 1982; Rasmussen and
Means, 1989). Moreover, antisense OM oligonucleotides inhibit growth of the T14 cell line in a dose-dependent manner similar to that observed by the same
authors with calmodulin anti-sense probes. Although
the functional significance of these observations remains
to be established, they suggest that oncomodulin may
influence cell-cycle progression in neoplasms.
In view of the well-documented parsimony of the
OC, the abundance of OM and its apparent absence
from a broad range of other guinea pig tissues (Senarita
et al., 1995) strongly imply an obligatory role for the
protein in the physiology of the OC, It is possible that
OM functions as an OC-specific Ca2+-dependent modulator. Although the presence of the nonfunctional AB
domain would prevent calmodulin-like interactions with
target peptides (Strynadka and James, 1989; McPhalen
et al., 1991), OM may participate in some other mode
of protein-protein interaction. In principle, Ca2+-dependent regulatory capacity requires only that the
apo- and Ca2+-bound states adopt significantly different
average conformations. At resting state Ca2+ levels, the
EF site of OM is presumably occupied by Mg2+, and
the CD site is vacant. An increase in cytosolic Ca2+
should therefore result in occupation of the CD site
by Ca2+ and the replacement of Mg2+ by Ca2+ at the
EF site. Although the conformational rearrangement
attendant to the Ca2+-Mg2+ exchange event is likely
to be minor (Declercq et al., 1991; Blancuzzi et al.,
1993), microcalorimetric studies (Cox et al., 1990;
Henzl et al., 1996) indicate that the binding of Ca2+
at the CD site of OM provokes a significant conformational change. The substantial enthalpy change for this
process (L~.H°' = -3.4 kcal/mol) (Henzl et al., 1996) presumably reflects the increase in van der Waals contacts
that accompanies rearrangement of the polypeptide
chain.
Alternatively, OM may function as a highly special-

110

M. T. Henzl et al. / Hearing Research 106 (1997) 105-111

ized Ca2+ buffer in the OC. As a consequence of the
steep K+ gradient between the endolymph and perilymph and the highly positive endolymphatic potential,
the OC must contend with a massive K+ current. This
challenging electrochemical environment may have required the recruitment of a Ca 2+ buffer having atypical
metal ion-binding properties. Whether OM is functioning as a regulatory protein or Ca2+ buffer is the focus of
continuing investigation.
This report marks the first observation of OM in a
normal, postnatal mammalian tissue. Having demonstrated the presence of OM within the OC, its distribution becomes a matter of immediate interest. Pack and
Slepecky (1995) have previously shown that expression
of the a-PV isoform is restricted to the inner hair cells
of guinea pig and gerbil. By contrast, preliminary immunohistochemical studies employing an isoform-specific monoclonal antibody against OM indicate that
the protein is expressed exclusively in the outer hair cells
of rat, gerbil, and mouse (Sakaguchi et al., 1997). If
confirmed, this finding would constitute further evidence that oncomodulin is not merely serving as a
Ca 2+ buffer in the Oc.
Acknowledgments

The authors wish to thank Dr. Edward Wilcox,
NIDCD/NIH, for generously providing an aliquot of
the Organ of Corti cDNA library produced in his laboratory. This work was supported by NIDCD/NIH
Grants DC01374 (LT.) and DC014l4 (R.T.).
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