Identification and Characterization of a Sodium/Calcium
Exchanger, NCX-1, in Osteoclasts and
Its Role in Bone Resorption
Baljit S. Moonga,* Robert Davidson,† Li Sun,* Olugbenga A. Adebanjo,* James Moser,‡
Mohammad Abedin,§ Neeha Zaidi,* Christopher L.-H. Huang,
¶
and Mone Zaidi*
,1
*Mount Sinai Bone Program, Departments of Medicine and Geriatrics, Mount Sinai School of Medicine, and Bronx
Veteran’s Affairs (VA) Geriatric Research Education and Clinical Center (GRECC), New York, New York;
†David Kriser School of Dentistry, New York University, New York, New York; ‡Department of Surgery,
University of Pittsburgh, Pittsburgh, Pennsylvania; §VA Medical Center, Philadelphia, Pennsylvania;
and
¶
Physiological Laboratory, University of Cambridge, Cambridge, United Kingdom
Received April 17, 2001
We provide the first demonstration for a Na
/Ca
2
ex-
changer, NCX-1, in the osteoclast. We speculate that by
using Na
exchange, NCX-1 couples H
extrusion with
Ca
2
fluxes during bone resorption. Microspectrofluo-
rimetry of fura-2-loaded osteoclasts revealed a rapid and
sustained, but reversible, cytosolic Ca
2
elevation upon
Na
withdrawal. This elevation was abolished by the
cytosolic introduction (by gentle permeabilization) of a
highly specific Na
/Ca
2
exchange inhibitor peptide,
XIP, but not its inactive analogue, sXIP. Confocal mi-
croscopy revealed intense plasma membrane immuno-
fluorescence with an isoform-specific monoclonal anti-
NCX-1 antibody applied to gently permeabilized
osteoclasts. Electrophysiological studies using excised
outside-in membrane patches showed a low-
conductance, Na
-selective, dichlorobenzamil-sensitive,
amiloride-insensitive channel that we tentatively as-
signed as being an NCX. Finally, to examine for physio-
logical relevance, an osteoclast resorption (pit) assay
was performed. There was a dramatic reduction of bone
resorption following NCX-1 inhibition by dichloroben-
zamil and XIP (but not with S-XIP). Together, the results
suggest that a functional NCX, likely NCX-1, is involved
in the regulation of osteoclast cytosolic Ca
2
and bone
resorption. © 2001 Academic Press
Key Words: Na
/Ca
2
exchange; osteoclasts; osteopo-
rosis; bone resorption.
The osteoclast is unique in that it is exposed to high
levels of ambient Ca
2+
in the millimolar range [1].
Mechanisms exist through which rising ambient Ca
2+
concentrations are ‘sensed’ and transduced into intra-
cellular Ca
2+
signals that regulate osteoclastic bone
resorption [2, 3]. These responses are mediated
through type 2 ryanodine receptor-gated Ca
2+
channels
expressed at the plasma membrane [4]. We are, how-
ever, unclear of mechanisms by which resorbed Ca
2+
is
extruded to restore cytosolic Ca
2+
to basal levels. A
Ca
2+
-ATPase has been identified on the osteoclast’s
dorsal surface; however, little is known of alternative
or parallel mechanisms of osteoclast Ca
2+
extrusion [5].
The primary functional role of Na
+
flux in the oste-
oclast has typically been associated with H
+
exchange
[6 –9]. Active osteoclasts involved in the resorptive phase
of bone remodeling show a massive H
+
extrusion via a
V-type H
+
-ATPase [10]. This step is essential for bone
resorption. Changes in intracellular pH resulting from
active proton pumping are readily corrected by a Cl
-
channel, a Cl
-
/HCO
3
-
exchanger, and an amiloride-
sensitive Na
+
/H
+
exchanger [6, 11–14]. We report func-
tional, electrophysiological and immunochemical evi-
dence for a Na
+
/Ca
2+
exchanger, likely an NCX-1, in the
osteoclast. We hypothesize that, as in cardiac muscle,
osteoclastic NCX-1 might couple with the Na
+
/H
+
ex-
changer to assist in the regulation of cytosolic Ca
2+
[15].
This would mean that in addition to Ca
2+
-ATPase [5],
there is a second mechanism for Ca
2+
extrusion following
Ca
2+
influx through the surface ryanodine receptor/
extracellular Ca
2+
sensor [2, 4]. Unlike Ca
2+
-ATPase,
however, this mechanism is linked to the proton extru-
sion that is a primary determinant of the rate and extent
of bone resorption.
MATERIALS AND METHODS
Microspectrofluorimetric measurements of cytosolic Ca
2+
. Osteo-
clasts were freshly isolated as described previously [2]. Coverslips
containing freshly isolated cells washed in -MEM and treated with
1
To whom correspondence should be addressed at Mount Sinai
Bone Program, P.O. Box 1055, Endocrinology, Mount Sinai School of
Medicine, One Gustave Levy Place, New York. NY 10029. Fax:
212-426-8312. E-mail: mone.zaidi@mssm.edu.
Biochemical and Biophysical Research Communications 283, 770 –775 (2001)
doi:10.1006/bbrc.2001.4870, available online at http://www.idealibrary.com on
770 0006-291X/01 $35.00
Copyright © 2001 by Academic Press
All rights of reproduction in any form reserved.