1 Mount Sinai Bone Program, Departments of Medicine and Geriatrics, Mount Sinai School of Medicine, and the Geriatric Research, Education, and Clinical Center, Bronx Veterans Affairs Medical Center, New York 10029; 2 Division of Basic Sciences, New York University Dental School, New York, New York 10010; and 3 Department of Physiology, Cambridge University, Cambridge CB2 3EG, United Kingdom
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We predict that the type 2 ryanodine receptor isoform (RyR-2) located in the osteoclastic membrane functions as a Ca2+ influx channel and as a divalent cation (Ca2+) sensor. Cytosolic Ca2+ measurements revealed Ca2+ influx in osteoclasts at depolarized membrane potentials. The cytosolic Ca2+ change was, as expected, not seen in Ca2+-free medium and was blocked by the RyR modulator ryanodine. In contrast, at basal membrane potentials (~25 mV) ryanodine triggered extracellular Ca2+ influx that was blocked by Ni2+. In parallel, single-channel recordings obtained from inside-out excised patches revealed a divalent cation-selective ~60-pS conductance in symmetric solutions of Ba-aspartate [Ba-Asp; reversal potential (Erev) ~0 mV]. In the presence of a Ba2+ gradient, i.e., with Ba-Asp in the pipette and Na-Asp in the bath, channel conductance increased to ~120 pS and Erev shifted to 21 mV. The conductance was tentatively classified as a RyR-gated Ca2+ channel as it displayed characteristic metastable states and was sensitive to ruthenium red and a specific anti-RyR antibody, Ab34. To demonstrate that extracellular Ca2+ sensing occurred at the osteoclastic surface rather than intracellularly, we performed protease protection assays using pronase. Preincubation with pronase resulted in markedly attenuated cytosolic Ca2+ signals triggered by either Ni2+ (5 mM) or Cd2+ (50 µM). Finally, intracellular application of antiserum Ab34 potently inhibited divalent cation sensing. Together, these results strongly suggest the existence of 1) a membrane-resident Ca2+ influx channel sensitive to RyR modulators; 2) an extracellular, as opposed to intracellular, divalent cation activation site; and 3) a cytosolic CaM-binding regulatory site for RyR. It is likely therefore that the surface RyR-2 not only gates Ca2+ influx but also functions as a sensor for extracellular divalent cations.
osteoclast; calcium receptor; calcium channels; osteoporosis; calcium
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
IT HAS BECOME CLEAR OVER the last decade that Ca2+ can function as an extracellular or "first" messenger (6). Ca2+ receptors generally fall into a closely related family of G protein-coupled, seven-pass membrane receptors (7). Several genes for this receptor have been cloned and sequenced (7, 26, 27), and the list of cells sensitive to changes in ambient (extracellular) Ca2+ concentration ([Ca2+]) is expanding. This list includes renal tubular cells, neurons, gastric cells, enterocytes, osteoclasts, osteoblasts, and testicular cells. It is generally accepted that the bone-resorbing osteoclast monitors changes in its ambient [Ca2+] through a Ca2+ sensor (19, 40) that does not appear to be the traditional Ca2+ receptor.
In the osteoclast, high extracellular Ca2+ triggers a rise in cytosolic [Ca2+] through both transmembrane Ca2+ influx and intracellular Ca2+ release (4, 31, 39). The elevated cytosolic [Ca2+] results in cell-matrix detachment, margin retraction, reduced enzyme and acid secretion, and bone resorption inhibition (12, 19-21a, 40). These correlates are the basis of a negative-feedback mechanism through which an osteoclast monitors its own activity using resorbed Ca2+ as an extracellular signal (39). Furthermore, the agonist effects of Ca2+ are mimicked by several "surrogate" cations that follow a rank order of potency: Cd2+ > Ni2+ > Ca2+ > Ba2+ = Sr2+ > Mg2+ (29-31). Although we have assumed that these cations act at the osteoclastic surface, this has never been proven. It is critical to confirm this, as the cations may alternatively permeate the cell membrane and release stored Ca2+. We hypothesize that these effects occur through interaction with intracellular ryanodine receptors (RyRs) and/or inositol 3,4,5-trisphosphate receptors (IP3Rs).
We have shown recently that a type 2 RyR isoform (RyR-2) resides in the osteoclastic plasma membrane and functions in extracellular Ca2+ sensing (43). We previously observed intense, strictly peripheral immunostaining with a highly specific anti-RyR-2 antibody raised to a putatively extracellular epitope (43). Importantly, the antibody potentiated the Ca2+ release triggered by Ni2+, suggesting that Ni2+ interacted with the surface RyR (43). Furthermore, several known RyR modulators, including ryanodine (41), ruthenium red (1), caffeine (33), and cADP-ribose (1), all attenuated cation-induced Ca2+ release. Notably, the effect of ryanodine was modulated by membrane voltage, indicating that the triggering events for Ca2+ release occurred at or near the plasma membrane (41). Furthermore, Ni2+ displaced [3H]ryanodine bound to osteoclasts, strengthening the link between cation sensing and RyR activation (43). These sets of biophysical evidence suggest that surface RyR-2 may be a functional component of the extracellular Ca2+-sensing system.
Ordinarily located in endoplasmic reticular membranes, RyRs gate Ca2+ release from intracellular stores into the cytosol (5). Our demonstration that RyR-2 is situated in the osteoclastic plasma membrane (43) represents the only known plasma membrane location for an RyR. In contrast, for IP3Rs there is strong electrophysiological evidence for surface localization, whereas their function still remains unclear (22, 37). Recently, both RyRs and IP3Rs have been assigned a new role in nuclear Ca2+ homeostasis on the inner nuclear membrane (15, 28). They are thought to modulate nuclear Ca2+ influx in response to cytosolic Ca2+ and IP3 changes.
Here, we have characterized the putative functional domains of the
surface RyR-2 in osteoclasts. We first provide microfluorimetric and
electrophysiological evidence for Ca2+ (divalent cation)
influx through a surface RyR. We find that 1) RyR modulation
blocks Ca2+ influx; 2) ryanodine itself triggers
Ca2+ influx; and 3) there exists a ruthenium
red- and RyR antibody-sensitive, divalent cation-selective conductance
in inside-out osteoclastic membrane patches. Taken together, the
results are compelling enough to assign a tentative role for the
surface RyR-2 as a Ca2+ influx channel. Second, we show
that the divalent cations, such as Ni2+ and
Cd2+, trigger cytosolic Ca2+ release through
interaction with a surface, rather than an intracellular, site.
Finally, we find that intracellular application of the anti-RyR antibody highly specific for the cytosolic calmodulin (CaM)-binding RyR
domain (38) attenuates Ni2+-induced
Ca2+ release. This provides additional evidence that the
surface RyR-2 is itself the cation sensor. Topologically, this makes
sense because the known intraluminal, low-affinity
Ca2+-binding site of the surface RyR-2 (3)
should be expressed extracellularly in a plasma membrane configuration.
Nevertheless, a possibility remains that there is a separate sensor for
extracellular Ca2+ (18), perhaps one that is
linked to IP3 generation, the activation of which triggers
Ca2+ release, which is followed by Ca2+ influx
through the surface RyR-2 (Fig. 1,
scheme 2).
|
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isolation and culture of osteoclasts.
Osteoclasts from decapitated neonatal Wistar rats or New Zealand
rabbits were extracted by curetting long bones into 1 ml of
HEPES-buffered -MEM (GIBCO BRL, Gaithersburg, MD), supplemented with
heat-inactivated fetal bovine serum (5% vol/vol, Sigma, St. Louis,
MO). The bones were minced for ~1 min. The supernatant from rabbit
bones was vortexed at low speed for 20 s and centrifuged at 2,000 rpm for 10 min at 4°C. After centrifugation, the supernatant was
discarded, and the pellet was resuspended in 5 ml of
-MEM. The cells
were subsequently plated onto 35-mm plastic culture dishes (Fisher, St.
Louis, MO) and incubated overnight (5% CO2). The medium
was then changed three times, and the cells were further incubated for
24 h. Osteoclasts that were at least 40 µm in diameter, displayed obvious spreading and a ruffled border, and contained at
least three nuclei were used for electrophysiological studies (see
below). Isolated rat osteoclasts were used the same day for microspectrofluorometric studies. Briefly, the cells were incubated in
-MEM and allowed to settle for 60 min on 0-grade, 22-mm glass coverslips (Fisher; see the next subsection). Contaminating cells were
removed by gently rinsing the coverslips.
Microspectrofluorimetric measurements of cytosolic
Ca2+.
For measuring the cytosolic [Ca2+] in single rat
osteoclasts, coverslips containing freshly isolated osteoclasts were
incubated for 30 min at 37°C with 10 µM fura 2 acetoxymethyl ester
(Molecular Probes, San Diego, CA) in serum-free medium. They were then
washed in -MEM and transferred to a Perspex bath positioned on the
stage of a microspectrofluorimeter. The latter was previously
constructed from an inverted microscope (Diaphot, Nikon, Telford, UK).
The cells were exposed alternatively to excitation wavelengths of either 340 or 380 nm approximately every second. Instead of pipetting compounds into the experimental chamber, we used a constant-flow superfusion system that avoided experimental artifacts.
Electrophysiological measurements.
Ionic currents were monitored in both rat and rabbit osteoclasts using
the patch-clamp recording technique in cell-attached and inside-out
excised configurations (16). Pipettes were filled with
Ba-aspartate (Asp; 110 mM Ba-Asp and 10 mM HEPES), and the bath
contained either Ba-Asp or Na-Asp (153 mM Na-Asp, 0.01 mM Ca-Asp, and
10 mM HEPES/KOH). Cells were viewed on an inverted microscope (at
×600) equipped with Nomarski or contrast modulation optics (model
IMT-2, Olympus, Tokyo, Japan). Patch pipettes were pulled from
borosilicate capillary tubing (Drummond Scientific, Broomall, PA),
coated with Sylgard (Dow-Corning, Midland, MI), and fire-polished. To
standardize patch geometry, only pipettes with resistances ranging from
4 to 12 M were used. Current-voltage (I-V)
relationships and voltage dependences were examined either during
steady-state conditions or during the application of instantaneous voltage steps or voltage ramps (at 20°C).
Antibody experiments. We used the antibody Ab34 to examine the role of the CaM-binding site of the RyR in Ca2+ sensing in the osteoclast. The antibody was raised previously in rabbit against the consensus CaM-binding region of the three RyR isoforms. Thus the antibody does not discriminate between the receptor isoforms (43).
Osteoclasts from both rabbit and rat were gently made permeable with Triton X-100 (0.1% vol/vol, Sigma) for 30 s in PBS (10 mM). Triton X-100 was then removed, and the cells were immediately exposed to antibody Ab34 (1:100) in PBS. Sixty minutes after the application of PBS, cell impermeability was restored by replacing the PBS solution with medium 199 supplemented with 10% FCS. The cells were incubated at 37°C for ~5 h. To confirm the integrity and impermeability of the cells, they were loaded with fura 2 and monitored for cytosolic [Ca2+] changes (due to influx across a [Ca2+] gradient) and fura 2 efflux. For up to 2 h after the loading with fura 2, there was no significant change in cytosolic [Ca2+], no dye quenching (monitored for excitation of 380 nm), and no trypan blue uptake, confirming the integrity of the cells. Under these aforementioned permeability conditions, we have previously shown that Ab34 permeates the cell (as evident by immunocytochemical staining) (43). It is apparent that incubating the cells in physiological media with FCS restored cellular impermeance. ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ni2+ is required for cytosolic
Ca2+ influx in response to
depolarization.
Figure 2 illustrates typical results from
test maneuvers that investigated the effects of valinomycin given with
Ni2+ preceding elevations of extracellular
[K+] (Fig. 2, AC), as well as
control procedures that investigated the effects of the component
agents given by themselves (Fig. 2, D
F)
(extracellular [Ca2+] = 1.25 mM). Hyperpolarization of
the osteoclastic membrane by valinomycin (5 µM in 5 mM
K+), accompanying or followed by the application of 50 µM
[Ni2+], resulted in a gradually increasing cytosolic
[Ca2+] (
= 436 ± 125 nM, n = 4) (Fig. 2A). This was qualitatively similar to and not
significantly different (P = 0.288) from the response
to valinomycin itself (
= 418.5 ± 89.8 nM,
n = 11) (Fig. 2E). Subsequent elevation of
extracellular [K+] to 100 mM in the presence of both
valinomycin, which would be expected to result in cellular
depolarization, and Ni2+ produced a sharp, sustainable rise
in cytosolic [Ca2+] to ~1,500 nM (Fig. 2B).
This sustained rise was reversible when extracellular
[K+] was switched back to 5 mM [Fig. 2C,
arrow (iv)]. However, both Ni2+ and valinomycin, as
opposed to their separate application, were required if a subsequent
addition of [K+] was to modify measured levels of
cytosolic [Ca2+] (Fig. 2, D
G). In
the absence of valinomycin, 100 mM [K+] produced a much
smaller cytosolic [Ca2+] change (mean
= 76.2 nM), likely indicating that the cells were not depolarized (Fig.
2D; note the difference of scale between Fig. 2D
and Fig. 2, B and C). Addition of 50 µM
[Ni2+] alone in the absence of valinomycin provided only
a small change in cytosolic Ca2+ (32)
(
= 92 ± 17 nM) (Fig. 2F). In the absence of
50 µM [Ni2+], although the gradual valinomycin-induced
cytosolic Ca2+ rise persisted, the K+-induced
sharp rise was not sustained (Fig. 2G), in contrast to Fig.
2, B and C, where the K+-induced
increase in [Ca2+] was sustained. This latter finding
suggested that Ni2+, a known Ca2+-receptor
agonist, is required for the sharp and sustained rise of cytosolic
[Ca2+] in depolarized osteoclasts.
|
|
Depolarization-induced Ca2+ influx is
ryanodine sensitive.
The effect of ryanodine on the cytosolic [Ca2+] elevation
in depolarized cells was evaluated microspectrofluorimetrically in Figs. 4 and
5. Such results were observed
when osteoclasts, exposed to 5 µM [valinomycin] (i) followed by 50 µM [Ni2+] (ii), which led to prolonged elevation of
cytosolic [Ca2+], were subjected to an addition of 100 mM
[K+] together with 4 µM [ryanodine] (iii): this led
to an abolition of the expected rise in cytosolic Ca2+
triggered by 100 mM [K+] (Fig. 4A). The latter
was particularly clear in comparisons with Fig. 4B, which
illustrates a similar experiment in which 50 µM [Ni2+]
was added to valinomycin-pretreated osteoclasts (i) to elicit the
initial gradual elevation of cytosolic [Ca2+], and this
was followed by addition of 100 mM-[K+] (ii) to produce a
further sharp increase in cytosolic [Ca2+]. Thus the
latter increase was also aborted by addition of 4 µM [ryanodine]
(iii). The smaller changes in cytosolic [Ca2+] triggered
by 100 mM [K+] without valinomycin were also blocked by
ryanodine (4 µM) (Fig. 4C). These results suggest that the
changes in cytosolic [Ca2+] were sensitive to ryanodine
application.
|
|
Electrophysiological evidence for an RyR-like cation conductance.
To confirm and further explore the characteristics of the detected
Ca2+ influx pathway, we performed electrophysiological
studies on cell-attached and inside-out excised patches.
Ba2+ was used as a divalent carrier ion in the pipette, and
the bath contained either Ba-Asp (110 mM) or Na-Asp (153 mM; see
MATERIALS AND METHODS). The cells were held at voltages
between 0 and 60 mV. A divalent cation-permeable channel was
identified in 9 of 52 (18%) osteoclasts (Fig.
6). This suggests that either channel density was relatively low or that the channels were mostly inactive. The kinetic behavior of this channel was variable: it frequently displayed one or more substates (partially open states) during the
course of recording. Note that at a membrane potential of
60 mV, a
substate was observed. The latter feature is reminiscent of recordings
obtained from RyRs in lipid bilayers (13). However, quite
typically, channel activity was continuous, with channel openings
occurring in both long- and short-duration bursts separated by
short-lived closed states. In several records, however, very long,
closed intervals were seen, suggesting that the channel resides in one
or more sustained inactive states. Furthermore, in at least three of
the patches, channel activity appeared to be sensitive to membrane
voltage. Thus the open probablility (Po) values
increased from 0.5 at
60 mV to
0.95 at
20 mV.
|
|
|
Evidence for a divalent cation-sensitive site on the osteoclastic
surface.
We have reported substantial biophysical data suggesting that the
surface RyR-2 may function as the Ca2+ and possibly
Ni2+ sensor (1, 31, 33, 41). To examine
whether such divalent cation-sensing occurred on the extracellular
osteoclastic surface rather than intracellularly, we performed protease
protection assays using the proteolytic enzyme pronase. Osteoclasts
were exposed to 5 mM [Ni2+] or 50 µM
[Cd2+] after preincubation with either 10 or 25 mg/l
pronase for intervals between 0 and 40 min. We used 5 mM
[Ni2+], rather than 50 µM [Ni2+], because
this was the maximally effective concentration and we expected gradual
reduction in the cytosolic Ca2+ response to zero from a
maximal response magnitude (31). Figures 9 and 10
show a time-dependent attenuation of the peak cytosolic response to the divalent cations applied, Ni2+ or
Cd2+, respectively, in pronase-pretreated cells. With 10 mg/l pronase (Fig. 9, A and C), maximal
attenuation occurred after a 20-min preincubation, which was well
before membrane damage set in, as evidenced by trypan blue uptake at
~28 min. With 25 mg/l pronase (Fig. 9, B and
D), maximal attenuation occurred within 5 min, whereas
trypan blue uptake was positive at 15 min. The results with
Cd2+ were identical to those with Ni2+ with an
~80% attenuation occurring within 10 min of pronase application (Fig. 10). Table 1 gives the mean
cytosolic [Ca2+] changes for Fig. 10C, thus
showing that the application of one divalent cation significantly
attenuates subsequent responses to the other.
|
|
|
Regulation of Ca2+ sensing through
the cytosolic CaM-binding domain of the RyR.
To test the hypothesis that a single surface-resident RyR-2 subserves
the dual roles of Ca2+ sensor and Ca2+ influx
channel, experiments were performed with Ab34, an
inhibitory antibody to RyR's cytosolically located CaM-binding sequence (43). The cytosolic introduction of
Ab34 by gentle permeabilization (42) markedly
attenuated the Ca2+ signals elicited by 5 mM
[Ni2+] (Fig. 11) (see
MATERIALS AND METHODS). That this action was intracellular was examined by comparing this response to that triggered by
extracellular Ab34 application. No attenuation of the
Ca2+ signal was observed with extracellular
Ab34 application, consistent with the cytosolic location of
the CaM-binding site. It is also noteworthy that the antibody has been
shown previously to stain permeabilized osteoclasts but not cells whose
plasma membranes are intact (43). These results provide
evidence for an intracellular CaM regulatory site on the RyR that
modulates divalent cation sensing.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have demonstrated previously that a type 2 RyR isoform positioned uniquely at the osteoclastic surface is involved in extracellular Ca2+ sensing (43). We now extend these studies to probe into its topology and functionality. We believe that the surface RyR-2 likely serves as a channel for the transcellular flux of Ca2+ during bone resorption. It is well known that Ca2+ concentrations in the resorptive hemivacuole rise to between 8 and 40 mM (34). This triggers an increase in cytosolic [Ca2+] followed by margin and podosomal retraction (12, 19). The latter should itself serve as a critical mechanism for the outward diffusion of the increasing ambient [Ca2+]. Nevertheless, it is conceivable that the surface RyR-2 also allows considerable Ca2+ influx. The rising cytosolic Ca2+ is then likely pumped out of the cell through the action of a Ca2+-ATPase and a Na+/Ca2+ exchanger that we have recently described (21).
This study provides the first evidence that the surface RyR acts as a divalent cation sensor and permits extracellular Ca2+ influx. Notably, an ~60-pS, divalent cation-selective, channel was observed that we tentatively identified as the RyR on the basis of two key observations: the existence of metastable states (13) and its complete blockade by the RyR modulator ruthenium red and by Ab34, an antibody raised to the CaM-binding region of the RyR family of receptors (34). The use of ruthenium red and Ab34 for these experiments using inside-out patches has also allowed us to confirm the hypothesized topology of the surface RyR-2 (43). Because of its highly charged nature, ruthenium red cannot permeate cell membranes. Therefore, it should not activate RyR domains that face the interior of the patch pipette (Fig. 8, inset). The known location of the ruthenium red-binding site is on the cytosolic portion of the RyR. As noted above, Ab34 binds the CaM-binding region located cytosolically in intact cells. Thus the blockade of Ba2+ conductance in inside-out patches, i.e., those with their cytosolic surface facing away from the pipette interior, would imply that the respective sites remain cytosolic in the surface membrane RyR-2. Information on topology in electrophysiological studies could not have been obtained using ryanodine as it permeates the plasma membrane freely and can cause contradictory effects on the channel Po depending on its concentration.
In microspectrofluorimetric experiments, application of ryanodine
blocked Ca2+ influx in osteoclasts at concentrations
similar to those that block cation permeation through skeletal muscle
RyRs reconstituted in lipid bilayers (36). Alternatively,
under certain conditions, such as a low concentration, ryanodine can
activate skeletal muscle RyRs to cause Ca2+ release
(36). In the osteoclast, it activates the RyR-2 to cause
both Ca2+ release and Ca2+ influx. The
induction of Ca2+ release by ryanodine was noted at resting
membrane potentials of around 25 mV (Fig. 5), whereas the blockage of
Ca2+ influx was noted in depolarized cells (Fig. 4).
Indeed, voltage sensitivity of RyRs has also been documented in lipid
bilayer studies (23), as well as by us in the osteoclast
(40).
The present demonstration of both excitatory and inhibitory effects of ryanodine on cytosolic [Ca2+] may be explained in terms of recent reports on the modulation of RyR conformation by changes in membrane potential (36a). Membrane potential, by altering the affinity of RyR for ryanodine, permits two different binding states that have been associated with the inhibitory and stimulatory effects of ryanodine (36a). New reports (19a, 36a) indicate that ryanodine does not lock the RyR into a ligand-insensitive open state. In agreement with the above findings, we show that ryanodine has dual actions depending on membrane potential and that the ryanodine-exposed RyR is modifiable by ligands such as Ni2+.
What might cause the surface RyR to become activated physiologically is not clear. Several possibilities exist (Fig. 1). Resorbed Ca2+ may itself activate RyR through its low-affinity Ca2+-binding site (Ref. 3; see immediately below and Fig. 1, scheme 1). Alternatively, Ca2+ may first activate a hypothetical, possibly G protein-coupled, Ca2+ sensing receptor (18), resulting in IP3 formation and Ca2+ release (Fig. 1, scheme 2). The elevated cytosolic [Ca2+] may then activate the surface RyR-2 via an action on its high-affinity cytosolic Ca2+-binding site (3). Additionally, capacitative Ca2+ influx may ensue either because of intracellular Ca2+ store depletion (42) or due to conformational coupling between the surface and intracellular RyRs (17, 22).
We have previously shown that, in addition to Ca2+ influx, cation-induced Ca2+ release is inhibited by several RyR modulators, including ryanodine (41), caffeine (33), and ruthenium red (1). It has remained unclear whether these modulators interfere with the surface or intracellular actions of the applied cations. To rule out the possibility that divalent cations, such as Ni2+ and Cd2+, to which the plasma membrane is generally considered impermeant (8), could permeate and trigger Ca2+ release by interacting with intracellular RyRs and IP3Rs, we conducted a protease protection assay. The proteolytic enzyme pronase, when applied at low concentrations, is known to cleave most cell surface proteins. Short of permeabilizing the cell membrane, pronase would be expected to abolish divalent cation sensing through any surface protein, including the surface RyR-2. We found that pronase abolished Ni2+- and Cd2+-induced Ca2+ release in a concentration- and time-dependent manner with membrane impermeability intact. This indicates that Ni2+ and Cd2+ act at the cell's surface, rather than intracellularly.
That the plasma membrane remained impermeant after pronase treatment
even to small cations and molecules was confirmed in four ways. Most
importantly, there were no significant shifts in basal cytosolic
[Ca2+], confirming that the cell membrane remained
impermeant even to a small divalent cation, Ca2+. Second,
any leakage of the Ca2+-sensitive fluorochrome fura 2 (molecular mass ~1 kDa) from the cell was assessed by continuous
monitoring of background fluorescence at ex of 380 nm.
Fluorescence remained constant over the course of the experiment,
virtually excluding dye extrusion and again confirming an intact plasma
membrane. Third, addition of Ni2+ or Cd2+ did
not result in a precipitous reduction in background fluorescence at
ex of 380 nm. Such a reduction would be expected if
either cation permeated the cell membrane and quenched fura 2. Fourth, trypan blue (molecular mass ~961 Da) uptake remained negative up to
28 min after incubation with 10 mg/l pronase and up to 15 min after
incubation with 25 mg/l pronase. Together, these results strongly
suggest that the cations Ni2+, Cd2+, and
possibly Ca2+ interact with a cell-surface moiety, likely
RyR-2, to release intracellularly stored Ca2+.
We could not use Ca2+ in these studies to activate the Ca2+ sensor because we would then be measuring a combination of events, namely, receptor activation, Ca2+ influx, and Ca2+ release. With Ni2+ and Cd2+ as surrogate cations, it becomes possible to separate these components. Note, however, that the Ni2+ concentrations between the two sets of experiments to demonstrate Ca2+ release and Ca2+ influx are different. We have shown extensively in previous studies (31, 39-43) that 50 µM Ni2+ triggers the Ca2+ sensor to cause Ca2+ release without blocking the channel. Therefore, in Figs. 2-4, we simply aim at triggering the RyR/Ca2+ sensor while allowing the Ca2+ influx under study to continue. In Figs. 9 and 10, however, we are using Ni2+ at 5 mM as this is the concentration that elicits Ca2+ release from intracellular stores and blocks Ca2+ influx. It is the ideal concentration for selectively examining Ca2+ release, as opposed to a combination of Ca2+ release and Ca2+ influx that would otherwise be difficult to separate. The concept of using surrogate divalent cations is not new. They have been elegantly used in early studies with parathyroid cells (see Ref. 39 for a review).
We used Cd2+ to replicate and substantiate the observed effects of Ni2+ in the pronase protection assay. In this assay, we examined Ca2+ release alone rather than the combination of Ca2+ release and Ca2+ influx because Ni2+ and Cd2+ both block RyRs (36). Both Cd2+ and Ni2+ are also surrogate agonists of the osteoclastic Ca2+ sensor/RyR (30, 39). Thus data presented in Table 1 show that Cd2+ and Ni2+ cross-react to desensitize each other's effects. Cd2+ also replicates the effect of Ni2+ in the pronase protection assay (Fig. 10). Furthermore, Cd2+ has a greater ionic radius and is used at a 100-fold lower concentration than Ni2+. Hence, Cd2+ is less likely than Ni2+ to permeate the cell membrane. Together, the results strengthen our hypothesis for a surface action of both cations.
Finally, intracellular introduction of Ab34 by gentle cell permeabilization (38) potently inhibited Ni2+-induced Ca2+ release. Expectedly, extracellular application of the antibody failed to attenuate Ca2+ release. This was consistent with the blockade of Ba2+ currents by the antibody in inside-out patches of osteoclastic plasma membranes, particularly as the antibody-binding site is expected to be cytosolic rather than extracellular. Taken together, these observations also suggest that the same epitope of the plasma membrane receptor was involved in modulating cytosolic Ca2+ release and Ca2+ influx. This antibody has previously been shown to stain permeabilized but not intact osteoclasts, suggesting a cytosolic action site (43). Whether the antibody also blocks the cytosolic CaM-binding site of organelle RyRs, however, remains to be determined. Nevertheless, these studies provide further and more compelling evidence that RyRs, in particular the surface RyR-2, play a role in Ca2+ sensing and Ca2+ influx in the osteoclast.
We thus demonstrate that the surface RyR-2 serves as both a Ca2+ influx channel and a divalent cation sensor. It would seem that the topology of the surface RyR-2, predicted from our previous studies (43) and ruthenium red data, would suit its proposed dual role. Notably, portions of a molecule that face the cytosol in its endoplasmic reticular configuration should remain cytosolic in its plasma membrane configuration. In the case of RyR, these cytosolic domains would include its high-affinity Ca2+-activation and CaM-responsive sites (38). Parts of the RyR that are normally intraluminal (3) should become extracellular. It is noteworthy that the cardiac muscle RyR-2 isoform possesses an intraluminal site that has a low millimolar affinity for Ca2+ (3). At least conceptually, this low-affinity divalent cation-binding site could represent the sensor for Ca2+ (Fig. 1, scheme 1). However, we cannot rule out the possibility of a separate Ca2+ sensor, one linked, for example, to the G protein-phospholipase C-IP3 pathway (Ref. 6; Fig. 1, scheme 2). Nevertheless, neither this sensor nor a similar molecule has been isolated from or identified in the osteoclast. Furthermore, the micromolar affinity of such a Ca2+ receptor is not consistent with the sensing of the millimolar ambient Ca2+ levels generated from hydroxyapatite dissolution. There are other possibilities. For example, a variant of the low-density lipoprotein receptor/Ca2+ sensor previously isolated from the cytotrophoblast (39) may play a role, although we find no evidence of this in preliminary studies. Alternatively, a new Ca2+-gated Ca2+ channel, polycystin-L, has been identified in the intestine (10), and a similar molecule may exist in the osteoclast. These interesting possibilities require further investigation.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Prof. Iain MacIntyre, Fellow of the Royal Society, for continuing support and Prof. F. A. Lai (Cardiff) for the antibody Ab34.
![]() |
FOOTNOTES |
---|
* S. Li and J. Iqbal contributed equally to this work.
This study was supported by grants to M. Zaidi from the National Institute on Aging (RO1-AG-14917-05) and the Department of Veterans Affairs (Merit Award and Geriatric Research, Education, and Clinical Center).
Address for reprint requests and other correspondence: M. Zaidi, Mount Sinai Bone Program, Div. of Endocrinology, PO Box 1055, Mount Sinai School of Medicine, 1 Gustave Levy Place, New York, NY 10029 (E-mail: mone.zaidi{at}mssm.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajprenal.00045.2000
Received 7 February 2000; accepted in final form 16 November 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Adebanjo, OA,
Igietseme J,
Huang CL,
and
Zaidi M.
The effect of extracellularly applied divalent cations on cytosolic Ca2+ in murine Leydig cells: evidence for a Ca2+ sensing receptor.
J Physiol (Lond)
513:
399-410,
1998
2.
Adebanjo, OA,
Shankar VS,
Pazianas M,
Simon B,
Lai FA,
Huang CLH,
and
Zaidi M.
Extracellularly applied ruthenium red and cADP-ribose elevate cytosolic Ca2+ in isolated rat osteoclasts.
Am J Physiol Renal Fluid Electrolyte Physiol
270:
F469-F475,
1996
3.
Anderson, K,
Lai FA,
Liu QY,
Erickson HP,
and
Meissner G.
Structural and functional characterization of the purified cardiac ryanodine receptor-calcium release channel.
J Biol Chem
264:
1329-1335,
1989
4.
Bax, CMR,
Shankar VS,
Moonga BS,
Huang CLH,
and
Zaidi M.
Is the osteoclast calcium "receptor" a receptor-operated calcium channel?
Biochem Biophys Res Commun
183:
619-625,
1992[ISI][Medline].
5.
Berridge, MJ.
Inositol trisphosphate and calcium signaling.
Nature
361:
315-325,
1993[ISI][Medline].
6.
Brown, EM.
Extracellular calcium sensing regulation of parathyroid cell function and other ions as extracellular first messenger.
Physiol Rev
71:
371-411,
1991
7.
Brown, EM,
Pollak M,
and
Seidman CE.
Calcium-ion-sensing cell-surface receptors.
N Engl J Med
333:
243-240,
1995.
8.
Caputo, C.
Nickel substitution for calcium and the time course of potassium conductances of single muscle fibers.
J Muscle Res Cell Motil
2:
167-182,
1981[ISI][Medline].
9.
Chattopadhyay, N,
Ye CP,
and
Yamaguchi T.
Evidence for extracellular calcium-sensing receptor mediated opening of an outward K+ channel in a human astrocytoma cell line.
Glia
26:
64-72,
1999[ISI][Medline].
10.
Chen, XZ,
Vassilev PM,
Basora N,
Peng JB,
Nonura H,
Segal Y,
Brown EM,
Reeders ST,
Hediger MA,
and
Zhou J.
Polycystin-L is a calcium-regulated cation channel permeable to calcium ions.
Nature
401:
383-386,
1999[ISI][Medline].
11.
Cima, RR,
Cheng I,
and
Klingensmith ME.
Identification and functional assay of an extracellular calcium-sensing receptor in Necturus gastric mucosa.
Am J Physiol Gastrointest Liver Physiol
273:
G1051-G1060,
1997
12.
Datta, HK,
MacIntyre I,
and
Zaidi M.
The effect of extracellular calcium elevation on morphology and function of isolated rat osteoclasts.
Biosci Rep
9:
747-751,
1990[ISI].
13.
Ding, J,
and
Kasai M.
Analysis of multiple conductance states observed in Ca2+ release channel of sarcoplasmic reticulum.
Cell Struct Funct
21:
7-15,
1996[ISI][Medline].
14.
Gama, L,
Baxendale-Cox LM,
and
Breitwieser GE.
Calcium sensing receptor in intestinal epithelium.
Am J Physiol Cell Physiol
273:
C1168-C1175,
1997[ISI][Medline].
15.
Gerasimenko, OV,
Gerasimenko JV,
Tepikin AV,
and
Petersen OH.
ATP-dependent accumulation and inositol trisphosphate- or cyclic ADP-ribose-mediated release of Ca2+ from the nuclear envelope.
Cell
80:
439-444,
1995[ISI][Medline].
16.
Hamill, OP,
Marty A,
Neher E,
Sakmann B,
and
Sigworth FJ.
Improved patch-clamp techniques for high-resolution current recording from cell and cell-free membrane patches.
Pflügers Arch
391:
85-100,
1981[ISI][Medline].
17.
Holda, JR,
Klishin A,
Sedova M,
Huser J,
and
Blatter L.
Capacitative calcium entry.
News Physiol Sci
13:
157-163,
1998
18.
Kameda, T,
Mano H,
Yamada Y,
Takai H,
Amizuka N,
Kobori M,
Izumi N,
Kawashima H,
Ozawa H,
Ikeda K,
Kameda A,
Hakeda Y,
and
Kumegawa M.
Calcium-sensing receptor in mature osteoclasts, which are bone-resorbing cells.
Biochem Biophys Res Commun
245:
419-422,
1998[ISI][Medline].
19.
Malgaroli, A,
Meldolesi J,
Zabonin-Zallone A,
and
Teti A.
Control of cytosolic free calcium in rat and chicken osteoclasts.
J Biol Chem
264:
14342-14347,
1989
20.
Masumiya, H,
Li P,
Zhang L,
and
Chen SR.
Ryanodine sensitizes the Ca2+ release channel (ryanodine receptor) to Ca2+ activation.
J Biol Chem
276:
39727-39735,
2001
21.
Miyauchi, A,
Hruska KA,
Greenfield EM,
Duncan R,
Alvarez J,
Barattolo R,
Colluci S,
Zambonin-Zallone A,
Teitelbaum SL,
and
Teti A.
Osteoclast cytosolic calcium, regulated by voltage-gated calcium channels, extracellular calcium, controls podosome assembly and bone resorption.
J Cell Biol
111:
2543-2552,
1990[Abstract].
21b.
Moonga, BS,
Davidson R,
Sun L,
Adebanjo OA,
Moser J,
Abedin M,
Zaidi N,
Huang CLH,
and
Zaidi M.
Identification and characterization of a sodium/calcium exchanger, NCX-1, in osteoclasts and its role in bone resorption.
Biochem Biophys Res Commun
283:
770-775,
2001[ISI][Medline].
21a.
Moonga, BS,
Moss DW,
Patchell A,
and
Zaidi M.
Intracellular regulation of enzyme secretion from rat osteoclasts and evidence for a functional role in bone resorption.
J Physiol (Lond)
42:
29-45,
1990.
22.
Mozhayeva, GN,
Naumov AP,
and
Kuryshev YA.
Inositol 1,4,5-trisphosphate activates two types of Ca2+-permeable channels in human carcinoma cells.
FEBS Lett
277:
233-234,
1990[ISI][Medline].
23.
Nakai, J,
Imagawa T,
Halamata Y,
Shigekawa M,
Takeshima H,
and
Numa S.
The primary structure and functional expression from cDNA of the cardiac ryanodine receptor/calcium release channel.
FEBS Lett
271:
169-177,
1990[ISI][Medline].
24.
Pazianas, M,
Adebanjo AO,
Shankar V,
James SY,
Colston KW,
Maxwell JD,
and
Zaidi M.
Extracellular calcium-sensing by the enterocyte: prediction of a novel divalent cation "receptor."
Biochem Biophys Res Commun
210:
448-453,
1995.
25.
Randriamanpita, C,
and
Tsein RY.
Emptying of intracellular Ca2+ stores releases a novel small messenger that stimulated Ca2+ influx.
Nature
364:
809-814,
1993[ISI][Medline].
26.
Ricardi, D,
Pak J,
and
Lee WS.
Cloning and functional expression of a rat kidney extracellular calcium/polyvalent cation-sensing receptor.
Proc Natl Acad Sci USA
92:
3161-3165,
1995[Abstract].
27.
Ruat, M,
Molliver M,
Snowman A,
and
Synder SH.
Calcium sensing receptor: molecular cloning in rat and localization to nerve terminals.
Proc Natl Acad Sci USA
92:
3161-3165,
1995[Abstract].
28.
Santella, L,
and
Carafoli E.
Calcium signaling in the cell nucleus.
FASEB J
11:
1091-1109,
1997
29.
Shankar, VS,
Alam ASMT,
Bax CMR,
Bax BE,
Pazianas M,
Huang CLH,
and
Zaidi M.
Activation and inactivation of the osteoclast Ca2+ receptor by the trivalent cation, La3+.
Biochem Biophys Res Commun
187:
907-912,
1992[ISI][Medline].
30.
Shankar, VS,
Bax CMR,
Alam ASMT,
Bax BE,
Huang CLH,
and
Zaidi M.
The osteoclast Ca2+ receptor is highly sensitive to activation by transition metal cations.
Biochem Biophys Res Commun
187:
913-918,
1992[ISI][Medline].
31.
Shankar, VS,
Bax CMR,
Bax BE,
Alam ASMT,
Simon B,
Pazianas M,
Moonga BS,
Huang CLH,
and
Zaidi M.
Activation of the Ca2+ "receptor" on the osteoclast by Ni2+ elicits cytosolic Ca2+ signals: evidence for receptor activation and inactivation, intracellular Ca2+ redistribution and divalent cation modulation.
J Cell Physiol
155:
120-129,
1993[ISI][Medline].
32.
Shankar, VS,
Huang CL,
Adebanjo O,
Simon B,
Alam ASMT,
Moonga BS,
Pazianas M,
Scott RH,
and
Zaidi M.
Effect of membrane potential on surface Ca2+ receptor activation in rat osteoclasts.
J Cell Physiol
162:
1-8,
1995[ISI][Medline].
33.
Shankar, VS,
Pazianas M,
Huang CLH,
Simon B,
Adebanjo O,
and
Zaidi M.
Caffeine modulates Ca2+ receptor activation in isolated rat osteoclasts and induces intracellular Ca2+ release.
Am J Physiol Renal Fluid Electrolyte Physiol
268:
F447-F454,
1995
34.
Silver, IA,
Murrills RJ,
and
Etherington DJ.
Microelectrode studies on acid microenvironment beneath adherent macrophages and osteoclasts.
Exp Cell Res
175:
266-276,
1988[ISI][Medline].
35.
Sundaresan, S,
Weiss J,
Bauer-Dantoin AC,
and
Jameson JL.
Expression of ryanodine receptors in the pituitary gland: evidence for a role in gonadotropin-releasing hormone signaling.
Endocrinology
138:
2056-2065,
1997
36.
Sutko, JL,
Airey JA,
Welch W,
and
Ruest L.
The pharmacology of ryanodine and related compounds.
Pharm Rev
49:
55-98,
1997.
36a.
Tanna, B,
Welch W,
Ruest L,
Sutko JL,
and
Williams AJ.
The interaction of a neutral ryanodine with the ryanodine receptor channel provides insights into the mechanisms by which ryanodine binding is modulated by voltage.
J Gen Physiol
116:
1-9,
2000
37.
Vaca, L,
and
Kunze DL.
IP3-activated Ca2+ channels in the plasma membrane of cultured vascular endothelial cells.
Am J Physiol Cell Physiol
269:
C733-C738,
1995[Abstract].
38.
Wagenknecht, T,
and
Radermacher M.
Three-dimensional architecture of the skeletal muscle ryanodine receptor.
FEBS Lett
369:
43-46,
1995[ISI][Medline].
39.
Zaidi, M,
Alam ASMT,
Huang CLH,
Pazianas M,
Bax CMR,
Bax BE,
Moonga BS,
Bevis PJR,
and
Shankar VS.
Extracellular Ca2+ sensing by the osteoclast.
Cell Calcium
14:
271-277,
1993[ISI][Medline].
40.
Zaidi, M,
Datta HK,
Patchell A,
Moonga BS,
and
MacIntyre I.
"Calcium-activated" intracellular calcium elevation: a novel mechanism of osteoclast regulation.
Biochem Biophys Res Commun
163:
1461-1465,
1989[ISI][Medline].
41.
Zaidi, M,
Shankar VS,
Alam ASMT,
Moonga BS,
Pazianas M,
and
Huang CLH
Evidence that a ryanodine receptor triggers signal transduction in the osteoclast.
Biochem Biophys Res Commun
188:
1332-1336,
1992[ISI][Medline].
42.
Zaidi, M,
Shankar VS,
Bax CMR,
Bax BE,
Bevis PJR,
Pazianas M,
Alam ASMT,
and
Huang CLH
Linkage of extracellular and intracellular control of cytosolic Ca2+ in rat osteoclasts in the presence of thapsigargin.
J Bone Miner Res
8:
961-967,
1993[ISI][Medline].
43.
Zaidi, M,
Shankar VS,
Tunwell R,
Adebanjo OA,
MacKrill J,
Pazianas M,
O'Connell D,
Simon BJ,
Rifkin BR,
Ventikaraman AR,
Huang CLH,
and
Lai FA.
A ryanodine receptor-like molecule expressed in the osteoclast plasma membrane functions in extracellular Ca2+ sensing.
J Clin Invest
96:
1582-1590,
1995[ISI][Medline].
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |