JOURNAL OF BONE AND MINERAL RESEARCH

Volume 7, Supplement 2, 1992

Mary Ann Liebert, Inc., Publishers

Voltage, Calcium, and Stretch Activated Ionic Channels

and Intracellular Calcium in Bone Cells

DIRK L. YPEY,' ADAM F. WIDEMA,',' KARIN M. HOLD,3 ARNOUD VAN DER LAARSE,3
JAN H. RAVESLOOT,',' ARIE VAN DER PLAS,' and PETER J. NIJWEIDE2

ABSTRACT
Embryonic chick bone cells express various types of ionic channels in their plasma membranes for as yet un-
resolved functions. Chick osteoclasts (OCL) have the richest spectrum of channel types. Specific for OCL is
a K*channel, which activates (opens) when the inside negative membrane potential (V,) becomes more neg-
ative (hyperpolarization). This is consistent with findings of others on rat OCL. The membrane conductance
constituted by these channels is called the inward rectifying K*conductance (&I), or inward rectifier, be-
cause the hyperpolarization-activatedchannels cause cell-inward K*current to pass more easily through the
membrane than outward K' current. Besides &i channels, OCL may express two other types of voltage-ac-
tivated K*channels. One constitutes the transient outward rectifying K*conductance (&& which is acti-
vated upon making the membrane potential less negative (depolarization) but has a transient nature. This
conductance favors transient K' conduction in the cell-outward direction. The &to also occurs in a small
percentage of cells in osteoblast (OBL) and periosteal fibroblast (PFB) cultures. The other OCL K*conduc-
tance, the &a, is activated by both membrane depolarization and a rise in [Ca'*]i. &cP channels are also
present in the other chick bone cell types, that is, OBL, osteocytes (OCY), and PFB. Furthermore, in excised
patches of all bone cell types, channels have been found that conduct anions, including CI- and phosphate
ions. These channels are only active around V, = 0 mV. While searching for a membrane mechanism for
adaptation of bone to mechanical loading, we found stretch-activatedchannels in chick osteoclasts; other in-
vestigators have found stretch-activated cation channels (K*or aselective) in rat and human osteogenic cell
lines. In contrast to other studies on cell lines or OBL from other species, we have not found any of the
classic macroscopic voltage-activated calcium conductances (Gcr) in any of the chick bone cells under our
experimental conditions. However, our fluorescence measurements of [Ca'*]i in single cells indicate the pres-
ence of CaZ+conductive pathways through the plasma membrane of osteoblastic cells and osteoclasts, consis-
tent with other studies. We discuss possible roles for &I, &a, and anion channels in acid secretion by
OCL and for stretch-activated channels in OCL locomotion.

INTRODUCTION tion of extra- and intracellular substances, and by mechan-

ical stress of the membrane."' Examples of channel-acti-
are tunnel-shaped transmembrane pro-
I ONIC CHANNELS
teins, serving as more or less selective conduction path-
ways for ions across the plasma membrane and across
vating substances are neurotransmitters, hormones, auto-
crine substances, and second messengers.(3)
Changes in the number of open channels of a certain
membranes surrounding intracellular organelles.(') Open- type may have two consequences for the cell. First, the
ing and closure of these conduction pathways may be con- membrane potential of the cell may change. This may in-
trolled by the transmembrane potential, by the concentra- fluence the flow of other ions through other channels as

'Department of Physiology, Leiden University, The Netherlands.

'Department of Cell Biology and Histology, Leiden University, The Netherlands.
'Department of Cardiology, University Hospital, Leiden, The Netherlands.

s377
 !a78 YPEY ET AL.

well, since the membrane potential change may cause fibroblasts (PFB) from 18 day chick embryonic calvariae
opening or closure of other channels and alters the driving were isolated and cultured on glass coverslips as described
force for the ions moving through the channels that are al- In some cases pure OCY cultures were
ready open. Thus, passive transmembrane transport of a used.‘”) OCL were usually taken for experiments a few
given type of ion is principally coupled to transport of hours after isolation. OBL/OCY were used after 1-3 days
other types of ions through changes in the membrane po- of culture, OCY could be recognized from their slender
tential, which we find of importance to understand the role branched processes, contacting the processes of other
of ionic channels in bone cell functions. Second, the intra- OCY, as in vivo.
cellular concentration of ions may change, which in turn
may provide a signal to the cell to change its functional ac-
Patch-clamp technique
tivity. For example, an influx of Ca2+ions into the cell may Details of the technique we used have been given be-
control protein secretion,(4)fluid and salt secretion,(s)cell fore,(15*20)but for convenience we explain here the metho-
movement,(6)or other metabolic processes. dology, based on Hamill et al.(13)and using the results re-
In recent years investigatorshave begun to study the role viewed in Figs. 1 and 2.
of ionic channels in bone cell function^(^.'-^^) using a The actual measuring probe of the patch-clamp tech-
powerful membrane electrophysiologic technique, known nique is the patch pipette electrode. This is a glass micro-
as the patch-clamp technique. (l3.l4) This technique, for pipette, drawn from a 1-2 mm glass capillary to an open
which Neher and Sakmann recently received the Nobel tip diameter of approximately 1 pn. The pipette is filled
prize in Medicine and Physiology, allows the measurement with a physiologic salt solution in which an Ag/AgCl wire
of the microscopic, picoampere (l0-l2 A) currents through is inserted to connect the ion-conducting solution inside
single ionic channel proteins in small patches of membrane the pipette to the input of a current or voltage amplifier.
but can also be used to measure the macroscopic currents To obtain a measurement circuit, a reference Ag/AgCl
through or potentials across the plasma membrane of the electrode in the physiologic salt solution bathing the cells is
whole cell. The technique allowed the discovery of various connected to the other input of the amplifier (see insets,
types of cation and anion channels in bone cells. Most Fig. 1). Using a remote-controlled manipulator, the patch
channels are voltage activated‘l5)but some are sensitive to pipette can be placed on the membrane of a cell under the
membrane stretch. (16) These results open the possibility microscope. Upon touching the membrane, slight suction
that biophysical, that is, electrical and mechanical, mem- is applied to the inside of the pipette to obtain firm attach-
brane properties are important determinants in the control ment and sealing of the membrane to the mouth of the
of bone cell functions, such as bone formation by osteo- pipette. Once the seal is perfect, as decided from the high
blasts, bone resorption by osteoclasts, autocrine communi- value of the seal resistance R, (> 1 GQ 1 Gfl = lo9 fl),
cation between osteoblasts and osteoclasts, and intercellu- measured between the inside of the pipette and the bath,
lar communication between osteocytes and osteoblasts picoampere ionic currents through ionic channels in the
through gap junctions.‘”) The stretch sensitivity of ionic patch can be measured while clamping the membrane
channel activity may even imply a mechanism for bone patch at different voltage displacements from the resting
adaptation to changes in mechanical loading. membrane potential with the voltage source in the patch-
Another way to study the role of ionic membrane cur- clamp amplifier. The measurement configuration obtained
rents in bone cell functions is to measure the resulting in- in this way is called the cell-attached patch configuration
tracellular ion concentration changes using fluorescence (CAP; see Fig. 1A). It allows the recording of single-chan-
techniques. This technique has been greatly improved in nel activity from a patch while the intact cell is still at-
recent years(’9)and can even be used to measure ion con- tached to the patch. Therefore, this configuration is most
centration changes in (parts of) single cells (microfluores- useful to study the functional role of identified channels
cence), thus completing patch-clamp measurements on sin- under physiologic conditions. By rapidly pulling the sealed
gle cells. patch pipette from the cell, the sealed patch can be “ex-
It is the purpose of the present paper to review the cised” from the cell and becomes an inside-out patch (IOP;
patch-clamp studies on chick bone cells from our group in see Fig. lC), with the inside face of the membrane exposed
the light of studies of other groups on other species, to ex- to the bathing fluid. This configuration allows well-con-
plain the methodology used (the patch-clamp and micro- trolled studies of the effects of cytoplasmic factors on
fluorescence technique), to add some new comlementary channel activity. Another widely used measurement con-
results, and to integrate the data into a functional context. figuration is the whole-cell (WC) configuration (see Fig.
The new results presented include stretch-activated ionic lB), which is obtained by breaking a cell-attached patch by
channels in osteoclasts and [Caf+Jimeasurements with fluo- applying an extra suction pulse to the pipette. In the WC
rescence imaging techniques relevant for the control of the cytoplasrna of the cell is perfused by the pipette solu-
[Ca2+],-activatedK+channels and many other cellular func- tion, which allows study of the collective behavior of all
tions. the ionic channels in the plasma membrane under defined
intracellular conditions.
METHODS AND METHODOLOGY The perfusion of the whole cell with an artificial solu-
Isolation and culture of cells tion may be a disadvantage if the ionic channels studied re-
quire diffusable intracellular factors. However, this disad-
Osteoclasts (OCL) from 18 day chick embryonic long vantage can be overcome by using a CAP configuration,
bones and osteoblasts (0BL)-osteocytes ( 0 0 , periosteal the perforated patch, in which the CAP has been strongly
IONIC CHANNELS AND INTRACELLULAR CALCIUM IN BONE CELLS 5379

-50 0 so lbo

C 0
FIG.1. Calcium-dependent K' channels and conductance in osteoblasts, measured in the various patch-clamp config-
urations. (A, B, and D)The bathing solution was a high-sodium extracellular solution (SECS); the pipette was filled
with a high-potassium intracellular-like solution (ICS). (A) Current-voltage plot of cell-attached patch channel activity
during application of a voltage ramp (6 superimposed sweeps). ICS with pCa = 5.1. (B) Repeated whole-cell recording
from the same cell at two different intracellularpCa (indicated), applied by using two successive patch pipettes filled with
different calcium-buffered ICS. (C)Increased inside-out patch channel activity upon increasing the Ca'* concentration
at the original intracellular face of the membrane patch by changing the- of the bathing solution (ICS) from 7 to 3.2.
The pipette contained SECS to establish normal Na' and K' concentration asymmetry across the patch membrane. The
applied pipette potential was 0 mV. (D)Outside-out patch channel activity, recorded as a current-voltage plot from 17
superimposed sweeps. ICS in pipette with pCa = 5.1. (Reproduced and adapted with permission from Ref. 20, Ravesloot
et al., 1990).

permeabilized by incorporation of nystatin, an antibiotic anion current, is plotted as positive (upward) current (Figs.
forming monovalent cation channels, into the patch mem- 1, 2, and 3).
brane. In the present patch-clamp experiments, carried out at
The fourth frequently used patch-clamp configuration is room temperature, the cells were usually bathed in a stan-
that of the outside-out patch (OOP; see Fig. 1D).which is dard extracellular salt solution (SECS) consisting of (mM)
obtained by pulling a vesicle from the WC configuration. 150 NaCl, 5 KCl, 1 CaCll, 1 MgCll, and 10 HEPEW
The OOP is actually a micro-WC and allows the resolution NaOH @H 7.2). Intracellular-likeionic conditions, for ex-
of the single-channel behavior underlying the WC conduc- ample during WC or OOP experiments, were obtained by
tance behavior. Since channel opening and closing are re- filling the pipettes with an intracellular salt solution (ICS)
flected by membrane conductance changes and because consisting of (mM) 8-10 NaCl, 140-145 KCl, 1 MgCl,,
these openings and closings are usually membrane poten- 0.024 CaCL, 0.01-10 EGTA to obtain a p C a in the range
tial controlled, channel activity is usually measured at dif- of 3.2-8, and 10 HEPESIKOH (PH 7.2). CAP pipettes
ferent voltages, applied as voltage steps (Fig. 2) or as vol- were filled with either SECS or ICS.
tage ramps (see Fig. 1A). By convention, cell outward-di- Other details of measurement and data analysis have al-
rected ionic current, that is, outward cation or inward ready been described. (ls*lo)
 YPEY ET AL.

h .A ,100 mV

-7-
0

FIG.2. Voltage-activated whole-cell conductances in a chick embryonic osteoclast (OCL), consistent with Ravesloot et
al.(18)The current records (I,) are responses to hyperpolarizing and depolarizing voltage steps from a holding potential
V, = -70 mV to the voltages (V,) indicated. Hyperpolarizing steps activate GKi, depolarizing steps to > -20 mV acti-
vate G K ~Steps
~ . to >40 mV activate G K ~Successive
. steps were applied with intervals of 5.5 s, starting at -160 mV and
increasing with 20 mV increments. The bath contained SECS and the pipette ICS with a pCa = 7.8.

FIG. 3. Stretch-activated CAP channels in an embryonic chick osteoclast. (A) Current records before (left), during
-45 cm H,O pressure (middle), and after return to zero pressure (right). The CAP was 100 mV hyperpolarized to increase
the channel current amplitude. Increased channel activity is sustained during underpressure, although time-dependent
changes may occur. (B). Current-voltage relationship of the stretch-activated channel at -40 cm H,O pressure. Both
the bath and the pipette contained SECS.
IONIC CHANNELS AND INTRACELLULAR CALCIUM IN BONE CELLS s381

Microfluorescence measurements of [Caz+Ji During the measurements the cells were adhered to a
on single cells glass coverslip serving as the bottom of an incubation
chamber on the stage of the microscope.(23)The cells were
The concentration of free intracellular calcium ions bathed in a standard incubation solution composed of
[Ca1+liwas measured at 37°C on individual cells loaded (mM) 125 NaCl, 5 KCl, 1 MgCl,, 1.5 CaCla, 1 KHaPO4, 10
with the fluorescent calcium indicator fura-2, using a fluo- NaHC03, 20 HEPES (PH 7.4), 5.5 glucose, and 2.5 probe-
rescence microscope and calcium imaging equipment. necid. Probenecid (Sigma, St. Louis, MO) was used to pre-
Fura-2 is a dual-excitation fluorochrome; that is, it is ex- vent fura-2 extrusion from the cell and to prevent cornpart-
cited at two successive wavelengths (380 and 340 nm) while mentalization.04)Test solutions contained no added Caa+
the emitted fluorescence is measured for both excitations or 6 mM Ca”. To load the cells with fura-2, the cells were
at >480 nm. Emission at 340 nm excitation is proportional incubated before the experiment for 30 minutes at 37°C in
to fura-2 bound to Ca”, and emission at 380 nm excitation the standard incubation solution containing 2 pM fura-2-
is proportional to the free fura-2 concentration in the cell. AM (Boehringer, Mannheim, Germany), a membrane-per-
Grynkiewicz et al.(19) developed a formula to calculate meant derivative that is hydrolyzed to fura-2 by intracellu-
[Ca1+Iifrom dual-excitation emitted fluorescence, which is lar esterases.
independent of the precise intracellular concentration of Dual-wavelength excitation was controlled by a filter
fura-2: changer box connected to a personal computer. The F380
and F340 microscope images of the cells were captured by
[Ca’+li = &S(R - R~,.J/(R,, - R) (nM) a sensitive black-and-white video camera (Hamamatsu,
Hersching, Germany), connected to a video frame grabber
where Kd is the dissociation constant of fura-2 binding to board (PCVISION; Difa, Breda, The Netherlands) in an
Ca”, @ = F380,,/F38Omi,, with F380,, the fluores- IBM-compatible AT personal computer. Frame grabbing
cence F at 380 nm excitation with all fura-2 dissociated to calculate fluorescence ratios and [Caa+Iifrom the F340
from Cal+ions and F38OdnF a t 380 nm excitation with all and F380 images was controlled by video image analysis
fura-2 bound to Cal+ ions. R = F340/F380, that is, the software (TIM; Difa, Breda, The Netherlands), supported
ratio of emitted fluorescence excited at 340 nm (F340)and by custom-made application and commercial (EQCAL;
that excited at 380 nm (F380). R, is the maximal R mea- Biosoft, Cambridge, UK) software. Before calculation of
sured after saturation of intracellular fura-2 with Ca” by R, the F340 and F380 images were corrected for the dark
adding 2 p M ionomycin (Calbiochem, La JoUa, CA) to the current signal of the video camera. A standard calibration
incubation solution, which allows equilibration of intracel- run for the determination of R, and Rd,,was used to
lular with extracellular Ca” (1.5 mM). R ~ ,is, the minimal obtain better comparison between the cultures. The cali-
R measured during complete dissociation of fura-2 from bration run was regularly repeated to establish that the
Ca2+by adding 20 mM EGTA just after the measurement measurement conditions were constant. Although this cal-
of R., Under our conditions Kd = 224 nM,(19)fl = 4.2, culation procedure implies some uncertainty on the precise
R- = 0.34, and R , = 3.44. [Caa+Ii,it does not influence our conclusions about the rel-

TABLE1. CELLMEMBRANE
CONDUCTANCES
G FOR VARIOUS IONSIN DIFFERENT
BONECELLTYPES SPECIES~
AND ANIMAL

Conductance (activation by)

GKi GKto GKCo Ga GNa Gco GxATP Gxsa

Cell type (hyperpol) (depol) (depol) (voltage) (depol) GN= (depol) (agonut) (stretch)
Chick osteoblasts
Chick osteocytes
Chick PFB
Chick osteoclasts
Rat osteoblasts
Rat ROS 17/2.8
Rat UMR-106
Rat osteoclasts
Pig chondrocytes
Human G292 + (81
~ ~

.The first one to two subscript letter@)(K, (I,Na, Ca, x ) indicate the type of ion conducted ((I for anion; x for unknown or unspecified
ion selectivity). GK abbreviations(GKi, GKtp, GKCa) are explained in the text. The activation mechanism is indicated as voltage activated
(if not further specified), depolarization advated, hyperpolarization activated, agonist activated, or stretch activated (su). The depolari-
zation-activated GNa is the classic TTX (tetrodotoxin)-blockable sodium conductance. The conductances listed under voltage-activated
GNaand Gcs and stretch-activated Gs, are heterogeneous. A plus sign means that the conductance or its underlying channels have been
rcproducibly found in that cell, although not necessarily with 100% frequency. The minus sign means that the conductance(or channels)
has never been found, so far, in that particular cell. Empty places indicate absence of data. A question mark is suggestive, but there is no
definitive evidence from voltage clamp analysis. Numbers refer to the reference list, with (0) referring to the present paper and (*) to un-
published data from our group.
s382 YPEY ET AL.

ative changes in intracellular [Cal+] measured within the observations suggest that [Cal+]iin cells with silent CAP
same cell and compared between cells. G K channels
~ ~ under resting conditions (Fig. 1A) may be
as low as 100 nM. This is confirmed with intracellular
[Ca1+Iimeasurements (see later).
RESULTS Figure 1D shows voltage ramp-induced channel currents
Voltage-activatedconductances and channels: from an OOP pulled from a WC like that in Fig. 1B. At
A short review the applied &ai = 5.1, the channels are active at V, >
-50 mV, which makes it possible to observe or estimate
In the next two sections we review the voltage-activated the voltage at which the channel current reverses from out-
channels and conductances in embryonic chick bone cells, ward to inward. This reversal potential E, cannot be deter-
based on results from our own studies. This review also mined precisely because the inward current becomes
serves to explain and illustrate the power of the patch- smaller than the outward current, but E, must be at <
clamp technique in analyzing transmembrane ionic cur- -50 mV. This proves K+conduction through the channel,
rents in single bone cells. When appropriate, we compare since the asymmetric K+gradient is the only gradient (with
the observations with studies from other groups on other a calculated equilibrium potential of -84 mV) that can
species. Without attempting to be complete, the conduc- provide such a negative E,.
tances and channels discussed in this paper are summarized In about one-third of CAPSon OBL, OCY, or PFB, the
in Table 1. G K achannels were already active under resting conditions
( Vpip = 0), which may indicate an increased [Ca1+Ii.These
Calcium-dependent outward-rectifyingK +
channels could be made less active by decreasing external
conductance ( G K c ~ [Ca”] to nominally zero by EGTA (data not shown).
A recently described K+channel in pig chondrocytes in
The G K has ~ been found in all types of embryonic CAP(1o)is very similar to the chick bone cell %ca channel
chick bone cells we investigated.(11.15*17.10) It is a K+ con- in CAP. The existence of a GKQ in osteoblast-like cells is
ductance activated by depolarization as well as by an in- also consistent with predictions from earlier microelec-
crease in [Cal+]i. Its properties and the various patch- trode but our results also raised the question of
clamp configurations to establish these properties are re- whether normal intracellular conditions would also require
viewed in Fig. 1 for cells from OBL cultures. such high Cal+concentrations (> 1 pM) for significant acti-
Figure 1A shows that silent G K channels
~ can be acti- vation of the G K Cchannels.
~
vated in a CAP of an intact OBL by depolarizing the patch
more than 50 mV. The voltage across the patch is made Other voltage-dependent conductances
more positive by applying a linearly increasing voltage dif-
ference (a ramp) to the two electrodes. Thus, the x axis is Figure 2 reviews the principal voltage-activated conduc-
as voltage as well as a time axis, with 100 mV correspond- tances in freshly isolated osteoclasts in WC configuration,
ing to 5 s. The lower, almost horizontal black record is the described by Ravesloot et al.(I5) The conductances were ac-
small baseline current, occurring when no channels are tivated by voltage steps, illustrated in the lower part of Fig.
open. Current at increased levels above baseline indicate 2. Hyperpolarizing voltage steps from a holding potential
open channels. Analysis of many records (six in Fig. 1A) V,, = -70 mV evoke large inward currents that become
made it possible to conclude that the three increased levels time dependent at extreme hyperpolarization. The conduc-
correspond to three identical channels. The more the patch tance underlying these currents was identified as the in-
is depolarized, the more channels open and the longer they ward-rectifying K+ conductance (GK~),blocked by 5 mM
remain in the open state. More depolarization also pro- external Cs+. It is activated below its reversal potential E,
vides more driving force and increases the current through (= Ek, the equilibrium or Nernst potential for the K+ion
the channels. The slope between the first-level current distribution across the membrane), where it conducts K+
(I)-voltage (V) record and baseline is the single-channel ions from outside to inside. Depolarizing steps to V , >
conductance g, which was 220 pS for the conditions used -20 mV evoke transient outward currents. The underlying
(SECS in the bath and ICS in the pipette). transient outward-rectifying conductance ( G K ~preferen-
~)
A CAP with G Kchannels~ as in Fig. 1A was excised to tially conducts K+ions and is blocked by 4 mM external 4-
make an IOP, and its inside was exposed to ICS with in- aminopyridine. Steps to V, > -50 mV activate an out-
creased [Cal+] to demonstrate [Cal+]i dependence (Fig. ward-rectifying K+conductance ( G K ~ )The . noisy G Kcur- ~
1C). At pCa = 7, no or only a small amount of channel rents are superimposed on the declining G K currents.
~ ~
activity was present, but exposure to pCa = 3.2 caused The G K has~ never been observed by us and others in
tremendous, but reversible, channel activation. osteogenic cells of any species. The G K has ~ been
~ seen oc-
The [Ca1+Iidependence of the GKQ channels was also casionally in chick osteoblast cultures. (I1) The G K occurs
~
established from WC experiments, in which the current in all chick osteoblastic cells we have studied,‘”) and its
flowing at each applied voltage was measured (Fig. 1B) at underlying channels are calcium-dependent K+ chan-
low and high [Can]i in the pipette. At pCai = 7, the cur- n e l ~ . ‘ ~Thus,
~ . ~ a~ plot
) of the GKo current as a function of
rent activates at V, > 80 mV. However, application of the voltage step, after subtraction of the G K current,
~ ~ is
pCai = 3.2 to the same cell shifts activation to V, > 0 similar to that of Fig. 1B at the low [Cal+], which implies
mV. This shift is graded with [Ca1+lifor 3 < pCa < 6. The that G K =~
IONIC CHANNELS AND INTRACELLULAR CALCIUM IN BONE CELLS 5383

Figure lB, as well as Fig. 2, illustrates for our measure- to be able to find channels that are both voltage and
ment conditions, the absence of the classic types of depol- stretch activated.
arization-activated Ca" or Na+ conductances in chick em- In 18 GO-sealed osteocyte CAPs from pure osteocyte
bryonic osteoblasts and osteoclasts, consistent with experi- cultures and 7 osteoblast CAPs, we have not found chan-
mental results under optimal recording conditions.(11.16) nels activated by membrane stretch; neither did we find
Others did not find these conductances in rat osteo- clear evidence for direct or indirect effects of stretch on
clasts,(ll*as)but Chesnoy-Marchais and Fritsch") did find G K channel
~ activity, different from the results of Tani-
these conductances in rat osteoblasts. guchi and G~ggino,(~') who found results consistent with
The final voltage-activated channel in chick embryonic stretch-induced calcium entry affecting GKQ channels in a
bone cells to be mentioned is a large-conductance (380 pS) rabbit kidney cell line. In later series of experiments on
channel conducting such anions as C1- and phosphate osteoblastic cells we only occasionally found channel activ-
ions.(a6)This anion conductance (G,) channel has not yet ity increased by membrane stretch. The infrequent pres-
been seen in intact cells (CAP), only in excised (IOP) ence of this type of chmnel is in contrast to the findings of
patches,'") but has been best characterized in OBL, OCY, Duncan and Misler(16)and Davidson et al.(@)
and PFB, where the channel is active only between -30 In WC experiments on osteoclasts, we found no evi-
and 30 mV. These results raised the question of the physio- dence for membrane stretch-induced (40 cm H,O pressure)
logic intracellular factors activating the G, channel, which changes in the WC currents through the G K and ~ G K ~ ~
is distinctly different from the WC chloride conductance (three cells). In one of the cells, for example, the effects of
found in rat oste~blasts(~~ and rat osteoclasts.(lS)Various 40 cm HIO overpressure and 80 cm underpressure were
conductances and channels found in bone cells in different within the normal range of variation (5%) of control cur-
animal species are summarized in Table 1. rent peaks at -120 mV (GKJ and 30 mV ( G K , ~ )We . also
did not fiid significant effects on G Kchannels
~ in cell-at-
tached patches.
Some recent results However, in 11 of 35 CAPs (from 35 OCL), we found
The results to be discussed here are new but also serve to 40 pS channels (mean 41 pS, range 29-53 pS, estimated
review similar observations from other studies. from 12 I-Y plots from seven cells), which were reversibly
activated by membrane stretch (Fig. 3). At resting ( Ypip=
Stretch Sensitivity of Ion Channels: In mechanorecep 0 mv) and hyperpolarized (positive Vpip>conditions, chan-
tive sensory cells, mechanoreceptive ion channels are the nel currents were inward with SECS in the pipette and in
mechanoelectrical transducers for the generation of recep the bath, and the mean reversal potential (E,) was 36.0 mV
tor potentials as a fist step in the encoding process for (range -4 to 70 mV, based on 12 I-Y from seven cells)
neuronal information processing. Recently it has become more positive than the resting membrane potential. Under
clear that the mechanosensitivity of ion channels is a wide- these conditions, and G K channels
~ reverse at Y ,
spread property of cell membranes and is not restricted to more negative than the resting membrane potential (data
specific classes of sensory cells.(2)This notion stimulated not shown). Channel activity was not strongly dependent
the search for mechanosensitivechannels in bone cells that on the membrane potential.
could serve as sensors transducing mechanical stress to
changed bone cell activity during adaptation of bone to Intracellular Caa+Measurements: The observations on
loading.(a*16) The first results of Duncan and Misler(l') and the G Kchannels
~ in the various chick bone cells raised in-
Davidson et al.(a) are promising and prompted us to ex- teresting questions with respect to normal values of single-
tend their observations for the various classes of chicken cell [Ca'+]i, its variability in time and in the intracellular
bone cells in primary culture to arrive at a more general space, and its dependence on external [Ca'+].
picture of the possible role of mechanosensitivechannels in The microfluorescence experiments showed that [Caa+Ij
bone metabolism. of OCY in mixed OBL/OCY cultures depends on external
During current measurements in patch-clamp experi- Ca" (Fig. 4). At 1.5 mM external Ca", the stationary
ments, mechanical stress was applied to the cell membrane mean [Ca2+Ijwas 144 nM (range of singleell mean values
by applying under- or overpressure (&SO cm water) to the 94-188 nM, 12 cells from three cultures). Over a period of
inside of a patch pipette under cell-attached patch or 5 minutes, singleell mean values could fluctuate by 20%.
whole-cell conditions. External underpressure on a CAP No calcium oscillations were found under our conditions.
distends the patch membrane, and the membrane shrinks After 4-5 minutes of exposure to practically 0 mM external
again during overpressure. The opposite occurs during WC Ca", [Ca1+liwas reduced to 46% (range 35-55%) of its
measurements. control value. Recovery of [ C a l i after 4-5 minutes at 1.5
Since investigators have hypothesized that osteocytes mM external Ca'+ was to 84% (range 67-loSVo) of the con-
and osteoblasts could have a mechanoreceptor function in trol. After 4-5 minutes of exposure to 6 mM external Caa+,
the response of bone to loading,("-") we fiirst searched for [Ca1+Ij was slightly further increased to 98% (range
stretch sensitivity of ionic channels in these two bone cell 61-172070) of the original 1.5 mM control value. Individual
types. As a control we screened osteoclasts for stretch-sen- OBL/PFB-like cells and the mean [Caa+Iiof all (100-200)
sitive channels. While testing the membrane for stretch cells imaged from a section of the culture behaved in the
sensitivity, the membrane was depolarized or hyperpol- same way. The calculated [Ca1+liin single OCYs, both in
arized by 5&100 mV to facilitate finding the channels and OBL/OCY and in pure OCY cultures, was often (for ex-
 YPEY ET AL.

FIG.4. [Caa+Iiin embryonic chick bone cells and the effect of external Ca’+. An osteocyte from a 2 day OBL/OCY cul-
ture, first exposed to l .5 mM external Ca’+ (l), then to nominally zero mM Ca2+(2),then back to l .5 mM (3). and finally
to 6 mM external Ca’+. The images were taken 5 minutes after exposure to the external [Ca2+].Pixels with fluorescence
ratio values above the mean of the OCY under control conditions (1) are darkened in 1-4 to illustrate the effect of exter-
nal Ca” on [CaZ+li.Pixels with ratio values above the mean in 2 and 3 are hatched to illustrate maintenance of the intra-
cellular Ca” distribution during changed external Ca2+.The ratios in 1-4 were 0.64,0.52, 0.63, and 0.72, respectively.
Pixel values are ratio values x 64. The finely branched processes of the OCY are not visible in the imaged cell because of
a lack of resolution ( x 20 objective used).

ample, 80% of the cells in a pure OCY culture) seen to be het- cell body. This distribution was not drastically changed at
erogeneous (Fig. 4A). In the example OCY shown in Fii. 4A, zero external [Ca2+],as in OCY.
[Ca*]i values > 100 nM (the mean value of the cell, range 61- The intracellular inhomogeneity in apparent [Ca2+Ii,and
181 nM) are clustered midright and along the upper and lower the variability and dependence of [Caa+Iion external [Ca2+]
borders of the cell at 1.5 mM external Ca*. This distribution are consistent with the observed variability in G K chan-~
was not drastically changed at zero external [aa+],where the nel activity under resting conditions and the decrease in
mean [Ca*Ii = 55 nM, range 34142 nM (Fig. 4A2). channel activity with the removal of external Ca’+. How-
In OCL, the mean [Ca1+liequaled 104 nM (range of ever, pCa < 6, as required for GKC- activation in IOP and
mean singlecell values 59-184 nM, eight cells from five WC measurements, were usually not encountered in intact
cultures from three culture batches), consistent with micro- cells, except in a few cells of a culture, which were proba-
photometric measurements by Miyauchi et al. After 4-5 bly dying.
minutes of exposure to zero external [Ca2+],[Caa+Iiwas re-
duced to 51% of its control value (range 39-58%, five cells
from two cultures). All five cells had recovered after 4-5 DISCUSSION
minutes at 1.5 mM external Ca”, to a mean of 121%
(range 78-156%) of the original control value. The calcu- The present paper reviews properties of ionic channels in
lated [Ca2+]iwas not homogeneous within a single OCL embryonic chick bone cells in comparison to ionic channel
(Fig. 4B). In one example cell, the mean [Ca2+Iiwas 103 properties of bone cell membranes in other species. Fur-
nM at external [Ca’+] = 1.5 mM and the range was 39-277 thermore, two new results are presented. First, embryonic
nM (>500 intracellular measurement points), with the chick osteoclasts express stretch-activated channels, and
above-mean values patterned along the cell boundary and second, microfluorescence measurements revealed that the
in the lamellipods extending from the nuclei-containing calculated resting [Ca2+Iiof bone cells may be heterogene-
IONIC CHANNELS AND INTRACELLUIAR CALCIUM IN BONE CELLS 5385

ous within bone cell and is dependent on the extracellular The presence of stretch-activated channels in chick em-
[Ca’+]. bryonic OCL and its infrequent presence in chick OBL and
A survey of the principal conductances and channels OCY is surprising, since OBL and OCY have been pro-
found in bone cells is given in Table 1. The various con- posed to possess membrane stress mechanisms to trans-
ductances can be discriminated by ionic selectivity, mecha- duce changes in mechanical bone cell loading to changed
nism of activation (by voltage, agonists, or stretch), and bone metabolism.(8.16)Therefore, we speculate that the
kinetics (transient or stationary activity). Voltage-activated OBL/OCY stretch-activated channels are subject to vari-
channels comprise two classes, depolarization-activated able expression and that the OCL stretch-activated chan-
and hyperpolarization-activated channels. An example of nels rather play a role in OCL locomotion during resorp-
an agonist-activated conductance is the extracellular ATP- tive OCL activity. For example, OCL locomotive move-
activated conductance G X ~ with p as yet undefined ionic ments may stretch part of the cell membrane, which may
selectivity. (3a) lead to local stretch-induced opening of channels. If the
As Table 1 shows, bone cell ionic channels, both cation channels are aselective, consistent with the data, Caa+
and anion channels, are usually strongly dependent on the could enter into the cell and cause local cell detachment
membrane potential. This implies an important role for the due to decreased podosome expression.(I8) This could even
membrane potential in bone cell function regulation. Un- be part of a mechanism for cell displacement to continue
fortunately, the role is still unknown but is becoming more bone resorption at another spot after excavation of the
appreciated since membrane electrophysiology has become previous resorption lacuna. The stretch-activated channel
part of the research effort to clarify the cellular physiologic in chick OCL has similarities to that in cultured skeletal
mechanisms of bone tissue metabolism. Thus far, we have muscle of the same species.(38)
been mainly concerned with K’ channels, since these chan- We observed variability between culture batches in the
nels were dominant in embryonic chick bone cells. The im- distensibility of a cell membrane in WC or in CAP with
portance of K+ ions in podosome expression by OCL is applied pressure. Thus, the absence of stretch effects in
suggested by Miyauchi et al.cL8)Other investigators have some of the OCL and in the OCY and OBL may have been
focused on Caa+ channels in mammalian osteoblastic due to stiffness of the membrane. This stiffness may be
cells.(4,7.33*34) Richter and F e r ~ i e r , ‘ on
~ ~ )the other hand, under the control of cytoskeletal proteins. (38) Further-
found stationary weakly voltage-dependent Na+ channel more, certain ionic channels and intracellular processes
activity in the osteoblastic cell line ROS 17/2.8, different may be sensitive to shear ~ t r e ~ instead~ ( ~ ~of *to~planar
~ )
from the TTX-blockable Na+ channels in rat osteo- membrane stretching.
blasts.ta9)Our patch-clamp experiments did not reveal any Morris and Horn‘”’) proposed in their recent study that
of the classic voltage-activated Caa+conductances in chick stretch-activated ion channels in membrane patches derive
bone cells, but the microfluorescence experiments suggest their stretch sensitivity from patch-pipette membrane inter-
the presence of Caa+conduction pathways in the plasma actions, leading to alterations of the cortical cytoskeleton
membrane, possibly small voltage-insensitive Caa+chan- under the plasma membrane. Such alterations may be in-
nels. (36) volved in osteoclast attachment to and detachment from
It may be expected that some bone cell functions depend the substratum during osteoclast locomotion.
on the coordinated action of K+and Ca” channels. In vol- Our measurements of [Caa+Iiin intact cells by image
tage-activated Caa+channels, the K’ channels may provide analysis were a rust step in the analysis of the role of G K C ~
a hyperpolarizing force to the membrane for deactivation channels in bone cell function. The high [Caa+Iirequired
of Ca” channels and recovery of inactivated Ca” chan- for G Kactivation
~ in WC and IOP is still to be explained.
nels, and stationary Na+(35)and Caa+channels could pro- It may indicate an intracellular calcium amplification fac-
vide depolarizing forces to the membrane, leading to in- tor for G K activation
~ in the intact cell.
creased [Ca2+]i.In voltage-insensitive Ca” channels, the Thus far, the observations of the present study are in
K” channels may set the driving force for Ca” entry favor of a local, subcellular role for [Ca’+]i in bone cell ac-
through the plasma membrane.(371 tivity, as in neurons.‘41) The combined application of
The G K of ~ OCL is most suited for inward K+ conduc- patch-clamp and calcium-imaging techniques is a promis-
tion. This K+uptake by the cell could occur, for example, ing approach to study and analyze the local effects of
from the resorption lacuna, in exchange for proton extru- membrane stretching and channel opening on local [Ca’+]i
sion, when the electrogenic effects of the. proton pump and on cell behavior.
drive the membrane potential to below the resting mem-
brane potential.‘l’) The G K Cmay ~ serve to replenish the
resorption lacuna with K+ ions when the [Ca2+Iibecomes
high under depolarized conditions. Finally, G, channels
could also serve to provide the electrogenic proton pump ACKNOWLEDGMENTS
with counterions for proton extrusion. The membrane po-
tential would then determine which types of ionic channels We thank Prof. Dr. E.H. Burger (Dept. Oral Cell Biol-
( G K ~G. K C ~G, K ~or~ G,)
, become active in controlling the ogy, Free University, Amsterdam) for her encouragement
pump efficiency. If we learn more about the molecular to search for stretch-sensitive channels. We acknowledge
control of the OCL secretion machinery, we will come the contributions of Mrs. A. Wiltink and Mr. W. Mulder
closer to a complete biophysical model of osteoclastic pro- to the experiments on stretch-activated channels. This
ton secretion. work was supported by The Netherlands Organization for
5386 YPEY ET AL.

Scientific Research (NWO) through grants (for Ravesloot regulated by voltage-gated calcium channels and extracellular
and Weidema) from the Foundation for Biophysics. We calcium, controls podosome assembly and bone resorption. J
are particularly grateful to Mr. G.J.Verschragen for de- Cell Biol 111:2543-2552.
signing and constructing the filter change controlling dual- 19. Grynkiewicz G, Poenie M, Tsien RY 1985 A new generation
of calcium indicators with greatly improved fluorescence
wavelength excitation and to Mr. J.P.Buys for writing the
properties. J Biol Chem 260:3440-3450.
application software. 20. Ravesloot JH, Van Houten RJ, Ypey DL, Nijweide PJ 1990
Identification of Caa*activated K+channels in cells of embry-
onic chick osteoblast cultures. J Bone Miner Res 51201-
1210.
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Department of Physiology
Faculty of Medicine
Leiden University
PO Box 9604
2300RC Leiden, The Netherlands