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INTRODUCTION |
In many cells, emptying of the endoplasmic reticulum
(ER)1 represents an
initial signal that triggers activation of the so-called capacitative
Ca2+ entry through non-voltage gated pathway(s) (CCE) (1).
Remarkably, the CCE represents the main mechanism for Ca2+
entry in non-excitable cells and achieves long lasting elevation of
[Ca2+]cyto. Although the actual protein(s)
responsible for CCE is/are still under debate and matter of intense
investigation, it has been clearly described that CCE is prevented by
an elevation of Ca2+ at the mouth of the channel(s) (2-6).
On the other hand, the amount of Ca2+ that actually enters
the cells through CCE critically depends on activation of
Ca2+-activated K+ channels to achieve a
membrane hyperpolarization and, thus, provide the driving force for
Ca2+ entry (7, 8). Notably, in endothelial cells,
superficial ER (sER) domains create spatial Ca2+ gradients
beneath the plasma membrane (subplasmalemmal
Ca2+ control unit,
SCCU) that result in local activation of BKCa channels (9-12). The existence of such localized Ca2+ elevation
beneath the plasma membrane would explain, at least in part, the
"Ca2+ paradox" that during cell stimulation activation
of Ca2+-activated ion currents occurs simultaneously with
the Ca2+-inhibitable CCE. However, we previously observed
that, during a strong cell stimulation (i.e. 100 µM histamine), where BKCa channels get
activated also in regions far from the ER, a strong CCE still takes
place (10). These findings emphasize that, although the sER contributes
to Ca2+ influx by membrane hyperpolarization due to
Ca2+-activated K+ channels, another phenomenon,
i.e. local lowering/buffering of the subplasmalemmal
Ca2+ concentration ([Ca2+]sub),
has to occur simultaneously to facilitate CCE activity. Consequently,
evidence was provided that mitochondria play a key role for CCE
activity in non-excitable cells. In these experiments, in which
mitochondria were depolarized by uncouplers of mitochondrial oxidative
phosphorylation (i.e. the carbonyl cyanide phenylhydrazones FCCP and CCCP), which results in inhibition of mitochondrial
Ca2+ uptake, the maintenance of CCE was prevented (2-4,
13). This phenomenon further referred to as "mitochondrial
Ca2+ buffering" is thought to facilitate CCE
by lowering subplasmalemmal Ca2+ at the mouth of this
Ca2+-inhibitable Ca2+-entry pathway (14).
However, these carbonyl cyanide-based mitochondrial uncouplers prevent
mitochondrial Ca2+ signaling in a rather indirect way via
abolishment of the H+ gradient, which results in a change
of the mitochondrial pH and depolarization of the inner mitochondria
membrane. Furthermore, these compounds have been found to affect the
Ca2+ release from the ER (15) and result in a
depolarization of the plasma membrane (16). In view of the potential
unspecific properties of mitochondrial uncouplers, ultimate proofs for
the concept of mitochondrial Ca2+ buffering are
necessary. Therefore, this study was designed to find further
and direct evidence of mitochondrial Ca2+ buffering during
cell stimulation in the human umbilical vein endothelial cell-derived
cell line EA.hy926.
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EXPERIMENTAL PROCEDURES |
Materials--
Cell culture chemicals were obtained from
Invitrogen (Vienna, Austria) and fetal calf serum was from PAA
Laboratories (Linz, Austria). Fura-2/AM was from Molecular Probes
Europe (Leiden, Netherlands). FCCP (carbonyl
cyanide-4-trifluoromethoxyphenylhyrazone) and histamine were from Sigma
Chemicals (St. Louis, MO). Restriction enzymes and T4 DNA ligase were
from New England BioLabs (Frankfurt, Germany) and the EndoFree Plasmid
Maxi Kit was from Qiagen (Hilden, Germany). All other chemicals were
from Roth, Karlsruhe, Germany.
Cell Culture--
The human umbilical vein endothelial cell
line, EA.hy926 (17) at passage
45 was used. Cells were grown on
glass coverslips in Dulbecco's minimum essential medium containing
10% fetal calf serum and 1% HAT (5 mM hypoxanthine, 20 µM aminopterin, 0.8 mM thymidine).
Plasmids and Transfection--
YC4-ER (18, 19) and mtDsRed were
cloned into pcDNA3 (Invitrogen, Groningen, Netherlands). For double
transfection YC4-ER and mtDsRed were inserted into the two multiple
cloning sites of the transfection vector pBudCE4.1 (Invitrogen). Cells
of approximately 80% confluency were transiently transfected with
1.5-3 µg of purified plasmid DNA using TransFastTM
Transfection Reagent (Promega, Mannheim, Germany).
Organelle Visualization--
Organelle organization was
visualized in cells transiently transfected with YC4-ER or mtDsRed as
described previously (10). Experiments were performed using a
deconvolution microscope recently described (11, 12). A Nikon inverted
microscope (Eclipse 300TE, Nikon, Vienna) was equipped with CFI Plan
Fluor 40× oil immersion objective (numerical aperture 1.3, Nikon,
Vienna, Austria), an epifluorescence system (150 W XBO, Optiquip,
Highland Mills, NY), a computer controlled z-stage (Ludl Electronic
Products, Hawthorne, NY), and a liquid-cooled charge-coupled
device camera (
30 °C, Quantix KAF 1400G2, Roper Scientific, Acton,
MA) that allowed image resolution of 0.171 µm pixel
1.
Excitation/emission wavelengths were selected using a
computer-controlled filter wheel (Ludl, Electronic Products, Hawthorne,
NY). All devices were controlled either by Metafluor 4.0 (Visitron
Systems, Puchheim, Germany) or ImagePro 3.0 (Media Cybernetics, Sliver
Spring, MA) for deconvolution imaging. For organelle visualization,
cells were illuminated alternatively at 440 nm (cameleon: 440AF21;
Omega Optical, Brattleboro, VT) or 575 nm (mtDsRed: 575DF25; Omega
Optical), respectively. Emission was monitored at 535 nm for cameleons
(535AF26; Omega Optical) and 528-633 nm for mtDsRed (528-633DBEM;
dichroic XF53; Omega Optical). Image analysis and deconvolution were
performed as previously described using Image Pro 3.0 (Media
Cybernetics, Silver Spring, MD) and constrained iterative
deconvolution (Microtome, VayTek, Fairfield, IA)
(10).
Ca2+ Measurements--
Experiments were performed in
Hepes-buffered solution containing (in mM) 138 NaCl, 5 KCl,
2.0 Ca2Cl, 1 MgCl2, and 10 Hepes acid, pH
adjusted to 7.4. Nominal Ca2+-free solution contained (in
mM) 138 NaCl, 5 KCl, 1 MgCl2, 1 EGTA, and 10 Hepes acid, pH adjusted to 7.4. [Ca2+]cyto
values were measured using Fura-2, YC4-ER, and
ratiometric-pericam-mt as described previously (10, 20). To
monitor [Ca2+]cyto, cells were illuminated
alternatively at 360 ± 15 and 380 ± 15 nm (Fura-2: 360HT15
and 380HT15; Omega Optical, Brattleboro, VT), and emission was
monitored at 510 nm (510WB40; Omega Optical) as described previously
(11).
Cytosolic Ca2+ Measurements during Cell Membrane
Potential Clamp--
As previously described (9, 21), cytosolic
Ca2+ measurements were combined with the patch clamp
technique (whole cell configuration) to control the cell membrane
potential during cell stimulation. Experiments were performed using the
Hepes-buffered solution mentioned above. The pipette solution contained
(in mM) 130 KCl, 5 MgATP, 0.2 Na2GTP, 1 MgCl2, 10 Hepes, with pH adjusted to 7.2 with KOH.
Single Channel Recordings--
Cell-attached and inside-out
configurations of the patch clamp technique were used (22).
Borosilicate glass pipettes (resistance of 6-10 M
) were pulled with
a Narishige puller (Narishige Co. Ltd., Tokyo, Japan). Currents were
recorded with an EPC-7 amplifier (List Medical, Darmstadt, Germany)
filtered at 1 kHz (900C9L8L, Frequency Devices, Haverhill, MA),
digitized by a digidata 1320 interface (Axon Instruments, Union City,
CA), and sampled by a PC running with pClamp 8.0 (Axon Instruments) at
5 kHz. Analysis of single currents was performed using Fetchan and
pStat (Axon Instruments). The channel open state probability
(Po) was expressed as the time spent in the open
state (to) divided by the total time of the
recording (t): Po = to/t. Po was
usually calculated on a 2-s sweep. When several identical channels
(N) were simultaneously open on the same patch, the open
probability of one channel was calculated as follows:
Po = (to1 + 2to2 + 3to3 + ... + NtoN)/Nt, where
toN is the time spent by a channel at the open
level N. The pipette solution contained (in mM)
130 KCl, 1 MgCl2, 10 Hepes (pH 7.4 with KOH).
Ca2+ Calibration of BKCa Channel
Activity--
The Ca2+ dependence of the BKCa
channel was tested within 0.5-30 µM free
Ca2+ (calculated by MaxChelator; Dr. C. Patton, Hopkins
Marine Station, Stanford University, CA). Consistent with our recent
work (10) and with that of Barrett et al. (23), a
correlation of the activity of BKCa channels
(Po) with the cell membrane potential
(Vwc) and [Ca2+]pm was
extracted out of a series of in situ calibration procedures (see Fig. 7A).
Standard Patch Protocol--
The standard experimental bath
solution contained (in mM) 130 KCl, 1 MgCl2, 2 CaCl2, 10 Hepes (pH 7.45 with KOH; Fig. 6A). According to the Ca2+ calibration of the BKCa
channels, the [Ca2+]pm at each individual
pipette location was calculated with the obtained
Po using the following equation,
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(Eq. 1)
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where EC
is the Ca2+ sensitivity
of the channel. Based on our calibration published recently, log
EC
is
5.566 (10).
Physiological Patch Protocol--
In experiments using the
physiological patch approach, the bath solutions contained (in
mM) 130 NaCl, 5.6 KCl, 1 MgCl2, 2 CaCl2, 8 Hepes (pH 7.45 with NaOH). For more detail see
Fig. 6. The procedure by which [Ca2+]pm was
obtained using the physiological patch approach is shown in detail in
Fig. 7B. Based on the linear relationship between Vpm and the Ca2+ sensitivity of the
BKCa channel (EC
), the obtained
Po and the actual Vpm the
Ca2+ concentration at the mouth of the BKCa
channel ([Ca2+]pm) under physiological patch
clamp conditions was calculated as explained in Fig. 7B.
Statistics--
Analysis of variance was performed, and
statistical significance was evaluated using Scheffe's post hoc
F test. The level of significance was defined as
p < 0.05.
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RESULTS |
Pipette Positioning in Proximity and Distance of
Visualized Organelles--
Based on the expression of
organelle-targeted DsRed (mtDsRed) and cameleon (YC4-ER) the
organization of mitochondrial and ER network was visualized (Fig.
1). The patch pipette was placed far from
any organelle (L1, Fig. 1), in the vicinity of mitochondrial rich domains (L2), or at locations close to ER structures
(L3). As recently described, the activity of
BKCa channels was utilized to estimate the Ca2+
concentration at the inner side of the patch membrane
([Ca2+]pm) in the proximity of the mouth of
the channel (see "Experimental Procedures") (10).

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Fig. 1.
Visualization of mitochondria and endoplasmic
reticulum in endothelial cells for defined pipette positioning.
Cells were transiently transfected with a vector encoding
mtDsRed (red) and YC4-ER (green). The pipette was
either localized in distance of any organelle (L1), close to
mitochondria (L2), or in the vicinity of sER domains
(L3) as illustrated in the respective panels.
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Mitochondria Effectively Buffer Cytosolic Ca2+ Rises
and Ca2+ Entry Beneath the Plasma Membrane--
In
standard experimental bath solution, cells were stimulated with 100 µM histamine at a patch holding potential of +40 mV while
the cell membrane potential was approximately 0 mV. Based on the
monitored channel activity, the [Ca2+]pm at
each individual pipette location was calculated as explained in detail
under "Experimental Procedures." With the pipette located far from
mitochondria (i.e.
5 µm; L1 and
L2), BKCa activity, expressed as its maximal
Po (normalized) during stimulation, was 0.290 ± 0.068 (n = 10), corresponding to ~1600
nM [Ca2+]pm (Fig.
2A). In agreement with our
previous report, there was no considerable difference between the
L1 and the L3 pipette location in response to
histamine. In the vicinity of mitochondria (L2) BKCa activity was reduced by 91% (maximal
Po 0.025 ± 0.013, n = 8, p < 0.05 versus L1 and
L3) and represented an estimated
[Ca2+]pm of ~160 nM (Fig.
2B).

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Fig. 2.
Mitochondria buffer subplasmalemmal
Ca2+ elevation in their neighborhood. In the cell
attached configuration of the patch clamp technique, the activation of
single BKCa channels at pipette locations L1 and
L3 (A) and L2 (B) in
response to 100 µM histamine was recorded in isometric
K+ conditions (i.e. 130 mM KCl in
bath and pipette solution). The holding potential was +40 mV. Activity
is shown before application of histamine (a) and during the
time of maximal stimulation by 100 µM histamine
(b and c). The presence of functional
BKCa channels was tested after each individual recording in
the inside-out configuration. Closed and open levels of the channel are
indicated to the immediate right of each recording by
"c" and "o," respectively. Based on the
monitored channel activity (Po) the
[Ca2+]pm on each individual pipette location
was calculated as previously described using the following equation:
log[Ca2+]pm = log EC log[(0.8305 Po)/(Po + 0.02181)],
where EC is the Ca2+ sensitivity of the
channel. Based on our calibration published recently, log
EC is 5.566. The mean Po
and estimated [Ca2+]pm for the pipette
location chosen are given in the left panels.
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The capacity of mitochondria to buffer neighboring
subplasmalemmal Ca2+ and, thus, to prevent BKCa
channel stimulation by histamine was further experienced under
circumstances where the L2 pipette location was chosen
initially but subplasmalemmal mitochondria occasionally moved away from
the pipette (L1) during the experiment. If mitochondria were
in the proximity of the patch (L2; Fig.
3A, left panel), the channel activation in response to 100 µM histamine
was very small (Po, 0.015 ± 0.011, n = 4; Fig. 3, B and C,
tracings b) and comparable with resting cells
(Po, 0.003 ± 0.002, n = 4;
Fig. 3, B and C, tracings a). However,
after the respective mitochondrial domain moved approximately 4 µm
from the patch location (Fig. 3A, right panel), a
further histamine stimulation induced strong BKCa channel
activation (Po, 0.252 ± 0.073, n = 4; p < 0.05 versus previous L2 position; Fig. 3, B and C,
tracings c).

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Fig. 3.
Mitochondrial movement affects
BKCa channel activation. A, as illustrated,
mitochondria occasionally moved apart from the pipette location ( 4
µm) during experiments. Such large movement was unpredictable and did
not occur frequently (n = 4). B, channel
activity in isometric K+ is shown before application of
histamine (a), after stimulation with 100 µM
histamine where the mitochondria were close to the pipette location
(L2) (b), and after the mitochondria are 4
µm apart from the pipette location (L1)
(c). C, representative time course of
BKCa channel activation in the proximity of mitochondria
(L2) and after the mitochondrial movement
(L1).
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If mitochondrial Ca2+ uptake was prevented by 2 µM FCCP, the activity of neighboring BKCa
channels in response to histamine increased 8-fold (maximal
Po, 0.130 ± 0.035, n = 5, p < 0.05 versus close to mitochondria
without FCCP; Fig. 4, A and
B). Notably, addition of 2 µM FCCP failed to
initiate BKCa channel activation at L2 (data not shown).
The time courses of BKCa channel activation did not differ
in any conditions tested and represented a transient activation of
BKCa channels (Fig. 4C) (10).

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Fig. 4.
Effect of FCCP on mitochondrial buffer
function under isometric K+ conditions. In isometric
K+, activation of BKCa channels in response to
100 µM histamine was monitored in the vicinity of
mitochondria (L2) in the presence of 2 µM FCCP
(A). The right panel represents channel activity
before cell stimulation (a) and at the time of maximal
stimulation after addition of 100 µM histamine
(b and c). Maximal elevation of
[Ca2+]pm was calculated as described in Fig.
3 and is given in the left schema. B, statistical
analysis on BKCa channel activation in response to 100 µM histamine at locations L1 and L3, L2, and L2 in
the presence of 2 µM FCCP. *, p < 0.05 versus L1 and L3 (L1 and L3, n = 10; L2,
n = 8; and L2 + FCCP, n = 5).
C, representative time courses of BKCa channel
activation to 100 µM histamine at various pipette
locations (L1 and L3, L2) and in the absence or presence of 2 µM FCCP.
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Limitations of the Standard Patch Protocol--
Notably, in the
protocol above, high bath K+ concentration was used to
prevent changes in the membrane potential during the recording. Under
such conditions, plasma membrane K+ currents and, thus,
membrane hyperpolarization that represents the driving force for
Ca2+ entry are prevented (7, 8). Indeed, under
physiological conditions endothelial cells hyperpolarized from
30.7 ± 3.1 to
62.0 ± 2.8 mV (n = 14)
upon stimulation with 100 µM histamine. The pivotal role
of the driving force for Ca2+ entry in endothelial cells
was tested in experiments where the patch clamp technique (whole cell
configuration) and single cell fluorometry were used simultaneously.
Cells were stimulated with histamine and clamped at 0,
30,
60, or
90 mV. Although the initial transient remained unaffected, the
Ca2+ plateau (measured 3 min after the onset of the
response) increased with the amount of the hyperpolarization applied
(Fig. 5). These findings demonstrate that
in the experiments presented above (standard patch protocol) a very
limited Ca2+ entry occurs due to the membrane
depolarization in isometric K+, whereas under physiological
conditions Ca2+ influx is facilitated by a significant
membrane hyperpolarization.

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Fig. 5.
Contribution of agonist-evoked membrane
hyperpolarization on capacitative Ca2+ entry. Using
conventional whole cell recording technique, the cells were clamped at
0, 30, 60, and 90 mV. The Ca2+ plateau was measured 3 min after the onset of the response elicited by histamine and plotted
as a function of the holding potential (n = 6-8; *,
p < 0.05 versus +0 mV).
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The Physiological Patch Protocol--
To estimate the impact of
the driving force for Ca2+ entry on autacoid-induced
BKCa stimulation, the effect of histamine on the activity
of BKCa channels that were far from any organelle were
compared under isometric (i.e. 130 mM) and
physiological (i.e. 5.6 mM) extracellular
K+ conditions (Fig.
6A). To allow a comparison of
the BKCa channel activation under both patch clamp
protocols, in the physiological patch protocol, a patch membrane
potential was applied that compensated the actual cell membrane
potential to achieve +40 mV at the channels in the patch. In 130 mM K+-containing buffer, histamine induced a
transient BKCa channel stimulation, whereas under
physiological conditions the activation of BKCa channels by
histamine was biphasic and comprised an initial transient followed by a
long lasting channel activation (Fig. 6B). These data
clearly indicate that the conventional standard patch technique limits
significantly the quantity and duration of the response of a cell to
agonist stimulation.

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Fig. 6.
A, schematic explanation of the
"standard patch" and "physiological patch" approaches.
B, representative time courses of histamine (100 µM)-induced BKCa channel activation using the
standard patch (holding potential +40 mV) and physiological patch
(effective potential of the patch, Vpm, ~+40
mV) protocols (n = 5-6).
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Thus, it seems important to verify whether mitochondria buffer
subplasmalemmal Ca2+ elevation even under conditions where
hyperpolarization facilitates physiological Ca2+ entry. To
address this point, experiments in physiological bath K+
concentration were performed. Notably, an experimental protocol was
chosen (further referred to as "physiological patch") that allowed
the cell to hyperpolarize freely while single channel recordings were
performed. The experimental procedure and analysis to estimate the
Po of the distinct BKCa channel at
a certain pipette location, the whole cell membrane
potential (Vwc), and
[Ca2+]pm are explained in detail in Fig.
7. Using the amplitude of the current and
the current-voltage relationship of the BKCa channel, the
actual membrane potential of the patch (Vpm) was
calculated (Fig. 7B, steps 1 and 2).
With Vpm and the applied potential
(Vapplied) the whole cell membrane potential
(Vwc) was estimated (Fig. 7B, step 3). Following the open probability of the
BKCa channels (Fig. 7, step 4), the
Ca2+ concentration at the mouth of the channel
([Ca2+]pm) was calculated according to the
equation given (Fig. 7B, step 5). Thus, the
physiological patch approach allows the measurements and quantitative
analysis of distinct subplasmalemmal Ca2+ concentrations in
stimulated cells, which are not handicapped by artificially imposed
whole cell membrane potential.

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Fig. 7.
A, Ca2+ calibration of the
BKCa channel activity with respect to membrane
potential. In a series of inside-out experiments under
isometric K+ conditions, the activity of BKCa
channels was correlated with the Ca2+ concentration at the
mouth of the channel at different whole cell membrane potentials
(Vwc). Lines represent fitted curves
out of 4-12 experiments. B, principle of the physiological
patch procedure. Calculations of Vwc and
[Ca2+]pm are given. Under physiological
asymmetric ion distribution (5.6 mM K+
extracellular versus ~130 mM K+
intracellular) Vwc was free to change following
histamine-induced activation of BKCa channels, resulting in
a membrane hyperpolarization to 60 mV or more. Because a conclusive
measurement of the activity of the BKCa needs effective
potential at the patch (Vpm) from approximately
+40 mV, a patch holding potential (Vapplied)
ranging between +80 and +120 mV was applied through the patch pipette
(Vpm = Vwc + Vapplied). Recordings: typical tracings before
application of histamine (a) and during maximal stimulation
by 100 µM histamine (b). Arrow 1 (I/V): the current (I, pA) through a
single BKCa channel was extracted out of every single
tracing. Arrow 2: from the linear I/V
relationship (pA/mV) the effective potential of the patch
(Vpm, mV) for a period of 2 s was
calculated. Arrow 3: based on the effective potential of the
patch (Vpm) and the applied holding potential
(Vapplied) the membrane potential of
the cell (Vwc) was calculated and plotted as a
function of time (Vwc = Vpm Vapplied).
Arrow 4 (Po): the open state
probability (Po) was calculated for each
individual sweep. To compare experiments with different numbers of
BKCa channels in the patch, normalized
Po (norm. Po)
was calculated (see "Experimental Procedures") and expressed as a
function of time. Arrow 5 ([Ca2+]pm): based on a series of calibration
experiments (A) a linear relationship between
Vpm and the Ca2+ sensitivity of the
BKCa channel was found (log EC = 0.052·Vpm 3.501). For estimation of the
Ca2+ concentration at the mouth of the BKCa
channel ([Ca2+]pm) under physiological patch
clamp conditions the following equation was used,
log[Ca2+]pm = ( 0.052 × Vpm 3.501) log[(Po(max) Po)/(Po + Po(min))], where Po(max)
and Po(min) are the maximal and minimal
activities measured of the respective channel.
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Opposite Subplasmalemmal Ca2+ Gradients under
Physiological Conditions--
According to this procedure, evaluations
of the effect of histamine on BKCa channel activity,
Vwc, and [Ca2+]pm
under physiological conditions were completed in pipette locations L1, L2, and L3, and representative
tracings of a respective recording are provided in Fig.
8 (L1, panel A;
L2, panel B; L3, panel C). For further statistical evaluation and comparison, two distinct time
periods (P1 and P2) were chosen as indicated in Fig. 8, where P1 represents the initial and transient phase and
P2 the long lasting plateau phase where CCE takes
place. Far from any organelle (L1), 100 µM
histamine enhanced [Ca2+]pm in P1 and P2
approximately 35 and 12 times, respectively (Fig. 8A and
Table I). The response was biphasic and a
strong transient P1 was followed by a long lasting Ca2+
elevation during P2. Intriguingly, histamine-induced elevations of
[Ca2+]pm were completely buffered by
neighboring mitochondria (L2), and only a small and
transient Ca2+ elevation was observed (Fig. 8B
and Table I). In contrast, in the vicinity of sER (L3),
[Ca2+]pm rises upon histamine stimulation by
approximately 63 in P1 and 13 times in P2 (Fig. 8C and Table
I).

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Fig. 8.
Mitochondria effectively buffer neighboring
subplasmalemmal Ca2+ under physiological conditions.
Analysis of BKCa channel activity
(Po), whole cell membrane potential
(Vwc), and [Ca2+]pm at
pipette location L1 (A), L2 (B), and L3 (C).
D, schematic overview of the spatial distribution of
subplasmalemmal Ca2+ elevations in response to 100 µM histamine. For statistical details see Table I.
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Table I
Activation of BKCa channels in response to 100 µM
histamine at various patch pipette locations under physiological patch
conditions
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DISCUSSION |
In this work we report that superficial domains of the
mitochondria and the ER create opposite Ca2+ gradients upon
cell stimulation with the inositol 1,4,5-trisphosphate-generating agonist histamine. Using the combination of high resolution
fluorescence microscopy for visualization of organelle-targeted
fluorescent proteins and electrophysiology for locally defined single
channel recordings, we found that superficial mitochondria effectively buffer subplasmalemmal Ca2+ during cell stimulation despite
a large increase in cytosolic Ca2+. On the contrary,
superficial ER domains were observed to generate a high
Ca2+ gradient beneath the cell membrane that results in
cell hyperpolarization by activation of BKCa channels.
One essential achievement of the present study was the simultaneous
visualization of mitochondria and ER domains to allow an exact
positioning of the patch pipette. Because the transfection efficiency
of endothelial cells is rather low and the amount of expression of each
individual fluorescent protein would need to be equal, a vector for
double transfection was used. As shown in Fig. 1, a clear separation
between mitochondria and ER could be realized by transfecting the cells
with the vector pBudCE4.1 encoding ER-targeted YC4-ER and
mitochondrial-targeted DsRed. Thus, this approach allowed distinct
pipette positioning in respect to the ER and the mitochondria. Using
the standard patch approach (i.e. 130 mM
K+ outside) a strong activation of the BKCa
channels in response to 100 µM histamine was found at the
ER (L3) and far from any organelle (L1). These findings are
consistent with our previous report in which 100 µM
histamine stimulated BKCa channels in pipette locations L1
and L3, whereas only at low histamine concentration (i.e. 10 µM) a spatial subplasmalemmal Ca2+ elevation
occurred between the plasma membrane and the sER (i.e. L3)
(10). Localized Ca2+ events have been often reported in
excitable and non-excitable cells (see Ref. 24 for review). Such
localized elevations of subplasmalemmal
[Ca2+]pm have been clearly shown in
pancreatic acinar cells (25), cardiac myocytes (26), smooth muscle
(27), or HeLa cells (28) where Ca2+ signaling has been
found to constitute a multitude of local, highly controlled processes
that include ion channels, pumps, and organelles. All these reports
dealt with local elevation of Ca2+ in restricted areas of
the cell. Although such high Ca2+ gradients have been found
to constitute distinct triggers for the spatial modulation of
Ca2+-activated mechanisms (24), these studies fail to
explain how during a cell stimulation that is accompanied with a large
elevation in the cytosolic Ca2+ concentration
Ca2+-sensitive ion channels are still active despite the
inhibitory action of Ca2+ on this pathway.
As in most other non-excitable cells, Ca2+ enters
endothelial cells through the so-called CCE pathway. Notably, the CCE
is sensitive to elevation of Ca2+ at the mouth of one or
more of the channels (2-6, 29). However, in this study and our
previous work (10) we demonstrate that the subplasmalemmal
Ca2+ concentration elevates up to 1.6 µM free
Ca2+ under standard patch clamp conditions, and, although
such high Ca2+ concentration is known to prevent CCE, a
large CCE took place during strong cell stimulation (10). Because this
paradox situation was found in many cells, a phenomenon of local
subplasmalemmal Ca2+ lowering was postulated. As mechanisms
of such spatial subplasmalemmal Ca2+-buffering plasma
membrane Ca2+ pumps (30) and/or Ca2+ buffering
by the mitochondria (2-6, 31-33) were suggested. The mitochondrial Ca2+ buffer function was predominantly
investigated using uncouplers of mitochondrial oxidative
phosphorylation (i.e. the carbonyl cyanide phenylhydrazones
FCCP and CCCP) that result in inhibition of mitochondrial
Ca2+ uptake (34) due to the depolarization of the
mitochondria and consequently prevent CCE activity, monitored by
conventional fluorometric Ca2+ measurements or whole cell
currents (2-6, 31-33). However, because the uncouplers of
mitochondrial oxidative phosphorylation have been reported to initiate
Ca2+ release from the ER (15) and to depolarize the plasma
membrane (16), mitochondrial Ca2+ buffering during cell
stimulation needed to be studied directly.
Thus, our present findings, that in isometric K+ bath
conditions BKCa channel activation in response to 100 µM histamine was strongly reduced in the proximity of
mitochondria, indicate for the first time that the increase in
[Ca2+]pm in response to histamine was
effectively reduced by superficial mitochondria. This direct
demonstration of mitochondrial "Ca2+ buffering" was
further confirmed in experiments where BKCa channel activity was restored after the superficial mitochondria displaced from
the patch during the experiments. Such mitochondrial movements have
been reported frequently (for review see Refs. 35 and 36) and are
thought to result from mitochondrial movements along the microtubular
network (37). Considering our recent findings that the BKCa
channels are ubiquitously distributed in EA.hy926 cells (10), these
data indicate that moving organelles affect the activity of neighboring
plasma membrane channel. Thus, it seems possible that superficial
organelles create their own distinct microenvironment along their way.
The contribution of mitochondria to local Ca2+ buffering
monitored by using the BKCa channels as Ca2+
sensors was further supported by our findings that FCCP restored BKCa channel activation in patches close to mitochondria.
Because mitochondrial Ca2+ buffering was measured in these
experiments directly under controlled conditions (i.e.
defined patch localization and clamped membrane potential), these data
point to an elevation of [Ca2+]pm in response
to histamine due to the lack of mitochondrial Ca2+
sequestration under FCCP treatment. Also, although these data are
consistent with previous experiments where uncouplers of mitochondrial oxidative phosphorylation were used to investigate the contribution of
mitochondrial Ca2+ buffering to CCE (2-6, 31-33), this is
the first time that FCCP was demonstrated to prevent subplasmalemmal
mitochondrial Ca2+ buffering upon agonist stimulation on
the single cell level.
Remarkably, the activation of BKCa channels far from any
organelle (L1), close to sER (L3) or next to mitochondria (L2) in the
presence of FCCP, was found to be transient. This finding is quite
surprising considering the long lasting cytosolic Ca2+
elevation and membrane hyperpolarization found in these cells in
response to 100 µM histamine (9, 10, 21). The simplest explanation for the transient BKCa channel activation in
the standard patch protocol is that, in standard bath solution
(i.e. isometric K+) very little or no
Ca2+ entry takes place as Ca2+ influx
critically depends on the driving force that is most prominently provided by the activation of Ca2+-activated K+
channels (8). This assumption is further supported by our data
presented herein and previous reports that in endothelial cells
Ca2+ entry depends critically on membrane hyperpolarization.
Thus, out of these findings we conclude that experiments in which the
standard patch approach was used do not allow a proper evaluation of
the kinetics and the magnitude of spatial Ca2+ gradients
due to the strong reduction of CCE under the depolarizing conditions
used. These findings raise a number of important questions: What is the
subplasmalemmal Ca2+ concentration achieved by cell
stimulation under physiological conditions? Do mitochondria still
buffer Ca2+ under physiological conditions where CCE
occurs? And, finally, is our SCCU concept still accurate, although one
can expect higher transmembrane Ca2+ movements?
These aspects were verified in our experiments using a physiological
patch that allowed the cell to manipulate its membrane potential freely
while one can still follow single channel activity. Under these
conditions, the driving force for Ca2+ entry is not
diminished by artificial membrane depolarization, and, thus, a
physiological CCE occurs. We believe that this approach, which reveals
the actual patch potential (Vpm), whole cell
membrane potential (Vwc), and
[Ca2+]pm, represents a landmark for progress
in the evaluation of cellular Ca2+ homeostasis.
Convincingly, under physiological but not
standard patch conditions Ca2+ entry occurs,
which was indicated by the second long lasting activation of the
BKCa located far from any organelle (Figs. 6B, 8A, and 8C). This biphasic activation of the
BKCa channels occurred despite a long lasting cell membrane
hyperpolarization (Fig. 8, A and C), which was in
the same range as that obtained in conventional current clamp protocol
(i.e. approximately 30 mV). At pipette location L1, the
estimated [Ca2+]pm elevations in response to
histamine were also biphasic and revealed up to ~3.5 and ~1.2
µM during the initial transient (P1) and long lasting
phase (P2), respectively. These levels of
[Ca2+]pm correspond precisely to that found
using membrane targeted ratiometric-pericam in pancreatic islet
-cells (38) and confirm our approach of monitoring
[Ca2+]pm by BKCa channels.
When locating the pipette at sER domains (L3) the subplasmalemmal
Ca2+ elevation in response to histamine exceeded that found
in L1 (up to ~6.3 and ~1.3 µM during P1 and P2,
respectively), whereas the onset of the second phase was faster. These
data further support our previous concept on the specific role of the
sER for local Ca2+ elevation (SCCU) (9-12). Moreover, by
introducing the physiological patch approach we demonstrate that even
under strong cell stimulation the SCCU builds up a subplasmalemmal
Ca2+ gradient in which the Ca2+ concentration
is higher than in areas without sER.
In our standard patch experiments the mitochondria have been found to
buffer effectively neighboring Ca2+ in the subplasmalemmal
area indicated by the lack of BKCa channel activation upon
100 µM histamine administration (Fig. 2). Using the
physiological patch approach, it was of interest whether or not
mitochondria are still able to buffer subplasmalemmal Ca2+
in their neighborhood, although we have found a 3- to 6-fold higher
subplasmalemmal Ca2+ concentration at L1 and L3 compared
with our standard patch experiments. Remarkably, despite such high
subplasmalemmal Ca2+ elevation to histamine far from any
organelle and close to ER domains, superficial mitochondria were still
capable of buffering [Ca2+]pm during
histamine stimulation to 0.25 and 0.10 µM in P1 and P2,
respectively. These data demonstrate that, during strong cell activation, mitochondria buffer subplasmalemmal Ca2+
elevation by about 95 and 98% compared with the L1 and L3 pipette positions. Furthermore, during P2, the phase where the
Ca2+-sensitive CCE takes place (29), subplasmalemmal
mitochondria keep neighboring subplasmalemmal Ca2+ at basal
levels. This is the first time that mitochondrial the "Ca2+ buffering" function was demonstrated directly
under physiological conditions and without any pharmacological tools.
Furthermore, these data convincingly prove the concept that superficial
mitochondria indeed create a local microdomain of low Ca2+
that might sustain the activity of the Ca2+-inhibitable CCE pathway.
Our findings, that even under physiological conditions, superficial
organelles are able to create opposite Ca2+ gradients and
build their own Ca2+ dynamics in their microenvironment,
have important implications because Ca2+ operates as a
crucial messenger for numerous pivotal functions in the cell. In
endothelial cells, Ca2+ regulates the production of
vasoactive compounds (for review see Ref. 39) and the activation of
transcription factors (e.g. NF
B) (40) and ion channels
(41). Due to the opposite characteristics of Ca2+
gradients at superficial organelles during cell stimulation presented herein, the mechanisms for the versatility of Ca2+ as a
ubiquitous second messenger becomes more transparent.