Mitochondria Efficiently Buffer Subplasmalemmal Ca2+ Elevation during Agonist Stimulation*

Roland MalliDagger , Maud Frieden§, Karin OsibowDagger , and Wolfgang F. GraierDagger

From the Dagger  Department of Medical Biochemistry & Medical Molecular Biology, University of Graz, Graz 8010, Austria and the § Department of Physiology, University of Geneva, 1211 Geneva 4, Switzerland

Received for publication, December 19, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In endothelial cells, local Ca2+ release from superficial endoplasmic reticulum (ER) activates BKCa channels. The resulting hyperpolarization promotes capacitative Ca2+ entry (CCE), which, unlike BKCa channels, is inhibited by high Ca2+. To understand how the coordinated activation of plasma membrane ion channels with opposite Ca2+ sensitivity is orchestrated, the individual contribution of mitochondria and ER in regulation of subplasmalemmal Ca2+ concentration ([Ca2+]pm) was investigated. For organelle visualization, cells were transfected with DsRed and yellow cameleon targeted to mitochondria and ER. The patch pipette was placed far from any organelle (L1), close to ER (L3), or mitochondria (L2) and activity of BKCa channels was used to estimate local [Ca2+]pm. Under standard patch conditions (130 mM K+ in the bath), histamine increased [Ca2+]pm at L1 and L3 to ~1.6 µM, whereas close to mitochondria [Ca2+]pm remained unchanged. If mitochondria moved apart from the pipette or in the presence of carbonyl cyanide-4-trifluoromethoxyphenylhyrazone, [Ca2+]pm at L2 increased in response to histamine. Under standard patch conditions Ca2+ entry was negligible due to cell depolarization. Using a physiological patch approach (5.6 mM K+ in the bath), changes in [Ca2+]pm to histamine could be monitored without cell depolarization and, thus, in conditions where Ca2+ entry occurred. Here, histamine induced an initial transient Ca2+ elevation to >= 3.5 µM followed by a long lasting plateau at ~1.2 µM in L1 and L3, whereas mitochondria kept neighboring [Ca2+]pm low during stimulation. Thus, superficial mitochondria and ER generate local domains of low and high Ca2+ allowing simultaneous activation of BKCa and CCE, despite their opposite Ca2+ sensitivity.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 MOmega ) 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,


<UP>log</UP>[<UP>Ca<SUP>2+</SUP></UP>]<SUB><UP>pm</UP></SUB><UP> = log EC</UP><SUP><UP>Ca</UP></SUP><SUB><UP>50</UP></SUB><UP> − log</UP>[(<UP>0.8305 − </UP>P<SUB><UP>o</UP></SUB>)<UP>/</UP>(P<SUB><UP>o</UP></SUB><UP> + 0.02181</UP>)] (Eq. 1)
where EC<UP><SUB>50</SUB><SUP>Ca</SUP></UP> is the Ca2+ sensitivity of the channel. Based on our calibration published recently, log EC<UP><SUB>50</SUB><SUP>Ca</SUP></UP> 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<UP><SUB>50</SUB><SUP>Ca</SUP></UP>), 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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

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<UP><SUB>50</SUB><SUP>Ca</SUP></UP> - log[(0.8305 - Po)/(Po + 0.02181)], where EC<UP><SUB>50</SUB><SUP>Ca</SUP></UP> is the Ca2+ sensitivity of the channel. Based on our calibration published recently, log EC<UP><SUB>50</SUB><SUP>Ca</SUP></UP> is -5.566. The mean Po and estimated [Ca2+]pm for the pipette location chosen are given in the left panels.

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).

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.

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).

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).

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<UP><SUB>50</SUB><SUP>Ca</SUP></UP> = -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.

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


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -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. NFkappa 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.

    ACKNOWLEDGEMENTS

We thank Beatrix Petschar and Anna Schreilechner for excellent technical assistance; Prof. R. Y. Tsien, Dr. A. Miyawaki, and Prof. T. Pozzan for providing the cameleon and mtDsRed constructs; and Dr. C. J. S. Edgell for providing the EA.hy926 cells.

    FOOTNOTES

* This work was supported by the Austrian Funds (Grants SFB 714 and P-14586-PHA to W. F. G.), the Austrian National Bank (Grants P7542 to W. F. G. and P7902 to R. M., respectively), and the Swiss National Funds (Grant 31-56902.99). The Department of Medical Biochemistry & Medical Molecular Biology is a member of the Institutes of Basic Medical Sciences at the University of Graz and was supported by the infrastructure program (Grant UGP4) of the Austrian ministry of education, science and culture.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.

To whom correspondence should be addressed: Dept. Medical Biochemistry & Medical Molecular Biology, University of Graz, Harrachgasse 21/III, Graz A-8010, Austria. Tel.: 43-316-380-7560; Fax: 43-316-380-9615; E-mail: wolfgang.graier@uni-graz.at.

Published, JBC Papers in Press, January 15, 2003, DOI 10.1074/jbc.M212971200

    ABBREVIATIONS

The abbreviations used are: ER, endoplasmic reticulum; BKCa, large conductance Ca2+-activated K+ channels; [Ca2+]cyto, free cytosolic Ca2+; [Ca2+]pm, Ca2+ concentration at the inner side of the patch membrane; CCE, capacitative Ca2+ entry; CCCP, carbonyl cyanide-3-chlorophenylhyrazone; EC<UP><SUB>50</SUB><SUP>Ca</SUP></UP>, Ca2+ sensitivity of the BKCa channel; sER, superficial endoplasmic reticulum; FCCP, carbonyl cyanide-4-trifluoromethoxyphenylhyrazone; L1, L2, and L3, pipette locations far from any organelle, close to mitochondria, and close to sER; mtDsRed, mitochondrial-targeted DsRed; Po, open state probability of single BKCa channels; SCCU, subplasmalemmal Ca2+ control unit; YC4-ER, ER-targeted yellow cameleon 4.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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