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• W2007202933 abstract "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 BKCaand CCE, despite their opposite Ca2+sensitivity. 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 BKCaand CCE, despite their opposite Ca2+sensitivity. endoplasmic reticulum large conductance Ca2+-activated K+ channels free cytosolic Ca2+ Ca2+ concentration at the inner side of the patch membrane capacitative Ca2+ entry carbonyl cyanide-3-chlorophenylhyrazone Ca2+ sensitivity of the BKCa channel superficial endoplasmic reticulum carbonyl cyanide-4-trifluoromethoxyphenylhyrazone L2, and L3, pipette locations far from any organelle, close to mitochondria, and close to sER mitochondrial-targeted DsRed open state probability of single BKCa channels subplasmalemmal Ca2+ control unit ER-targeted yellow cameleon 4 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) (1Putney J.W. Adv. Pharmacol. 1991; 22: 251-269Crossref PubMed Scopus (59) Google Scholar). 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) (2Gilabert J.A. Parekh A.B. EMBO J. 2000; 19: 6401-6407Crossref PubMed Scopus (203) Google Scholar, 3Gilabert J.A. Bakowski D. Parekh A.B. EMBO J. 2001; 20: 2672-2679Crossref PubMed Scopus (103) Google Scholar, 4Hoth M. Button D.C. Lewis R.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10607-10612Crossref PubMed Scopus (232) Google Scholar, 5Hoth M. Fanger C.M. Lewis R.S. J. Cell Biol. 1997; 137: 633-648Crossref PubMed Scopus (461) Google Scholar, 6Parekh A.B. J. Biol. Chem. 1998; 273: 14925-14932Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). 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 (7Groschner K. Graier W.F. Kukovetz W.R. Biochim. Biophys. Acta. 1992; 1137: 162-170Crossref PubMed Scopus (56) Google Scholar, 8Kamouchi M. Droogmans G. Nilius B. Gen. Physiol. Biophys. 1999; 18: 199-208PubMed Google Scholar). Notably, in endothelial cells, superficial ER (sER) domains create spatial Ca2+ gradients beneath the plasma membrane (subplasmalemmalCa2+control unit, SCCU) that result in local activation of BKCa channels (9Frieden M. Graier W.F. J. Physiol. 2000; 524: 715-724Crossref PubMed Scopus (30) Google Scholar, 10Frieden M. Malli R. Samardzija M. Demaurex N. Graier W.F. J. Physiol. 2002; 540: 73-84Crossref PubMed Scopus (36) Google Scholar, 11Graier W.F. Paltauf-Doburzynska J. Hill B. Fleischhacker E. Hoebel B.G. Kostner G.M. Sturek M. J. Physiol. 1998; 506: 109-125Crossref PubMed Scopus (88) Google Scholar, 12Paltauf-Doburzynska J. Posch K. Paltauf G. Graier W.F. J. Physiol. 1998; 513: 369-379Crossref PubMed Scopus (41) Google Scholar). 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 (10Frieden M. Malli R. Samardzija M. Demaurex N. Graier W.F. J. Physiol. 2002; 540: 73-84Crossref PubMed Scopus (36) Google Scholar). 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 (2Gilabert J.A. Parekh A.B. EMBO J. 2000; 19: 6401-6407Crossref PubMed Scopus (203) Google Scholar, 3Gilabert J.A. Bakowski D. Parekh A.B. EMBO J. 2001; 20: 2672-2679Crossref PubMed Scopus (103) Google Scholar, 4Hoth M. Button D.C. Lewis R.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10607-10612Crossref PubMed Scopus (232) Google Scholar,13Hofer A.M. Landolfi B. Debellis L. Pozzan T. Curci S. EMBO J. 1998; 17: 1986-1995Crossref PubMed Scopus (91) Google Scholar). 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 (14Duchen M.R. Cell Calcium. 2000; 28: 339-348Crossref PubMed Scopus (269) Google Scholar). 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 (15Landolfi B. Curci S. Debellis L. Pozzan T. Hofer A.M. J. Cell Biol. 1998; 142: 1235-1243Crossref PubMed Scopus (171) Google Scholar) and result in a depolarization of the plasma membrane (16Park K.S. Jo I. Pak K. Bae S.W. Rhim H. Suh S.H. Park J. Zhu H. So I. Kim K.W. Pflügers Arch. 2002; 443: 344-352Crossref PubMed Scopus (71) Google Scholar). 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.DISCUSSIONIn 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 mmK+ outside) a strong activation of the BKCachannels 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 μmhistamine 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) (10Frieden M. Malli R. Samardzija M. Demaurex N. Graier W.F. J. Physiol. 2002; 540: 73-84Crossref PubMed Scopus (36) Google Scholar). Localized Ca2+ events have been often reported in excitable and non-excitable cells (see Ref. 24Berridge M.J. J. Physiol. 1997; 499: 291-306Crossref PubMed Scopus (916) Google Scholar for review). Such localized elevations of subplasmalemmal [Ca2+]pm have been clearly shown in pancreatic acinar cells (25Cancela J.M. Van Coppenolle F. Galione A. Tepikin A.V. Petersen O.H. EMBO J. 2002; 21: 909-919Crossref PubMed Scopus (158) Google Scholar), cardiac myocytes (26Lipp P. Egger M. Niggli E. J. Physiol. 2002; 542: 383-393Crossref PubMed Scopus (25) Google Scholar), smooth muscle (27Perez G.J. Bonev A.D. Nelson M.T. Am. J. Physiol. Cell Physiol. 2001; 281: C1769-C1775Crossref PubMed Google Scholar), or HeLa cells (28Rizzuto R. Pinton P. Brini M. Chiesa A. Filippin L. Pozzan T. Cell Calcium. 1999; 26: 193-199Crossref PubMed Scopus (149) Google Scholar) 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 (24Berridge M.J. J. Physiol. 1997; 499: 291-306Crossref PubMed Scopus (916) Google Scholar), 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 (2Gilabert J.A. Parekh A.B. EMBO J. 2000; 19: 6401-6407Crossref PubMed Scopus (203) Google Scholar, 3Gilabert J.A. Bakowski D. Parekh A.B. EMBO J. 2001; 20: 2672-2679Crossref PubMed Scopus (103) Google Scholar, 4Hoth M. Button D.C. Lewis R.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10607-10612Crossref PubMed Scopus (232) Google Scholar, 5Hoth M. Fanger C.M. Lewis R.S. J. Cell Biol. 1997; 137: 633-648Crossref PubMed Scopus (461) Google Scholar, 6Parekh A.B. J. Biol. Chem. 1998; 273: 14925-14932Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 29Madge L. Marshall I.C. Taylor C.W. J. Physiol. 1997; 498: 351-369Crossref PubMed Scopus (37) Google Scholar). However, in this study and our previous work (10Frieden M. Malli R. Samardzija M. Demaurex N. Graier W.F. J. Physiol. 2002; 540: 73-84Crossref PubMed Scopus (36) Google Scholar) 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 (10Frieden M. Malli R. Samardzija M. Demaurex N. Graier W.F. J. Physiol. 2002; 540: 73-84Crossref PubMed Scopus (36) Google Scholar). 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 (30Klishin A. Sedova M. Blatter L.A. Am. J. Physiol. 1998; 274: C1117-C1128Crossref PubMed Google Scholar) and/or Ca2+ buffering by the mitochondria (2Gilabert J.A. Parekh A.B. EMBO J. 2000; 19: 6401-6407Crossref PubMed Scopus (203) Google Scholar, 3Gilabert J.A. Bakowski D. Parekh A.B. EMBO J. 2001; 20: 2672-2679Crossref PubMed Scopus (103) Google Scholar, 4Hoth M. Button D.C. Lewis R.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10607-10612Crossref PubMed Scopus (232) Google Scholar, 5Hoth M. Fanger C.M. Lewis R.S. J. Cell Biol. 1997; 137: 633-648Crossref PubMed Scopus (461) Google Scholar, 6Parekh A.B. J. Biol. Chem. 1998; 273: 14925-14932Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 31Orlati S. Cavazzoni M. Rugolo M. Cell Calcium. 1996; 20: 399-407Crossref PubMed Scopus (4) Google Scholar, 32Krutetskaia Z.I. Lebedev O.E. Krutetskaia N.I. Tsitologiia. 1998; 40: 93-99PubMed Google Scholar, 33Glitsch M.D. Bakowski D. Parekh A.B. EMBO J. 2002; 21: 6744-6754Crossref PubMed Scopus (177) Google Scholar) 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 (34Sedova M. Blatter L.A. J. Biol. Chem. 2000; 275: 35402-35407Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) due to the depolarization of the mitochondria and consequently prevent CCE activity, monitored by conventional fluorometric Ca2+ measurements or whole cell currents (2Gilabert J.A. Parekh A.B. EMBO J. 2000; 19: 6401-6407Crossref PubMed Scopus (203) Google Scholar, 3Gilabert J.A. Bakowski D. Parekh A.B. EMBO J. 2001; 20: 2672-2679Crossref PubMed Scopus (103) Google Scholar, 4Hoth M. Button D.C. Lewis R.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10607-10612Crossref PubMed Scopus (232) Google Scholar, 5Hoth M. Fanger C.M. Lewis R.S. J. Cell Biol. 1997; 137: 633-648Crossref PubMed Scopus (461) Google Scholar, 6Parekh A.B. J. Biol. Chem. 1998; 273: 14925-14932Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 31Orlati S. Cavazzoni M. Rugolo M. Cell Calcium. 1996; 20: 399-407Crossref PubMed Scopus (4) Google Scholar, 32Krutetskaia Z.I. Lebedev O.E. Krutetskaia N.I. Tsitologiia. 1998; 40: 93-99PubMed Google Scholar, 33Glitsch M.D. Bakowski D. Parekh A.B. EMBO J. 2002; 21: 6744-6754Crossref PubMed Scopus (177) Google Scholar). However, because the uncouplers of mitochondrial oxidative phosphorylation have been reported to initiate Ca2+ release from the ER (15Landolfi B. Curci S. Debellis L. Pozzan T. Hofer A.M. J. Cell Biol. 1998; 142: 1235-1243Crossref PubMed Scopus (171) Google Scholar) and to depolarize the plasma membrane (16Park K.S. Jo I. Pak K. Bae S.W. Rhim H. Suh S.H. Park J. Zhu H. So I. Kim K.W. Pflügers Arch. 2002; 443: 344-352Crossref PubMed Scopus (71) Google Scholar), 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. 35Yaffe M.P. Nat. Cell. 1999; 1: 49-50Google Scholar and 36Yaffe M.P. Science. 1999; 283: 1493-1497Crossref PubMed Scopus (416) Google Scholar) and are thought to result from mitochondrial movements along the microtubular network (37Krendel M. Sgourdas G. Bonder E.M. Cell Motil. Cytoskeleton. 1998; 40: 368-378Crossref PubMed Scopus (50) Google Scholar). Considering our recent findings that the BKCachannels are ubiquitously distributed in EA.hy926 cells (10Frieden M. Malli R. Samardzija M. Demaurex N. Graier W.F. J. Physiol. 2002; 540: 73-84Crossref PubMed Scopus (36) Google Scholar), 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 (2Gilabert J.A. Parekh A.B. EMBO J. 2000; 19: 6401-6407Crossref PubMed Scopus (203) Google Scholar, 3Gilabert J.A. Bakowski D. Parekh A.B. EMBO J. 2001; 20: 2672-2679Crossref PubMed Scopus (103) Google Scholar, 4Hoth M. Button D.C. Lewis R.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10607-10612Crossref PubMed Scopus (232) Google Scholar, 5Hoth M. Fanger C.M. Lewis R.S. J. Cell Biol. 1997; 137: 633-648Crossref PubMed Scopus (461) Google Scholar, 6Parekh A.B. J. Biol. Chem. 1998; 273: 14925-14932Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 31Orlati S. Cavazzoni M. Rugolo M. Cell Calcium. 1996; 20: 399-407Crossref PubMed Scopus (4) Google Scholar, 32Krutetskaia Z.I. Lebedev O.E. Krutetskaia N.I. Tsitologiia. 1998; 40: 93-99PubMed Google Scholar, 33Glitsch M.D. Bakowski D. Parekh A.B. EMBO J. 2002; 21: 6744-6754Crossref PubMed Scopus (177) Google Scholar), 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 (9Frieden M. Graier W.F. J. Physiol. 2000; 524: 715-724Crossref PubMed Scopus (30) Google Scholar, 10Frieden M. Malli R. Samardzija M. Demaurex N. Graier W.F. J. Physiol. 2002; 540: 73-84Crossref PubMed Scopus (36) Google Scholar, 21Paltauf-Doburzynska J. Frieden M. Spitaler M. Graier W.F. J. Physiol. 2000; 524: 501-514Crossref Scopus (68) Google Scholar). 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 (8Kamouchi M. Droogmans G. Nilius B. Gen. Physiol. Biophys. 1999; 18: 199-208PubMed Google Scholar). 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 (V pm), whole cell membrane potential (V wc), and [Ca2+]pm, represents a landmark for progress in the evaluation of cellular Ca2+ homeostasis. Convincingly, under physiological but notstandard patch conditions Ca2+ entry occurs, which was indicated by the second long lasting activation of the BKCa located far from any organelle (Figs. 6 B,8 A, and 8 C). 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 β-cells (38Pinton P. Tsuboi T. Ainscow E.K. Pozzan T. Rizzuto R. Rutter G.A. J. Biol. Chem. 2002; 277: 37702-37710Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) 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) (9Frieden M. Graier W.F. J. Physiol. 2000; 524: 715-724Crossref PubMed Scopus (30) Google Scholar, 10Frieden M. Malli R. Samardzija M. Demaurex N. Graier W.F. J. Physiol. 2002; 540: 73-84Crossref PubMed Scopus (36) Google Scholar, 11Graier W.F. Paltauf-Doburzynska J. Hill B. Fleischhacker E. Hoebel B.G. Kostner G.M. Sturek M. J. Physiol. 1998; 506: 109-125Crossref PubMed Scopus (88) Google Scholar, 12Paltauf-Doburzynska J. Posch K. Paltauf G. Graier W.F. J. Physiol. 1998; 513: 369-379Crossref PubMed Scopus (41) Google Scholar). 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 (29Madge L. Marshall I.C. Taylor C.W. J. Physiol. 1997; 498: 351-369Crossref PubMed Scopus (37) Google Scholar), 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. 39Graier W.F. Sturek M. Kukovetz W.R. Endothelium. 1994; 1: 223-236Crossref Scopus (54) Google Scholar) and the activation of transcription factors (e.g. NFκB) (40Hu Q. Deshpande S. Irani K. Ziegelstein R.C. J. Biol. Chem. 1999; 274: 33995-33998Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) and ion channels (41Nilius B. Droogmans G. Physiol. Rev. 2001; 81: 1415-1459Crossref PubMed Scopus (751) Google Scholar). 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. 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) (1Putney J.W. Adv. Pharmacol. 1991; 22: 251-269Crossref PubMed Scopus (59) Google Scholar). 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) (2Gilabert J.A. Parekh A.B. EMBO J. 2000; 19: 6401-6407Crossref PubMed Scopus (203) Google Scholar, 3Gilabert J.A. Bakowski D. Parekh A.B. EMBO J. 2001; 20: 2672-2679Crossref PubMed Scopus (103) Google Scholar, 4Hoth M. Button D.C. Lewis R.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10607-10612Crossref PubMed Scopus (232) Google Scholar, 5Hoth M. Fanger C.M. Lewis R.S. J. Cell Biol. 1997; 137: 633-648Crossref PubMed Scopus (461) Google Scholar, 6Parekh A.B. J. Biol. Chem. 1998; 273: 14925-14932Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). 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