Mitochondria Efficiently Buffer Subplasmalemmal Ca2+Elevation during Agonist Stimulation

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 J.A. Parekh A.B. EMBO J. PubMed Scopus Google 2000; PubMed Scopus Google J. PubMed Scopus Google A.B. J. PubMed Scopus Google Scholar). the the of Ca2+ that the cells through CCE activation of Ca2+-activated K+ channels to a membrane hyperpolarization and, thus, the for Ca2+ entry PubMed Scopus Google Google Scholar). in endothelial cells, superficial ER domains Ca2+ the plasma membrane that in local activation of BKCa channels J. 2000; PubMed Scopus Google J. PubMed Scopus Google J. J. PubMed Scopus Google J. J. PubMed Scopus Google Scholar). The of Ca2+ elevation the plasma membrane at in the that during cell activation of Ca2+-activated ion with the during a cell μm where BKCa channels in far from the a CCE still J. PubMed Scopus Google Scholar). the sER to Ca2+ by membrane hyperpolarization due to Ca2+-activated K+ channels, local of the subplasmalemmal Ca2+ concentration has to to CCE was that mitochondria a for CCE activity in non-excitable In in mitochondria were by of the carbonyl and in of Ca2+ the of CCE was prevented (2Gilabert J.A. Parekh A.B. EMBO J. 2000; 19: 6401-6407Crossref PubMed Scopus (203) Google J.A. Parekh A.B. EMBO J. PubMed Scopus Google 2000; PubMed Scopus Google EMBO J. PubMed Scopus Google Scholar). to Ca2+ is to CCE by subplasmalemmal Ca2+ at the mouth of 2000; PubMed Scopus Google Scholar). carbonyl Ca2+ in a of the in a of the and depolarization of the inner mitochondria been to the Ca2+ release from the ER J. PubMed Scopus Google and in a depolarization of the plasma membrane J. PubMed Scopus Google Scholar). In of the of for the of Ca2+ was to and of Ca2+ during cell in the endothelial cell that superficial domains of the mitochondria and the ER opposite Ca2+ cell with the histamine. Using the of high for of and for single channel that superficial mitochondria subplasmalemmal Ca2+ during cell despite a large in cytosolic Ca2+. the superficial ER domains were to generate a high Ca2+ the cell membrane that in cell hyperpolarization by activation of BKCa of the was the simultaneous of mitochondria and ER domains to an of the patch the of endothelial cells is low and the of of individual to be a for was in a mitochondria and ER could be by the cells with the ER-targeted and mitochondrial-targeted Thus, approach pipette in to the ER and the Using the standard patch approach a activation of the in response to μm histamine was at the ER and far from any organelle with in BKCa channels in pipette locations L1 and L3, whereas at low histamine concentration a subplasmalemmal Ca2+ elevation the plasma membrane and the sER J. PubMed Scopus Google Scholar). Ca2+ been in and non-excitable cells J. PubMed Scopus Google for of subplasmalemmal [Ca2+]pm been clearly in cells EMBO J. PubMed Scopus Google J. PubMed Scopus Google J. PubMed Google or cells PubMed Scopus Google where Ca2+ has been to a of that ion channels, and with local elevation of Ca2+ in of the Although high Ca2+ been to triggers for the of Ca2+-activated J. PubMed Scopus Google to how during a cell that is with a large elevation in the cytosolic Ca2+ concentration ion channels still despite the of Ca2+ in non-excitable cells, Ca2+ endothelial cells through the so-called CCE the CCE is to elevation of Ca2+ at the mouth of or of the channels (2Gilabert J.A. Parekh A.B. EMBO J. 2000; 19: 6401-6407Crossref PubMed Scopus (203) Google J.A. Parekh A.B. EMBO J. PubMed Scopus Google 2000; PubMed Scopus Google J. PubMed Scopus Google A.B. J. PubMed Scopus Google J. PubMed Scopus Google Scholar). in and J. PubMed Scopus Google that the subplasmalemmal Ca2+ concentration to μm free Ca2+ under standard patch and, high Ca2+ concentration is to CCE, a large CCE during cell J. PubMed Scopus Google Scholar). was in many cells, a of local subplasmalemmal Ca2+ was of subplasmalemmal plasma membrane Ca2+ J. PubMed Google Ca2+ by the mitochondria (2Gilabert J.A. Parekh A.B. EMBO J. 2000; 19: 6401-6407Crossref PubMed Scopus (203) Google J.A. Parekh A.B. EMBO J. PubMed Scopus Google 2000; PubMed Scopus Google J. PubMed Scopus Google A.B. J. PubMed Scopus Google PubMed Scopus Google Google Parekh A.B. EMBO J. PubMed Scopus Google were The Ca2+ was of the carbonyl and that in of Ca2+ J. 2000; PubMed Scopus Google due to the depolarization of the mitochondria and CCE monitored by Ca2+ or cell (2Gilabert J.A. Parekh A.B. EMBO J. 2000; 19: 6401-6407Crossref PubMed Scopus (203) Google J.A. Parekh A.B. EMBO J. PubMed Scopus Google 2000; PubMed Scopus Google J. PubMed Scopus Google A.B. J. PubMed Scopus Google PubMed Scopus Google Google Parekh A.B. EMBO J. PubMed Scopus Google Scholar). the of been to Ca2+ release from the ER J. PubMed Scopus Google and to the plasma membrane J. PubMed Scopus Google Ca2+ during cell to be that in K+ conditions BKCa channel activation in response to μm histamine was in the of mitochondria, for the that the in [Ca2+]pm in response to histamine was by superficial of was in where BKCa channel activity was the superficial mitochondria from the patch during the been and PubMed Scopus Google and to from the PubMed Scopus Google Scholar). that the in cells J. PubMed Scopus Google that the activity of neighboring plasma membrane Thus, it that superficial their their contribution of mitochondria to local Ca2+ monitored by the BKCa channels was by that BKCa channel activation in close to Ca2+ was in under conditions patch and membrane to an elevation of [Ca2+]pm in response to histamine due to the of under with where of were used to the contribution of Ca2+ to CCE (2Gilabert J.A. Parekh A.B. EMBO J. 2000; 19: 6401-6407Crossref PubMed Scopus (203) Google J.A. Parekh A.B. EMBO J. PubMed Scopus Google 2000; PubMed Scopus Google J. PubMed Scopus Google A.B. J. PubMed Scopus Google PubMed Scopus Google Google Parekh A.B. EMBO J. PubMed Scopus Google is the that was to subplasmalemmal Ca2+ the single cell the activation of BKCa channels far from any organelle (L1), close to sER or to mitochondria (L2) in the presence of was to be is the long lasting cytosolic and membrane hyperpolarization in cells in response to μm histamine J. 2000; PubMed Scopus Google J. PubMed Scopus Google J. J. 2000; Scopus Google Scholar). The for the transient BKCa channel activation in the standard patch is in standard or Ca2+ entry Ca2+ the that is by the activation of Ca2+-activated Google Scholar). is by and that in endothelial cells Ca2+ entry membrane of that in the standard patch approach was used a of the and the of Ca2+ due to the of CCE under the conditions a of is the subplasmalemmal Ca2+ concentration by cell under physiological mitochondria still Ca2+ under physiological conditions where CCE is still Ca2+ were in a physiological patch that the cell to membrane still single channel Under the for Ca2+ entry is by membrane and, thus, a physiological CCE that the actual patch cell membrane and represents a for in the of Ca2+ under physiological patch conditions Ca2+ entry was by the long lasting activation of the BKCa far from any organelle and activation of the BKCa channels despite a long lasting cell membrane hyperpolarization and was in the that in pipette the [Ca2+]pm in response to histamine were and to and ∼1.2 μm during the initial transient and long lasting of [Ca2+]pm to that membrane targeted in J. PubMed Scopus Google and approach of [Ca2+]pm by BKCa the pipette at sER domains the subplasmalemmal Ca2+ elevation in response to histamine that in L1 to and μm during and whereas the of the was the of the sER for local Ca2+ elevation J. 2000; PubMed Scopus Google J. PubMed Scopus Google J. J. PubMed Scopus Google J. J. PubMed Scopus Google Scholar). by the physiological patch approach that under cell the a subplasmalemmal Ca2+ in the Ca2+ concentration is in without standard patch the mitochondria been to neighboring Ca2+ in the subplasmalemmal by the of BKCa channel activation μm histamine Using the physiological patch it was of or mitochondria still to subplasmalemmal their a to subplasmalemmal Ca2+ concentration at L1 and L3 with standard patch Remarkably, despite high subplasmalemmal Ca2+ elevation to histamine far from any organelle and close to ER superficial mitochondria were still of [Ca2+]pm during histamine to and μm in and during cell mitochondria subplasmalemmal by and with the L1 and L3 pipette during the where the CCE J. PubMed Scopus Google subplasmalemmal mitochondria neighboring subplasmalemmal Ca2+ at is the that the was under physiological conditions and without any the that superficial mitochondria a local of low the activity of the CCE that under physiological superficial to opposite Ca2+ and their Ca2+ in their Ca2+ a for in the In endothelial cells, Ca2+ the of Scopus Google and the activation of J. PubMed Scopus Google and ion channels PubMed Scopus Google Scholar). to the opposite of at superficial during cell the for the of Ca2+ a 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 J.A. Parekh A.B. EMBO J. PubMed Scopus Google 2000; PubMed Scopus Google J. PubMed Scopus Google A.B. J. PubMed Scopus Google Scholar). the the of Ca2+ that the cells through CCE activation of Ca2+-activated K+ channels to a membrane hyperpolarization and, thus, the for Ca2+ entry PubMed Scopus Google Google Scholar). in endothelial cells, superficial ER domains Ca2+ the plasma membrane that in local activation of BKCa channels J. 2000; PubMed Scopus Google J. PubMed Scopus Google J. J. PubMed Scopus Google J. J. PubMed Scopus Google Scholar). The of Ca2+ elevation the plasma membrane at in the that during cell activation of Ca2+-activated ion with the during a cell μm where BKCa channels in far from the a CCE still J. PubMed Scopus Google Scholar). the sER to Ca2+ by membrane hyperpolarization due to Ca2+-activated K+ channels, local of the subplasmalemmal Ca2+ concentration has to to CCE was that mitochondria a for CCE activity in non-excitable In in mitochondria were by of the carbonyl and in of Ca2+ the of CCE was prevented (2Gilabert J.A. Parekh A.B. EMBO J. 2000; 19: 6401-6407Crossref PubMed Scopus (203) Google J.A. Parekh A.B. EMBO J. PubMed Scopus Google 2000; PubMed Scopus Google EMBO J. PubMed Scopus Google Scholar). to Ca2+ is to CCE by subplasmalemmal Ca2+ at the mouth of 2000; PubMed Scopus Google Scholar). carbonyl Ca2+ in a of the in a of the and depolarization of the inner mitochondria been to the Ca2+ release from the ER J. PubMed Scopus Google and in a depolarization of the plasma membrane J. PubMed Scopus Google Scholar). In of the of for the of Ca2+ was to and of Ca2+ during cell in the endothelial cell that superficial domains of the mitochondria and the ER opposite Ca2+ cell with the histamine. Using the of high for of and for single channel that superficial mitochondria subplasmalemmal Ca2+ during cell despite a large in cytosolic Ca2+. the superficial ER domains were to generate a high Ca2+ the cell membrane that in cell hyperpolarization by activation of BKCa of the was the simultaneous of mitochondria and ER domains to an of the patch the of endothelial cells is low and the of of individual to be a for was in a mitochondria and ER could be by the cells with the ER-targeted and mitochondrial-targeted Thus, approach pipette in to the ER and the Using the standard patch approach a activation of the in response to μm histamine was at the ER and far from any organelle with in BKCa channels in pipette locations L1 and L3, whereas at low histamine concentration a subplasmalemmal Ca2+ elevation the plasma membrane and the sER J. PubMed Scopus Google Scholar). Ca2+ been in and non-excitable cells J. PubMed Scopus Google for of subplasmalemmal [Ca2+]pm been clearly in cells EMBO J. PubMed Scopus Google J. PubMed Scopus Google J. PubMed Google or cells PubMed Scopus Google where Ca2+ has been to a of that ion channels, and with local elevation of Ca2+ in of the Although high Ca2+ been to triggers for the of Ca2+-activated J. PubMed Scopus Google to how during a cell that is with a large elevation in the cytosolic Ca2+ concentration ion channels still despite the of Ca2+ in non-excitable cells, Ca2+ endothelial cells through the so-called CCE the CCE is to elevation of Ca2+ at the mouth of or of the channels (2Gilabert J.A. Parekh A.B. EMBO J. 2000; 19: 6401-6407Crossref PubMed Scopus (203) Google J.A. Parekh A.B. EMBO J. PubMed Scopus Google 2000; PubMed Scopus Google J. PubMed Scopus Google A.B. J. PubMed Scopus Google J. PubMed Scopus Google Scholar). in and J. PubMed Scopus Google that the subplasmalemmal Ca2+ concentration to μm free Ca2+ under standard patch and, high Ca2+ concentration is to CCE, a large CCE during cell J. PubMed Scopus Google Scholar). was in many cells, a of local subplasmalemmal Ca2+ was of subplasmalemmal plasma membrane Ca2+ J. PubMed Google Ca2+ by the mitochondria (2Gilabert J.A. Parekh A.B. EMBO J. 2000; 19: 6401-6407Crossref PubMed Scopus (203) Google J.A. Parekh A.B. EMBO J. PubMed Scopus Google 2000; PubMed Scopus Google J. PubMed Scopus Google A.B. J. PubMed Scopus Google PubMed Scopus Google Google Parekh A.B. EMBO J. PubMed Scopus Google were The Ca2+ was of the carbonyl and that in of Ca2+ J. 2000; PubMed Scopus Google due to the depolarization of the mitochondria and CCE monitored by Ca2+ or cell (2Gilabert J.A. Parekh A.B. EMBO J. 2000; 19: 6401-6407Crossref PubMed Scopus (203) Google J.A. Parekh A.B. EMBO J. PubMed Scopus Google 2000; PubMed Scopus Google J. PubMed Scopus Google A.B. J. PubMed Scopus Google PubMed Scopus Google Google Parekh A.B. EMBO J. PubMed Scopus Google Scholar). the of been to Ca2+ release from the ER J. PubMed Scopus Google and to the plasma membrane J. PubMed Scopus Google Ca2+ during cell to be that in K+ conditions BKCa channel activation in response to μm histamine was in the of mitochondria, for the that the in [Ca2+]pm in response to histamine was by superficial of was in where BKCa channel activity was the superficial mitochondria from the patch during the been and PubMed Scopus Google and to from the PubMed Scopus Google Scholar). that the in cells J. PubMed Scopus Google that the activity of neighboring plasma membrane Thus, it that superficial their their contribution of mitochondria to local Ca2+ monitored by the BKCa channels was by that BKCa channel activation in close to Ca2+ was in under conditions patch and membrane to an elevation of [Ca2+]pm in response to histamine due to the of under with where of were used to the contribution of Ca2+ to CCE (2Gilabert J.A. Parekh A.B. EMBO J. 2000; 19: 6401-6407Crossref PubMed Scopus (203) Google J.A. Parekh A.B. EMBO J. PubMed Scopus Google 2000; PubMed Scopus Google J. PubMed Scopus Google A.B. J. PubMed Scopus Google PubMed Scopus Google Google Parekh A.B. EMBO J. PubMed Scopus Google is the that was to subplasmalemmal Ca2+ the single cell the activation of BKCa channels far from any organelle (L1), close to sER or to mitochondria (L2) in the presence of was to be is the long lasting cytosolic and membrane hyperpolarization in cells in response to μm histamine J. 2000; PubMed Scopus Google J. PubMed Scopus Google J. J. 2000; Scopus Google Scholar). The for the transient BKCa channel activation in the standard patch is in standard or Ca2+ entry Ca2+ the that is by the activation of Ca2+-activated Google Scholar). is by and that in endothelial cells Ca2+ entry membrane of that in the standard patch approach was used a of the and the of Ca2+ due to the of CCE under the conditions a of is the subplasmalemmal Ca2+ concentration by cell under physiological mitochondria still Ca2+ under physiological conditions where CCE is still Ca2+ were in a physiological patch that the cell to membrane still single channel Under the for Ca2+ entry is by membrane and, thus, a physiological CCE that the actual patch cell membrane and represents a for in the of Ca2+ under physiological patch conditions Ca2+ entry was by the long lasting activation of the BKCa far from any organelle and activation of the BKCa channels despite a long lasting cell membrane hyperpolarization and was in the that in pipette the [Ca2+]pm in response to histamine were and to and ∼1.2 μm during the initial transient and long lasting of [Ca2+]pm to that membrane targeted in J. PubMed Scopus Google and approach of [Ca2+]pm by BKCa the pipette at sER domains the subplasmalemmal Ca2+ elevation in response to histamine that in L1 to and μm during and whereas the of the was the of the sER for local Ca2+ elevation J. 2000; PubMed Scopus Google J. PubMed Scopus Google J. J. PubMed Scopus Google J. J. PubMed Scopus Google Scholar). by the physiological patch approach that under cell the a subplasmalemmal Ca2+ in the Ca2+ concentration is in without standard patch the mitochondria been to neighboring Ca2+ in the subplasmalemmal by the of BKCa channel activation μm histamine Using the physiological patch it was of or mitochondria still to subplasmalemmal their a to subplasmalemmal Ca2+ concentration at L1 and L3 with standard patch Remarkably, despite high subplasmalemmal Ca2+ elevation to histamine far from any organelle and close to ER superficial mitochondria were still of [Ca2+]pm during histamine to and μm in and during cell mitochondria subplasmalemmal by and with the L1 and L3 pipette during the where the CCE J. PubMed Scopus Google subplasmalemmal mitochondria neighboring subplasmalemmal Ca2+ at is the that the was under physiological conditions and without any the that superficial mitochondria a local of low the activity of the CCE that under physiological superficial to opposite Ca2+ and their Ca2+ in their Ca2+ a for in the In endothelial cells, Ca2+ the of Scopus Google and the activation of J. PubMed Scopus Google and ion channels PubMed Scopus Google Scholar). to the opposite of at superficial during cell the for the of Ca2+ a In that superficial domains of the mitochondria and the ER opposite Ca2+ cell with the histamine. Using the of high for of and for single channel that superficial mitochondria subplasmalemmal Ca2+ during cell despite a large in cytosolic Ca2+. the superficial ER domains were to generate a high Ca2+ the cell membrane that in cell hyperpolarization by activation of BKCa channels. of the was the simultaneous of mitochondria and ER domains to an of the patch the of endothelial cells is low and the of of individual to be a for was in a mitochondria and ER could be by the cells with the ER-targeted and mitochondrial-targeted Thus, approach pipette in to the ER and the Using the standard patch approach a activation of the in response to μm histamine was at the ER and far from any organelle with in BKCa channels in pipette locations L1 and L3, whereas at low histamine concentration a subplasmalemmal Ca2+ elevation the plasma membrane and the sER J. PubMed Scopus Google Scholar). Ca2+ been in and non-excitable cells J. PubMed Scopus Google for of subplasmalemmal [Ca2+]pm been clearly in cells EMBO J. PubMed Scopus Google J. PubMed Scopus Google J. PubMed Google or cells PubMed Scopus Google where Ca2+ has been to a of that ion channels, and with local elevation of Ca2+ in of the Although high Ca2+ been to triggers for the of Ca2+-activated J. PubMed Scopus Google to how during a cell that is with a large elevation in the cytosolic Ca2+ concentration ion channels still despite the of Ca2+ in non-excitable cells, Ca2+ endothelial cells through the so-called CCE the CCE is to elevation of Ca2+ at the mouth of or of the channels (2Gilabert J.A. Parekh A.B. EMBO J. 2000; 19: 6401-6407Crossref PubMed Scopus (203) Google J.A. Parekh A.B. EMBO J. PubMed Scopus Google 2000; PubMed Scopus Google J. PubMed Scopus Google A.B. J. PubMed Scopus Google J. PubMed Scopus Google Scholar). in and J. PubMed Scopus Google that the subplasmalemmal Ca2+ concentration to μm free Ca2+ under standard patch and, high Ca2+ concentration is to CCE, a large CCE during cell J. PubMed Scopus Google Scholar). was in many cells, a of local subplasmalemmal Ca2+ was of subplasmalemmal plasma membrane Ca2+ J. PubMed Google Ca2+ by the mitochondria (2Gilabert J.A. Parekh A.B. EMBO J. 2000; 19: 6401-6407Crossref PubMed Scopus (203) Google J.A. Parekh A.B. EMBO J. PubMed Scopus Google 2000; PubMed Scopus Google J. PubMed Scopus Google A.B. J. PubMed Scopus Google PubMed Scopus Google Google Parekh A.B. EMBO J. PubMed Scopus Google were The Ca2+ was of the carbonyl and that in of Ca2+ J. 2000; PubMed Scopus Google due to the depolarization of the mitochondria and CCE monitored by Ca2+ or cell (2Gilabert J.A. Parekh A.B. EMBO J. 2000; 19: 6401-6407Crossref PubMed Scopus (203) Google J.A. Parekh A.B. EMBO J. PubMed Scopus Google 2000; PubMed Scopus Google J. PubMed Scopus Google A.B. J. PubMed Scopus Google PubMed Scopus Google Google Parekh A.B. EMBO J. PubMed Scopus Google Scholar). the of been to Ca2+ release from the ER J. PubMed Scopus Google and to the plasma membrane J. PubMed Scopus Google Ca2+ during cell to be Thus, that in K+ conditions BKCa channel activation in response to μm histamine was in the of mitochondria, for the that the in [Ca2+]pm in response to histamine was by superficial of was in where BKCa channel activity was the superficial mitochondria from the patch during the been and PubMed Scopus Google and to from the PubMed Scopus Google Scholar). that the in cells J. PubMed Scopus Google that the activity of neighboring plasma membrane Thus, it that superficial their their The contribution of mitochondria to local Ca2+ monitored by the BKCa channels was by that BKCa channel activation in close to Ca2+ was in under conditions patch and membrane to an elevation of [Ca2+]pm in response to histamine due to the of under with where of were used to the contribution of Ca2+ to CCE (2Gilabert J.A. Parekh A.B. EMBO J. 2000; 19: 6401-6407Crossref PubMed Scopus (203) Google J.A. Parekh A.B. EMBO J. PubMed Scopus Google 2000; PubMed Scopus Google J. PubMed Scopus Google A.B. J. PubMed Scopus Google PubMed Scopus Google Google Parekh A.B. EMBO J. PubMed Scopus Google is the that was to subplasmalemmal Ca2+ the single cell Remarkably, the activation of BKCa channels far from any organelle (L1), close to sER or to mitochondria (L2) in the presence of was to be is the long lasting cytosolic and membrane hyperpolarization in cells in response to μm histamine J. 2000; PubMed Scopus Google J. PubMed Scopus Google J. J. 2000; Scopus Google Scholar). The for the transient BKCa channel activation in the standard patch is in standard or Ca2+ entry Ca2+ the that is by the activation of Ca2+-activated Google Scholar). is by and that in endothelial cells Ca2+ entry membrane Thus, of that in the standard patch approach was used a of the and the of Ca2+ due to the of CCE under the conditions a of is the subplasmalemmal Ca2+ concentration by cell under physiological mitochondria still Ca2+ under physiological conditions where CCE is still Ca2+ were in a physiological patch that the cell to membrane still single channel Under the for Ca2+ entry is by membrane and, thus, a physiological CCE that the actual patch cell membrane and represents a for in the of Ca2+ under physiological patch conditions Ca2+ entry was by the long lasting activation of the BKCa far from any organelle and activation of the BKCa channels despite a long lasting cell membrane hyperpolarization and was in the that in pipette the [Ca2+]pm in response to histamine were and to and ∼1.2 μm during the initial transient and long lasting of [Ca2+]pm to that membrane targeted in J. PubMed Scopus Google and approach of [Ca2+]pm by BKCa channels. the pipette at sER domains the subplasmalemmal Ca2+ elevation in response to histamine that in L1 to and μm during and whereas the of the was the of the sER for local Ca2+ elevation J. 2000; PubMed Scopus Google J. PubMed Scopus Google J. J. PubMed Scopus Google J. J. PubMed Scopus Google Scholar). by the physiological patch approach that under cell the a subplasmalemmal Ca2+ in the Ca2+ concentration is in without In standard patch the mitochondria been to neighboring Ca2+ in the subplasmalemmal by the of BKCa channel activation μm histamine Using the physiological patch it was of or mitochondria still to subplasmalemmal their a to subplasmalemmal Ca2+ concentration at L1 and L3 with standard patch Remarkably, despite high subplasmalemmal Ca2+ elevation to histamine far from any organelle and close to ER superficial mitochondria were still of [Ca2+]pm during histamine to and μm in and during cell mitochondria subplasmalemmal by and with the L1 and L3 pipette during the where the CCE J. PubMed Scopus Google subplasmalemmal mitochondria neighboring subplasmalemmal Ca2+ at is the that the was under physiological conditions and without any the that superficial mitochondria a local of low the activity of the CCE that under physiological superficial to opposite Ca2+ and their Ca2+ in their Ca2+ a for in the In endothelial cells, Ca2+ the of Scopus Google and the activation of J. PubMed Scopus Google and ion channels PubMed Scopus Google Scholar). to the opposite of at superficial during cell the for the of Ca2+ a and for and for the cameleon and and J. for the