Plasma Membrane Voltage-dependent Anion Channel Mediates Antiestrogen-activated Maxi Cl– Currents in C1300 Neuroblastoma Cells

The cell membrane large conductance voltage-dependent chloride channel (Maxi Cl– channel) has been recorded in different cell types following excision of membrane patches or stimulation by antiestrogens under whole-cell recording conditions. However, both its molecular nature and relevance to cell physiology await elucidation. Its electrophysiological properties resemble those of the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane. This observation has led to the controversial hypothesis that VDAC could be the molecular correlate of the plasma membrane Maxi Cl– channel. We have investigated the cellular localization of VDAC and its relationship with the antiestrogen-activated Maxi Cl– current in C1300 neuroblastoma cells. The presence of a plasma membrane VDAC was demonstrated by immunoblotting of membrane fractions with monoclonal antibodies against the VDAC and by reverse transcription-PCR using primers that hybridize to a VDAC sequence coding for an N-terminal leader peptide required for its plasma membrane sorting. Besides, VDAC colocalized with markers of plasma membrane lipid rafts (cholera toxin β subunit) but not caveolin-1. Transfection of C1300 cells with an antisense oligonucleotide directed against the specific membrane leader sequence of VDAC markedly reduced both VDAC immunostaining and antiestrogen-activated Maxi Cl– currents, suggesting that VDAC forms the plasma membrane Maxi Cl– channel or a part thereof. The cell membrane large conductance voltage-dependent chloride channel (Maxi Cl– channel) has been recorded in different cell types following excision of membrane patches or stimulation by antiestrogens under whole-cell recording conditions. However, both its molecular nature and relevance to cell physiology await elucidation. Its electrophysiological properties resemble those of the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane. This observation has led to the controversial hypothesis that VDAC could be the molecular correlate of the plasma membrane Maxi Cl– channel. We have investigated the cellular localization of VDAC and its relationship with the antiestrogen-activated Maxi Cl– current in C1300 neuroblastoma cells. The presence of a plasma membrane VDAC was demonstrated by immunoblotting of membrane fractions with monoclonal antibodies against the VDAC and by reverse transcription-PCR using primers that hybridize to a VDAC sequence coding for an N-terminal leader peptide required for its plasma membrane sorting. Besides, VDAC colocalized with markers of plasma membrane lipid rafts (cholera toxin β subunit) but not caveolin-1. Transfection of C1300 cells with an antisense oligonucleotide directed against the specific membrane leader sequence of VDAC markedly reduced both VDAC immunostaining and antiestrogen-activated Maxi Cl– currents, suggesting that VDAC forms the plasma membrane Maxi Cl– channel or a part thereof. Chloride channels of large conductance were first identified in excised patches from the plasma membrane of skeletal muscle 20 years ago (1Blatz A.L. Magleby K.L. Biophys. J. 1983; 43: 237-241Abstract Full Text PDF PubMed Scopus (191) Google Scholar). The search for their functional and molecular has with the different with their the the of cell J. PubMed Scopus Google J. Full Text PDF PubMed Google and PubMed Scopus Google J. PubMed Scopus Google Scholar). This channel has been by antiestrogens J. PubMed Scopus Google J. PubMed Scopus Google J. Scopus Google PubMed Scopus Google and by J. PubMed Scopus Google molecular of Maxi Cl– channels Maxi Cl– large conductance voltage-dependent chloride Maxi large conductance toxin β voltage-dependent anion plasma membrane reverse Maxi Cl– large conductance voltage-dependent chloride Maxi large conductance toxin β voltage-dependent anion plasma membrane reverse has been to The that Maxi Cl– channel electrophysiological properties resemble those of the mitochondrial voltage-dependent anion channel (VDAC) Scopus Google to that the channels were and the J. PubMed Scopus Google PubMed Scopus Google Scholar). This hypothesis was suggesting the presence of VDAC the plasma membrane J. PubMed Scopus Google J. PubMed Scopus Google but was by J. PubMed Scopus Google Scholar). The suggesting the presence of VDAC in has from the of a VDAC that a leader sequence for its to the plasma membrane PubMed Scopus Google and the presence of VDAC in J. Full Text Full Text PDF PubMed Scopus Google Scholar). the have the presence of VDAC in the plasma membrane of C1300 neuroblastoma cells its to the Maxi Cl– by antiestrogens in and neuroblastoma from the were J. Scopus Google Scholar). cells were or with for were from was from of and cells were with of and the of cell and was The cell was to a for and for was from the and for of The was and in membrane were using a J. 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PubMed Scopus Google Scholar). was from C1300 cells using the to the was to We the following for of PubMed Scopus Google and were a a of and cells were and the of an were recorded using the or whole-cell J. Scopus Google Scholar). The was for an and and were with a and and were to the for whole-cell were in a was the the of the membrane in were using a or were and patches were and for to both and were and were and for from to in and of to the were the different were by of by the for a of presence of VDAC C1300 cells was by The monoclonal in was against an N-terminal peptide PubMed Scopus Google Scholar). a from a membrane or of C1300 cells. of the for was from both the of with cellular of the membrane with was with antibodies against the mitochondrial or that membrane for the mitochondrial but for the of the an the presence of VDAC has been PubMed Scopus Google in and with the of VDAC different the plasma membrane the and the The of a to for its to the plasma of the outer mitochondrial has the to its localization J. PubMed Scopus Google Scholar). However, the of an in the a of VDAC with an N-terminal leader sequence for its to the plasma membrane PubMed Scopus Google Scholar). The presence of a in C1300 cells was by using primers specific to the the leader sequence of the was identified and to be by of suggesting the presence of VDAC in the plasma membrane has been its from membrane lipid rafts and functional in J. Full Text Full Text PDF PubMed Scopus Google Scholar). VDAC was in membrane in C1300 using markers for lipid were the of to with lipid rafts and a in lipid rafts with membrane J. Full Text Full Text PDF PubMed Scopus Google Full Text Full Text PDF PubMed Scopus Google Scholar). of C1300 cells with and in and in or toxin to in and an of and VDAC the plasma membrane VDAC was with caveolin-1. suggesting the presence of VDAC the plasma and to cell types J. Full Text Full Text PDF PubMed Scopus Google VDAC in C1300 cells to lipid has been in cells from cells PubMed Scopus Google its localization with the of cell in the of to the in with C1300 neuroblastoma cells. The of of VDAC with in C1300 cells be to the of the cells the presence of the the plasma membrane. However, using C1300 by the of not the localization of or using antibodies against in cells be to the presence of VDAC in of C1300 of VDAC to membrane and C1300 cells were and with (VDAC) and immunostaining with and and the of of VDAC and caveolin-1. with and of for and VDAC were by of different large conductance chloride Maxi Cl– has been recorded under both and whole-cell from C1300 cells to antiestrogens and J. Scopus Google Scholar). The of Maxi Cl– channels to the of antiestrogens with an plasma membrane and the of J. Scopus Google Scholar). The plasma membrane Maxi Cl– channel of the electrophysiological and of the mitochondrial VDAC Scopus Google that VDAC be the molecular correlate of Maxi Cl– channels PubMed Scopus Google Scholar). This hypothesis from recording of Maxi Cl– channels from cells VDAC PubMed Scopus Google PubMed Scopus Google and their functional with VDAC antibodies PubMed Scopus Google Scholar). However, be the that cells for the channel of the of a of VDAC and Maxi Cl– channel in cells. to the antiestrogen-activated Maxi Cl– current in C1300 cells. that C1300 cells with antisense against the of the The oligonucleotide with the leader sequence required for VDAC to the plasma membrane PubMed Scopus Google Scholar). The antisense oligonucleotide was directed against an The of an a for the of cells. of cells with or VDAC in cells were by with the and and the of the by the of the in and the in and the in a be the and VDAC in those cells with antisense and but not in those cells with demonstrated that the antisense oligonucleotide reduced VDAC to the of Maxi Cl– antisense of C1300 cells. VDAC of C1300 cells with antisense against in the cells in of the cells. VDAC of C1300 cells with antisense in the cells in of the cells. of VDAC in C1300 cells with Maxi Cl– were recorded under and following the of to the in cells with antisense or antisense Transfection with the reduced the of the Maxi Cl– currents, with antisense not the channel The Maxi Cl– channel recorded from and cells to in We by and that VDAC was not by the of C1300 cells with antiestrogens not antisense of Maxi Cl– whole-cell Cl– recorded from a C1300 cell with and the of to the whole-cell Cl– recorded from a C1300 cell with and the of to the The cells for of were those the of current under the for for and for Cl– channels be following the excision of the membrane the channel (1Blatz A.L. Magleby K.L. Biophys. J. 1983; 43: 237-241Abstract Full Text PDF PubMed Scopus (191) Google J. Scopus Google Scholar). This was to the Maxi Cl– channel in cells to excised channel from cells or cells the Maxi Cl– channel of were The was to the whole-cell with the from channel was The of Maxi Cl– channels in the excised patches with in the of VDAC Maxi Cl– channel was in of patches from cells but in of patches from in the channel conductance in and in or the not However, that the whole-cell the of channels in the plasma membrane was the Maxi Cl– channel was reduced in of the by or membrane VDAC by to the plasma membrane Maxi Cl– channel was by from cells with in the presence or of of the Maxi Cl– of C1300 antisense of Maxi Cl– from a cell and a cell were and from a of of whole-cell Maxi Cl– recorded from C1300 cells under in the presence of and following the of anion channels membrane that a to and to have been identified in the mitochondrial outer a for the of J. Full Text Full Text PDF PubMed Scopus Google and PubMed Scopus Google have been in the mitochondrial to PubMed Scopus Google J. PubMed Scopus Google Scholar). their mitochondrial the presence of VDAC in the plasma membrane of different cell types PubMed Scopus Google J. PubMed Scopus Google PubMed Scopus Google J. Full Text Full Text PDF PubMed Scopus Google Scholar). the in to the of VDAC with membrane lipid rafts of C1300 neuroblastoma have the first molecular VDAC with plasma membrane Maxi Cl– channels by The of the molecular nature of the Maxi Cl– channel to its by and antiestrogens a the of and a of the with the channel to the of Maxi channels by PubMed Scopus Google J. PubMed Scopus Google Scholar). Chloride channels of large conductance were first identified in excised patches from the plasma membrane of skeletal muscle 20 years ago (1Blatz A.L. Magleby K.L. Biophys. J. 1983; 43: 237-241Abstract Full Text PDF PubMed Scopus (191) Google Scholar). The search for their functional and molecular has with the different with their the the of cell J. PubMed Scopus Google J. Full Text PDF PubMed Google and PubMed Scopus Google J. PubMed Scopus Google Scholar). This channel has been by antiestrogens J. PubMed Scopus Google J. PubMed Scopus Google J. Scopus Google PubMed Scopus Google and by J. PubMed Scopus Google Scholar). The molecular of Maxi Cl– channels Maxi Cl– large conductance voltage-dependent chloride Maxi large conductance toxin β voltage-dependent anion plasma membrane reverse Maxi Cl– large conductance voltage-dependent chloride Maxi large conductance toxin β voltage-dependent anion plasma membrane reverse has been to The that Maxi Cl– channel electrophysiological properties resemble those of the mitochondrial voltage-dependent anion channel (VDAC) Scopus Google to that the channels were and the J. PubMed Scopus Google PubMed Scopus Google Scholar). This hypothesis was suggesting the presence of VDAC the plasma membrane J. PubMed Scopus Google J. PubMed Scopus Google but was by J. PubMed Scopus Google Scholar). The suggesting the presence of VDAC in has from the of a VDAC that a leader sequence for its to the plasma membrane PubMed Scopus Google and the presence of VDAC in J. Full Text Full Text PDF PubMed Scopus Google Scholar). the have the presence of VDAC in the plasma membrane of C1300 neuroblastoma cells its to the Maxi Cl– by antiestrogens in cells. and neuroblastoma from the were J. Scopus Google Scholar). cells were or with for were from was from of and cells were with of and the of cell and was The cell was to a for and for was from the and for of The was and in membrane were using a J. 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PubMed Scopus Google Scholar). was from C1300 cells using the to the was to We the following for of PubMed Scopus Google and were a a of and cells were and the of an were recorded using the or whole-cell J. Scopus Google Scholar). The was for an and and were with a and and were to the for whole-cell were in a was the the of the membrane in were using a or were and patches were and for to both and were and were and for from to in and of to the were the different were by of by the for a of presence of VDAC C1300 cells was by The monoclonal in was against an N-terminal peptide PubMed Scopus Google Scholar). a from a membrane or of C1300 cells. of the for was from both the of with cellular of the membrane with was with antibodies against the mitochondrial or that membrane for the mitochondrial but for the of the an the presence of VDAC has been PubMed Scopus Google in and with the of VDAC different the plasma membrane the and the The of a to for its to the plasma of the outer mitochondrial has the to its localization J. PubMed Scopus Google Scholar). However, the of an in the a of VDAC with an N-terminal leader sequence for its to the plasma membrane PubMed Scopus Google Scholar). The presence of a in C1300 cells was by using primers specific to the the leader sequence of the was identified and to be by of suggesting the presence of VDAC in the plasma membrane has been its from membrane lipid rafts and functional in J. Full Text Full Text PDF PubMed Scopus Google Scholar). VDAC was in membrane in C1300 using markers for lipid were the of to with lipid rafts and a in lipid rafts with membrane J. Full Text Full Text PDF PubMed Scopus Google Full Text Full Text PDF PubMed Scopus Google Scholar). of C1300 cells with and in and in or toxin to in and an of and VDAC the plasma membrane VDAC was with caveolin-1. suggesting the presence of VDAC the plasma and to cell types J. Full Text Full Text PDF PubMed Scopus Google VDAC in C1300 cells to lipid has been in cells from cells PubMed Scopus Google its localization with the of cell in the of to the in with C1300 neuroblastoma cells. The of of VDAC with in C1300 cells be to the of the cells the presence of the the plasma membrane. However, using C1300 by the of not the localization of or using antibodies against in cells be to the presence of VDAC in of C1300 large conductance chloride Maxi Cl– has been recorded under both and whole-cell from C1300 cells to antiestrogens and J. Scopus Google Scholar). The of Maxi Cl– channels to the of antiestrogens with an plasma membrane and the of J. Scopus Google Scholar). The plasma membrane Maxi Cl– channel of the electrophysiological and of the mitochondrial VDAC Scopus Google that VDAC be the molecular correlate of Maxi Cl– channels PubMed Scopus Google Scholar). This hypothesis from recording of Maxi Cl– channels from cells VDAC PubMed Scopus Google PubMed Scopus Google and their functional with VDAC antibodies PubMed Scopus Google Scholar). However, be the that cells for the channel of the of a of VDAC and Maxi Cl– channel in cells. to the antiestrogen-activated Maxi Cl– current in C1300 cells. that C1300 cells with antisense against the of the The oligonucleotide with the leader sequence required for VDAC to the plasma membrane PubMed Scopus Google Scholar). The antisense oligonucleotide was directed against an The of an a for the of cells. of cells with or VDAC in cells were by with the and and the of the by the of the in and the in and the in a be the and VDAC in those cells with antisense and but not in those cells with demonstrated that the antisense oligonucleotide reduced VDAC to the of Maxi Cl– antisense of C1300 cells. VDAC of C1300 cells with antisense against in the cells in of the cells. VDAC of C1300 cells with antisense in the cells in of the cells. of VDAC in C1300 cells with Maxi Cl– were recorded under and following the of to the in cells with antisense or antisense Transfection with the reduced the of the Maxi Cl– currents, with antisense not the channel The Maxi Cl– channel recorded from and cells to in We by and that VDAC was not by the of C1300 cells with antiestrogens not antisense of Maxi Cl– whole-cell Cl– recorded from a C1300 cell with and the of to the whole-cell Cl– recorded from a C1300 cell with and the of to the The cells for of were those the of current under the for for and for Cl– channels be following the excision of the membrane the channel (1Blatz A.L. Magleby K.L. Biophys. J. 1983; 43: 237-241Abstract Full Text PDF PubMed Scopus (191) Google J. Scopus Google Scholar). This was to the Maxi Cl– channel in cells to excised channel from cells or cells the Maxi Cl– channel of were The was to the whole-cell with the from channel was The of Maxi Cl– channels in the excised patches with in the of VDAC Maxi Cl– channel was in of patches from cells but in of patches from in the channel conductance in and in or the not However, that the whole-cell the of channels in the plasma membrane was the Maxi Cl– channel was reduced in of the by or membrane VDAC by to the plasma membrane Maxi Cl– channel was by from cells with in the presence or of of the Maxi Cl– of C1300 antisense of Maxi Cl– from a cell and a cell were and from a of of whole-cell Maxi Cl– recorded from C1300 cells under in the presence of and following the of anion channels membrane that a to and to have been identified in the mitochondrial outer a for the of J. Full Text Full Text PDF PubMed Scopus Google and PubMed Scopus Google have been in the mitochondrial to PubMed Scopus Google J. PubMed Scopus Google Scholar). their mitochondrial the presence of VDAC in the plasma membrane of different cell types PubMed Scopus Google J. PubMed Scopus Google PubMed Scopus Google J. Full Text Full Text PDF PubMed Scopus Google Scholar). the in to the of VDAC with membrane lipid rafts of C1300 neuroblastoma have the first molecular VDAC with plasma membrane Maxi Cl– channels by The of the molecular nature of the Maxi Cl– channel to its by and antiestrogens a the of and a of the with the channel to the of Maxi channels by PubMed Scopus Google J. PubMed Scopus Google Scholar). The presence of VDAC C1300 cells was by The monoclonal in was against an N-terminal peptide PubMed Scopus Google Scholar). a from a membrane or of C1300 cells. of the for was from both the of with cellular of the membrane with was with antibodies against the mitochondrial or that membrane for the mitochondrial but for the of the an the presence of VDAC has been PubMed Scopus Google Scholar). The in and with the of VDAC different the plasma membrane the and the The of a to for its to the plasma of the outer mitochondrial has the to its localization J. PubMed Scopus Google Scholar). However, the of an in the a of VDAC with an N-terminal leader sequence for its to the plasma membrane PubMed Scopus Google Scholar). The presence of a in C1300 cells was by using primers specific to the the leader sequence of the was identified and to be by of suggesting the presence of VDAC in the plasma membrane has been its from membrane lipid rafts and functional in J. Full Text Full Text PDF PubMed Scopus Google Scholar). VDAC was in membrane in C1300 using markers for lipid were the of to with lipid rafts and a in lipid rafts with membrane J. Full Text Full Text PDF PubMed Scopus Google Full Text Full Text PDF PubMed Scopus Google Scholar). of C1300 cells with and in and in or toxin to in and an of and VDAC the plasma membrane VDAC was with caveolin-1. suggesting the presence of VDAC the plasma and to cell types J. Full Text Full Text PDF PubMed Scopus Google VDAC in C1300 cells to lipid has been in cells from cells PubMed Scopus Google its localization with the of cell in the of to the in with C1300 neuroblastoma cells. The of of VDAC with in C1300 cells be to the of the cells the presence of the the plasma membrane. However, using C1300 by the of not the localization of or using antibodies against in cells be to the presence of VDAC in of C1300 cells. The large conductance chloride Maxi Cl– has been recorded under both and whole-cell from C1300 cells to antiestrogens and J. Scopus Google Scholar). The of Maxi Cl– channels to the of antiestrogens with an plasma membrane and the of J. Scopus Google Scholar). The plasma membrane Maxi Cl– channel of the electrophysiological and of the mitochondrial VDAC Scopus Google that VDAC be the molecular correlate of Maxi Cl– channels PubMed Scopus Google Scholar). This hypothesis from recording of Maxi Cl– channels from cells VDAC PubMed Scopus Google PubMed Scopus Google and their functional with VDAC antibodies PubMed Scopus Google Scholar). However, be the that cells for the channel of the of a of VDAC and Maxi Cl– channel in cells. to the antiestrogen-activated Maxi Cl– current in C1300 cells. that C1300 cells with antisense against the of the The oligonucleotide with the leader sequence required for VDAC to the plasma membrane PubMed Scopus Google Scholar). The antisense oligonucleotide was directed against an The of an a for the of cells. of cells with or VDAC in cells were by with the and and the of the by the of the in and the in and the in a be the and VDAC in those cells with antisense and but not in those cells with demonstrated that the antisense oligonucleotide reduced VDAC to the of Maxi Cl– Maxi Cl– were recorded under and following the of to the in cells with antisense or antisense Transfection with the reduced the of the Maxi Cl– currents, with antisense not the channel The Maxi Cl– channel recorded from and cells to in We by and that VDAC was not by the of C1300 cells with antiestrogens not Maxi Cl– channels be following the excision of the membrane the channel (1Blatz A.L. Magleby K.L. Biophys. J. 1983; 43: 237-241Abstract Full Text PDF PubMed Scopus (191) Google J. Scopus Google Scholar). This was to the Maxi Cl– channel in cells to excised channel from cells or cells the Maxi Cl– channel of were The was to the whole-cell with the from channel was The of Maxi Cl– channels in the excised patches with in the of VDAC Maxi Cl– channel was in of patches from cells but in of patches from in the channel conductance in and in or the not However, that the whole-cell the of channels in the plasma membrane was the Maxi Cl– channel was reduced in of the by or membrane VDAC by to the plasma membrane Maxi Cl– channel was by from cells with in the presence or of of the Maxi Cl– of C1300 cells. anion channels membrane that a to and to have been identified in the mitochondrial outer a for the of J. Full Text Full Text PDF PubMed Scopus Google and PubMed Scopus Google have been in the mitochondrial to PubMed Scopus Google J. PubMed Scopus Google Scholar). their mitochondrial the presence of VDAC in the plasma membrane of different cell types PubMed Scopus Google J. PubMed Scopus Google PubMed Scopus Google J. Full Text Full Text PDF PubMed Scopus Google Scholar). the in to the of VDAC with membrane lipid rafts of C1300 neuroblastoma have the first molecular VDAC with plasma membrane Maxi Cl– channels by The of the molecular nature of the Maxi Cl– channel to its by and antiestrogens a the of and a of the with the channel to the of Maxi channels by PubMed Scopus Google J. PubMed Scopus Google Scholar). We for the

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