Mechanism of cardiac Na+-Ca2+ exchange current stimulation by MgATP: Possible involvement of aminophospholipid translocase

D. W. Hilgemann, A. Collins

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Abstract

1. The sensitivity of outward Na+-Ca2+ exchange current to charged amphiphiles and phospholipids was tested in giant excised inside-out membrane patches from guinea-pig and rabbit myocytes. 2. Screening of membrane surface potentials with dimethonium (10 mM), spermine (200 μM) and spermidine (100 μM) was without effect, while the positively charged ionic detergents hexadecyltrimethylammonium and dodecyltrimethylammonium strongly inhibited steady-state outward exchange current (0.1-10 μM). 3. Interventions expected to increase negative surface charge included treatment of the cytoplasmic surface with phospholipase D, application of dodecylsulphate. (1-10 μM), application of the short-chain phoshatidylserine derivative, dicapryl phosphatidylserine (C10PS) and inclusion of 1-3% phosphatidylserine in the hydrocarbon mixture used to coat electrodes. Each intervention strongly stimulated Na+-Ca2+ exchange current in a similar way to MgATP, reducing the fractional decay of outward exchange current (inactivation) during application of high cytoplasmic sodium. 4. The MgATP stimulated exchange current was inhibited with a K(i) of approximately 1 μM by pentalysine, which is known to associate with phosphatidylserine head groups. After deregulation of the exchanger by chymotrypsin, pentalysine wax without effect. 5. Inclusion in the pipette of 0.2 mM-pyridyldithioethylamine (an oxidizing inhibitor of aminophospholipid translocase) abolished stimulation of outward exchange current by MgATP without inhibiting basal outward exchange current or sodium pump current. 6. Application to the cytoplasmic side of 1.5 mM-diamide, which reportedly decreases membrane phospholipid asymmetry, apparently reversed the effect of MgATP. After treatment with diamide and subsequently with dithiothreitol, Na+Ca2+ exchange current was again stimulated by MgATP. Diamide was without effect when secondary exchange regulation had been previously removed by chymotrypsin. 7. Potassium current carried by the surface potential sensitive ionophore, nonactin, was stimulated by MgATP when extracellular surface charge had been neutralized. The effect was largest (40-90%) when low ionic strength cytoplasmic solutions were employed, consistent with an increase of negative membrane charge on the cytoplasmic side during MgATP application. 8. Potassium current carried by nonactin was inhibited by MgATP when cytoplasmic surface charge had been neutralized and extracellular solutions of low ionic strength were employed, consistent with a decrease of negative membrane charge on the extracellular side. 9. These results indicate that the stimulatory effect of MgATP on Na+-Ca2+ exchange current could involve changes of charged membrane lipids, that the effect probably involves a transmembrane, oxidation-sensitive protein, that pentalysine-sensitive sites are involved, that phosphatidylserine mimics the effect of MgATP, and that the effect extends to a simple surface potential-sensitive ionophore. All results are consistent with the activation by MgATP of an aminophospholipid translocase as the underlying mechanism.

Original languageEnglish (US)
Pages (from-to)59-82
Number of pages24
JournalJournal of Physiology
Volume454
StatePublished - 1992

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Phospholipid Transfer Proteins
Adenosine Triphosphate
pentalysine
Phosphatidylserines
Diamide
Membranes
Ionophores
Chymotrypsin
Osmolar Concentration
Phospholipids
Potassium
Sodium-Potassium-Exchanging ATPase
Phospholipase D
Spermidine
Spermine
Waxes
Dithiothreitol
Membrane Lipids
Hydrocarbons
Detergents

ASJC Scopus subject areas

  • Physiology

Cite this

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title = "Mechanism of cardiac Na+-Ca2+ exchange current stimulation by MgATP: Possible involvement of aminophospholipid translocase",
abstract = "1. The sensitivity of outward Na+-Ca2+ exchange current to charged amphiphiles and phospholipids was tested in giant excised inside-out membrane patches from guinea-pig and rabbit myocytes. 2. Screening of membrane surface potentials with dimethonium (10 mM), spermine (200 μM) and spermidine (100 μM) was without effect, while the positively charged ionic detergents hexadecyltrimethylammonium and dodecyltrimethylammonium strongly inhibited steady-state outward exchange current (0.1-10 μM). 3. Interventions expected to increase negative surface charge included treatment of the cytoplasmic surface with phospholipase D, application of dodecylsulphate. (1-10 μM), application of the short-chain phoshatidylserine derivative, dicapryl phosphatidylserine (C10PS) and inclusion of 1-3{\%} phosphatidylserine in the hydrocarbon mixture used to coat electrodes. Each intervention strongly stimulated Na+-Ca2+ exchange current in a similar way to MgATP, reducing the fractional decay of outward exchange current (inactivation) during application of high cytoplasmic sodium. 4. The MgATP stimulated exchange current was inhibited with a K(i) of approximately 1 μM by pentalysine, which is known to associate with phosphatidylserine head groups. After deregulation of the exchanger by chymotrypsin, pentalysine wax without effect. 5. Inclusion in the pipette of 0.2 mM-pyridyldithioethylamine (an oxidizing inhibitor of aminophospholipid translocase) abolished stimulation of outward exchange current by MgATP without inhibiting basal outward exchange current or sodium pump current. 6. Application to the cytoplasmic side of 1.5 mM-diamide, which reportedly decreases membrane phospholipid asymmetry, apparently reversed the effect of MgATP. After treatment with diamide and subsequently with dithiothreitol, Na+Ca2+ exchange current was again stimulated by MgATP. Diamide was without effect when secondary exchange regulation had been previously removed by chymotrypsin. 7. Potassium current carried by the surface potential sensitive ionophore, nonactin, was stimulated by MgATP when extracellular surface charge had been neutralized. The effect was largest (40-90{\%}) when low ionic strength cytoplasmic solutions were employed, consistent with an increase of negative membrane charge on the cytoplasmic side during MgATP application. 8. Potassium current carried by nonactin was inhibited by MgATP when cytoplasmic surface charge had been neutralized and extracellular solutions of low ionic strength were employed, consistent with a decrease of negative membrane charge on the extracellular side. 9. These results indicate that the stimulatory effect of MgATP on Na+-Ca2+ exchange current could involve changes of charged membrane lipids, that the effect probably involves a transmembrane, oxidation-sensitive protein, that pentalysine-sensitive sites are involved, that phosphatidylserine mimics the effect of MgATP, and that the effect extends to a simple surface potential-sensitive ionophore. All results are consistent with the activation by MgATP of an aminophospholipid translocase as the underlying mechanism.",
author = "Hilgemann, {D. W.} and A. Collins",
year = "1992",
language = "English (US)",
volume = "454",
pages = "59--82",
journal = "Journal of Physiology",
issn = "0022-3751",
publisher = "Wiley-Blackwell",

}

TY - JOUR

T1 - Mechanism of cardiac Na+-Ca2+ exchange current stimulation by MgATP

T2 - Possible involvement of aminophospholipid translocase

AU - Hilgemann, D. W.

AU - Collins, A.

PY - 1992

Y1 - 1992

N2 - 1. The sensitivity of outward Na+-Ca2+ exchange current to charged amphiphiles and phospholipids was tested in giant excised inside-out membrane patches from guinea-pig and rabbit myocytes. 2. Screening of membrane surface potentials with dimethonium (10 mM), spermine (200 μM) and spermidine (100 μM) was without effect, while the positively charged ionic detergents hexadecyltrimethylammonium and dodecyltrimethylammonium strongly inhibited steady-state outward exchange current (0.1-10 μM). 3. Interventions expected to increase negative surface charge included treatment of the cytoplasmic surface with phospholipase D, application of dodecylsulphate. (1-10 μM), application of the short-chain phoshatidylserine derivative, dicapryl phosphatidylserine (C10PS) and inclusion of 1-3% phosphatidylserine in the hydrocarbon mixture used to coat electrodes. Each intervention strongly stimulated Na+-Ca2+ exchange current in a similar way to MgATP, reducing the fractional decay of outward exchange current (inactivation) during application of high cytoplasmic sodium. 4. The MgATP stimulated exchange current was inhibited with a K(i) of approximately 1 μM by pentalysine, which is known to associate with phosphatidylserine head groups. After deregulation of the exchanger by chymotrypsin, pentalysine wax without effect. 5. Inclusion in the pipette of 0.2 mM-pyridyldithioethylamine (an oxidizing inhibitor of aminophospholipid translocase) abolished stimulation of outward exchange current by MgATP without inhibiting basal outward exchange current or sodium pump current. 6. Application to the cytoplasmic side of 1.5 mM-diamide, which reportedly decreases membrane phospholipid asymmetry, apparently reversed the effect of MgATP. After treatment with diamide and subsequently with dithiothreitol, Na+Ca2+ exchange current was again stimulated by MgATP. Diamide was without effect when secondary exchange regulation had been previously removed by chymotrypsin. 7. Potassium current carried by the surface potential sensitive ionophore, nonactin, was stimulated by MgATP when extracellular surface charge had been neutralized. The effect was largest (40-90%) when low ionic strength cytoplasmic solutions were employed, consistent with an increase of negative membrane charge on the cytoplasmic side during MgATP application. 8. Potassium current carried by nonactin was inhibited by MgATP when cytoplasmic surface charge had been neutralized and extracellular solutions of low ionic strength were employed, consistent with a decrease of negative membrane charge on the extracellular side. 9. These results indicate that the stimulatory effect of MgATP on Na+-Ca2+ exchange current could involve changes of charged membrane lipids, that the effect probably involves a transmembrane, oxidation-sensitive protein, that pentalysine-sensitive sites are involved, that phosphatidylserine mimics the effect of MgATP, and that the effect extends to a simple surface potential-sensitive ionophore. All results are consistent with the activation by MgATP of an aminophospholipid translocase as the underlying mechanism.

AB - 1. The sensitivity of outward Na+-Ca2+ exchange current to charged amphiphiles and phospholipids was tested in giant excised inside-out membrane patches from guinea-pig and rabbit myocytes. 2. Screening of membrane surface potentials with dimethonium (10 mM), spermine (200 μM) and spermidine (100 μM) was without effect, while the positively charged ionic detergents hexadecyltrimethylammonium and dodecyltrimethylammonium strongly inhibited steady-state outward exchange current (0.1-10 μM). 3. Interventions expected to increase negative surface charge included treatment of the cytoplasmic surface with phospholipase D, application of dodecylsulphate. (1-10 μM), application of the short-chain phoshatidylserine derivative, dicapryl phosphatidylserine (C10PS) and inclusion of 1-3% phosphatidylserine in the hydrocarbon mixture used to coat electrodes. Each intervention strongly stimulated Na+-Ca2+ exchange current in a similar way to MgATP, reducing the fractional decay of outward exchange current (inactivation) during application of high cytoplasmic sodium. 4. The MgATP stimulated exchange current was inhibited with a K(i) of approximately 1 μM by pentalysine, which is known to associate with phosphatidylserine head groups. After deregulation of the exchanger by chymotrypsin, pentalysine wax without effect. 5. Inclusion in the pipette of 0.2 mM-pyridyldithioethylamine (an oxidizing inhibitor of aminophospholipid translocase) abolished stimulation of outward exchange current by MgATP without inhibiting basal outward exchange current or sodium pump current. 6. Application to the cytoplasmic side of 1.5 mM-diamide, which reportedly decreases membrane phospholipid asymmetry, apparently reversed the effect of MgATP. After treatment with diamide and subsequently with dithiothreitol, Na+Ca2+ exchange current was again stimulated by MgATP. Diamide was without effect when secondary exchange regulation had been previously removed by chymotrypsin. 7. Potassium current carried by the surface potential sensitive ionophore, nonactin, was stimulated by MgATP when extracellular surface charge had been neutralized. The effect was largest (40-90%) when low ionic strength cytoplasmic solutions were employed, consistent with an increase of negative membrane charge on the cytoplasmic side during MgATP application. 8. Potassium current carried by nonactin was inhibited by MgATP when cytoplasmic surface charge had been neutralized and extracellular solutions of low ionic strength were employed, consistent with a decrease of negative membrane charge on the extracellular side. 9. These results indicate that the stimulatory effect of MgATP on Na+-Ca2+ exchange current could involve changes of charged membrane lipids, that the effect probably involves a transmembrane, oxidation-sensitive protein, that pentalysine-sensitive sites are involved, that phosphatidylserine mimics the effect of MgATP, and that the effect extends to a simple surface potential-sensitive ionophore. All results are consistent with the activation by MgATP of an aminophospholipid translocase as the underlying mechanism.

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