Inactivation of outward Na+-Ca2+ exchange current in guinea-pig ventricular myocytes

S. Matsuoka, D. W. Hilgemann

Research output: Contribution to journalArticle

61 Citations (Scopus)

Abstract

1. Outward Na+-Ca2+ exchange currents were measured in freshly dissociated guinea-pig myocytes to probe in intact cells the functional status of exchanger inactivation reactions, described previously in giant excised cardiac membrane patches. 2. When the cytoplasmic (pipette) solution contained 40 mM Na+ and 0.1 μM free Ca2+ (50 mM EGTA), the outward exchange current activated by extracellular Ca2+ decayed with time (time constant, 13.1 ± 2.6s; n=6), and an inward current transient was observed upon removal of extracellular Ca2+. Both the current decay and the subsequent inward current transient were remarkably diminished with a saturating (100 mM) pipette :Na+ concentration. 3. With 100 mM cytoplasmic Na+ and 140 mM extracellular Na+, a significant fraction of the exchanger population is predicted to be in an inactive state. Intracellular application of 2 mg ml-1 chymotrypsin and 5 μM sodium tetradecylsulphate, both of which decrease Na+-dependent inactivation in giant membrane patches, increased the outward exchange current by about 160-170%, suggesting that about 60-70% of exchangers might be inactivated. 4. With 100 mM cytoplasmic Na+ and no extracellular Na+ (replaced with 140 mM Li+), application of extracellular Ca2+ was predicted to reorient exchanger binding sites from the extracellular side to the cytoplasmic side and thereby favour inactivation. During such protocols, the outward exchange current decayed by 60-80% when activated by extracellular Ca2+. The current decayed similarly when extracellular Ca2+ and Na+ were applied together, whereby current magnitudes were about 3-fold smaller. 5. The decay of outward exchange current usually followed a biexponential time course (5.8 ± 3.5 and 27.3 ± 16.3 s, means ± S.D., n = 11). Intracellular application of 0.5-2 mg-1 trypsin attenuated the fast component more than the slow component, suggesting that the fast component reflects an inactivation process. 6. Current-voltage (I-V) relations of the outward exchange current became less steep during the inactivation protocols, but this flattening could not be correlated with inactivation. 7. Replacement of extracellular Li+ with N-methyl-D-glucamine (NMG), tetraethylammonium (TEA), sucrose or Cs+ resulted in a flattening of I-V relations and a decrease of the outward exchange current amplitude by approximately 3-fold, but the kinetics and extent of inactivation were not remarkably changed. Thus, the mechanism of inactivation appears to be independent of the mechanism(s) of activation by extracellular monovalent cations. 8. Preapplication of extracellular Na+ was predicted to orient binding sites to the cytoplasmic side and thereby induce inactivation in the absence of extracellular Ca2+. As predicted, exchange currents subsequently activated by extracellular Ca2+ were small and showed a small recovery phase, rather than current decay. 9. A consecutive model of the exchange cycle with inactivation taking place from fully Na+-loaded binding sites with cytoplasmic orientation described well most of the results on inactivation in whole myocytes. 10. It is concluded that, as in giant cardiac membrane patches, Na+-Ca2+ exchange function in intact myocytes is modulated by secondary inactivation reactions.

Original languageEnglish (US)
Pages (from-to)443-458
Number of pages16
JournalJournal of Physiology
Volume476
Issue number3
StatePublished - 1994

Fingerprint

Muscle Cells
Guinea Pigs
Binding Sites
Membranes
Monovalent Cations
Tetraethylammonium
Egtazic Acid
Chymotrypsin
Trypsin
Sucrose
Sodium
Population
caN protocol

ASJC Scopus subject areas

  • Physiology

Cite this

Inactivation of outward Na+-Ca2+ exchange current in guinea-pig ventricular myocytes. / Matsuoka, S.; Hilgemann, D. W.

In: Journal of Physiology, Vol. 476, No. 3, 1994, p. 443-458.

Research output: Contribution to journalArticle

@article{cb3dae5be6744535b9461632d6e2f201,
title = "Inactivation of outward Na+-Ca2+ exchange current in guinea-pig ventricular myocytes",
abstract = "1. Outward Na+-Ca2+ exchange currents were measured in freshly dissociated guinea-pig myocytes to probe in intact cells the functional status of exchanger inactivation reactions, described previously in giant excised cardiac membrane patches. 2. When the cytoplasmic (pipette) solution contained 40 mM Na+ and 0.1 μM free Ca2+ (50 mM EGTA), the outward exchange current activated by extracellular Ca2+ decayed with time (time constant, 13.1 ± 2.6s; n=6), and an inward current transient was observed upon removal of extracellular Ca2+. Both the current decay and the subsequent inward current transient were remarkably diminished with a saturating (100 mM) pipette :Na+ concentration. 3. With 100 mM cytoplasmic Na+ and 140 mM extracellular Na+, a significant fraction of the exchanger population is predicted to be in an inactive state. Intracellular application of 2 mg ml-1 chymotrypsin and 5 μM sodium tetradecylsulphate, both of which decrease Na+-dependent inactivation in giant membrane patches, increased the outward exchange current by about 160-170{\%}, suggesting that about 60-70{\%} of exchangers might be inactivated. 4. With 100 mM cytoplasmic Na+ and no extracellular Na+ (replaced with 140 mM Li+), application of extracellular Ca2+ was predicted to reorient exchanger binding sites from the extracellular side to the cytoplasmic side and thereby favour inactivation. During such protocols, the outward exchange current decayed by 60-80{\%} when activated by extracellular Ca2+. The current decayed similarly when extracellular Ca2+ and Na+ were applied together, whereby current magnitudes were about 3-fold smaller. 5. The decay of outward exchange current usually followed a biexponential time course (5.8 ± 3.5 and 27.3 ± 16.3 s, means ± S.D., n = 11). Intracellular application of 0.5-2 mg-1 trypsin attenuated the fast component more than the slow component, suggesting that the fast component reflects an inactivation process. 6. Current-voltage (I-V) relations of the outward exchange current became less steep during the inactivation protocols, but this flattening could not be correlated with inactivation. 7. Replacement of extracellular Li+ with N-methyl-D-glucamine (NMG), tetraethylammonium (TEA), sucrose or Cs+ resulted in a flattening of I-V relations and a decrease of the outward exchange current amplitude by approximately 3-fold, but the kinetics and extent of inactivation were not remarkably changed. Thus, the mechanism of inactivation appears to be independent of the mechanism(s) of activation by extracellular monovalent cations. 8. Preapplication of extracellular Na+ was predicted to orient binding sites to the cytoplasmic side and thereby induce inactivation in the absence of extracellular Ca2+. As predicted, exchange currents subsequently activated by extracellular Ca2+ were small and showed a small recovery phase, rather than current decay. 9. A consecutive model of the exchange cycle with inactivation taking place from fully Na+-loaded binding sites with cytoplasmic orientation described well most of the results on inactivation in whole myocytes. 10. It is concluded that, as in giant cardiac membrane patches, Na+-Ca2+ exchange function in intact myocytes is modulated by secondary inactivation reactions.",
author = "S. Matsuoka and Hilgemann, {D. W.}",
year = "1994",
language = "English (US)",
volume = "476",
pages = "443--458",
journal = "Journal of Physiology",
issn = "0022-3751",
publisher = "Wiley-Blackwell",
number = "3",

}

TY - JOUR

T1 - Inactivation of outward Na+-Ca2+ exchange current in guinea-pig ventricular myocytes

AU - Matsuoka, S.

AU - Hilgemann, D. W.

PY - 1994

Y1 - 1994

N2 - 1. Outward Na+-Ca2+ exchange currents were measured in freshly dissociated guinea-pig myocytes to probe in intact cells the functional status of exchanger inactivation reactions, described previously in giant excised cardiac membrane patches. 2. When the cytoplasmic (pipette) solution contained 40 mM Na+ and 0.1 μM free Ca2+ (50 mM EGTA), the outward exchange current activated by extracellular Ca2+ decayed with time (time constant, 13.1 ± 2.6s; n=6), and an inward current transient was observed upon removal of extracellular Ca2+. Both the current decay and the subsequent inward current transient were remarkably diminished with a saturating (100 mM) pipette :Na+ concentration. 3. With 100 mM cytoplasmic Na+ and 140 mM extracellular Na+, a significant fraction of the exchanger population is predicted to be in an inactive state. Intracellular application of 2 mg ml-1 chymotrypsin and 5 μM sodium tetradecylsulphate, both of which decrease Na+-dependent inactivation in giant membrane patches, increased the outward exchange current by about 160-170%, suggesting that about 60-70% of exchangers might be inactivated. 4. With 100 mM cytoplasmic Na+ and no extracellular Na+ (replaced with 140 mM Li+), application of extracellular Ca2+ was predicted to reorient exchanger binding sites from the extracellular side to the cytoplasmic side and thereby favour inactivation. During such protocols, the outward exchange current decayed by 60-80% when activated by extracellular Ca2+. The current decayed similarly when extracellular Ca2+ and Na+ were applied together, whereby current magnitudes were about 3-fold smaller. 5. The decay of outward exchange current usually followed a biexponential time course (5.8 ± 3.5 and 27.3 ± 16.3 s, means ± S.D., n = 11). Intracellular application of 0.5-2 mg-1 trypsin attenuated the fast component more than the slow component, suggesting that the fast component reflects an inactivation process. 6. Current-voltage (I-V) relations of the outward exchange current became less steep during the inactivation protocols, but this flattening could not be correlated with inactivation. 7. Replacement of extracellular Li+ with N-methyl-D-glucamine (NMG), tetraethylammonium (TEA), sucrose or Cs+ resulted in a flattening of I-V relations and a decrease of the outward exchange current amplitude by approximately 3-fold, but the kinetics and extent of inactivation were not remarkably changed. Thus, the mechanism of inactivation appears to be independent of the mechanism(s) of activation by extracellular monovalent cations. 8. Preapplication of extracellular Na+ was predicted to orient binding sites to the cytoplasmic side and thereby induce inactivation in the absence of extracellular Ca2+. As predicted, exchange currents subsequently activated by extracellular Ca2+ were small and showed a small recovery phase, rather than current decay. 9. A consecutive model of the exchange cycle with inactivation taking place from fully Na+-loaded binding sites with cytoplasmic orientation described well most of the results on inactivation in whole myocytes. 10. It is concluded that, as in giant cardiac membrane patches, Na+-Ca2+ exchange function in intact myocytes is modulated by secondary inactivation reactions.

AB - 1. Outward Na+-Ca2+ exchange currents were measured in freshly dissociated guinea-pig myocytes to probe in intact cells the functional status of exchanger inactivation reactions, described previously in giant excised cardiac membrane patches. 2. When the cytoplasmic (pipette) solution contained 40 mM Na+ and 0.1 μM free Ca2+ (50 mM EGTA), the outward exchange current activated by extracellular Ca2+ decayed with time (time constant, 13.1 ± 2.6s; n=6), and an inward current transient was observed upon removal of extracellular Ca2+. Both the current decay and the subsequent inward current transient were remarkably diminished with a saturating (100 mM) pipette :Na+ concentration. 3. With 100 mM cytoplasmic Na+ and 140 mM extracellular Na+, a significant fraction of the exchanger population is predicted to be in an inactive state. Intracellular application of 2 mg ml-1 chymotrypsin and 5 μM sodium tetradecylsulphate, both of which decrease Na+-dependent inactivation in giant membrane patches, increased the outward exchange current by about 160-170%, suggesting that about 60-70% of exchangers might be inactivated. 4. With 100 mM cytoplasmic Na+ and no extracellular Na+ (replaced with 140 mM Li+), application of extracellular Ca2+ was predicted to reorient exchanger binding sites from the extracellular side to the cytoplasmic side and thereby favour inactivation. During such protocols, the outward exchange current decayed by 60-80% when activated by extracellular Ca2+. The current decayed similarly when extracellular Ca2+ and Na+ were applied together, whereby current magnitudes were about 3-fold smaller. 5. The decay of outward exchange current usually followed a biexponential time course (5.8 ± 3.5 and 27.3 ± 16.3 s, means ± S.D., n = 11). Intracellular application of 0.5-2 mg-1 trypsin attenuated the fast component more than the slow component, suggesting that the fast component reflects an inactivation process. 6. Current-voltage (I-V) relations of the outward exchange current became less steep during the inactivation protocols, but this flattening could not be correlated with inactivation. 7. Replacement of extracellular Li+ with N-methyl-D-glucamine (NMG), tetraethylammonium (TEA), sucrose or Cs+ resulted in a flattening of I-V relations and a decrease of the outward exchange current amplitude by approximately 3-fold, but the kinetics and extent of inactivation were not remarkably changed. Thus, the mechanism of inactivation appears to be independent of the mechanism(s) of activation by extracellular monovalent cations. 8. Preapplication of extracellular Na+ was predicted to orient binding sites to the cytoplasmic side and thereby induce inactivation in the absence of extracellular Ca2+. As predicted, exchange currents subsequently activated by extracellular Ca2+ were small and showed a small recovery phase, rather than current decay. 9. A consecutive model of the exchange cycle with inactivation taking place from fully Na+-loaded binding sites with cytoplasmic orientation described well most of the results on inactivation in whole myocytes. 10. It is concluded that, as in giant cardiac membrane patches, Na+-Ca2+ exchange function in intact myocytes is modulated by secondary inactivation reactions.

UR - http://www.scopus.com/inward/record.url?scp=0028233988&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028233988&partnerID=8YFLogxK

M3 - Article

C2 - 7520059

AN - SCOPUS:0028233988

VL - 476

SP - 443

EP - 458

JO - Journal of Physiology

JF - Journal of Physiology

SN - 0022-3751

IS - 3

ER -