The giant cardiac membrane patch method

Stimulation of outward Na+-Ca2+ exchange current by MgATP

A. Collins, A. V. Somlyo, D. W. Hilgemann

Research output: Contribution to journalArticle

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Abstract

1. A giant patch method was used to study the stimulatory effect of cytoplasmic MgATP on outward Na+-Ca2+ exchange current in inside-out cardiac membrane patches (1-10 GΩ seals with 14-24 μm pipette tip diameters) excised from guinea-pig. rabbit and mouse myocytes. 2. To establish the validity of the method with respect to structure, bleb formation was examined with electron microscopy and with confocal fluorescence light microscopy. The blebs, which form as the sarcolemma detaches, excluded intracellular organelles and transverse tubules. The blebbed cells contained normal sarcomeres, sarcoplasmic reticulum, triads and diads. 3. To further establish the validity of the method for ion transport studies, measurements of Na+-K+ pump currents and charge movements are described briefly which demonstrate (i) free access to the cytoplasmic membrane side, (ii) MgATP dependence comparable to reconstituted pump (K(d), 94 μM), (iii) fast, rigorous concentration control and (iv) Na+-K+ pump densities in the range of whole-cell densities. 4. Stimulation of outward Na+Ca2+ exchange current by MgATP attenuated exchange current decay during step increments of cytoplasmic sodium, shifted the secondary activation of outward exchange currently cytoplasmic calcium to lower free calcium concentrations and, particularly in mouse cardiac sarcolemma, induced cytoplasmic calcium-independent current. 5. Upon removal of MgATP the stimulatory effect usually decayed with a t50 (half-time) of about 3 min. However, the reversal took place much more rapidly (t50, 5-20 s) in patches from individual guinea-pig and rabbit myocyte batches. When decay was rapid, secondary activation by cytoplasmic calcium was shifted to higher free cytoplasmic calcium concentrations (K(d), 10-65 μM-free calcium). 6. With repeated applications of MgATP the rate and magnitude of the stimulatory effect progressively decreased. 7. The K(d) for MgATP of the initial rate of stimulation of outward exchange current was 3 mM or greater. When decay was rapid, the steady-state dependence of exchange current on MgATP also had a K(d) of 3 mM or greater. 8. Stimulation of Na+-Ca2+ exchange current by MgATP occurred in the absence of cytoplasmic calcium with 9 mM-EGTA. 9. The stimulatory effect of 2 mM-MgATP was not inhibited by up to 200 μM of the protein kinase inhibitor 1-(5-isoquinoline sulphonyl)-2-methylpiperazine (H7), or by peptide inhibitors of cyclic AMP-dependent protein kinase, protein kinase C and calcium calmodulin-dependent protein kinase II. 10. The stimulatory effect of MgATP was not mimicked by MgATP-γ-S, and it was not reversed by acid phosphatase, alkaline phosphatase or an isolated cardiac protein phosphatase. Further, the effect was not enhanced nor was decay of the effect prolonged by 2 μM of the phosphatase inhibitor, okadaic acid. 11. We conclude that stimulation of Na+-Ca2+ exchange current in excised sarcolemmal patches by MgATP is not a calcium-dependent process and probably does not involve protein kinases.

Original languageEnglish (US)
Pages (from-to)27-57
Number of pages31
JournalJournal of Physiology
Volume454
StatePublished - 1992

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Adenosine Triphosphate
Membranes
Calcium
Calcium-Calmodulin-Dependent Protein Kinase Type 2
Sarcolemma
Blister
Protein Kinases
Muscle Cells
Guinea Pigs
Calcium-Calmodulin-Dependent Protein Kinase Kinase
Rabbits
Okadaic Acid
Sarcomeres
Phosphoprotein Phosphatases
Egtazic Acid
Ion Transport
Sarcoplasmic Reticulum
Protein Kinase Inhibitors
Acid Phosphatase
Fluorescence Microscopy

ASJC Scopus subject areas

  • Physiology

Cite this

The giant cardiac membrane patch method : Stimulation of outward Na+-Ca2+ exchange current by MgATP. / Collins, A.; Somlyo, A. V.; Hilgemann, D. W.

In: Journal of Physiology, Vol. 454, 1992, p. 27-57.

Research output: Contribution to journalArticle

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N2 - 1. A giant patch method was used to study the stimulatory effect of cytoplasmic MgATP on outward Na+-Ca2+ exchange current in inside-out cardiac membrane patches (1-10 GΩ seals with 14-24 μm pipette tip diameters) excised from guinea-pig. rabbit and mouse myocytes. 2. To establish the validity of the method with respect to structure, bleb formation was examined with electron microscopy and with confocal fluorescence light microscopy. The blebs, which form as the sarcolemma detaches, excluded intracellular organelles and transverse tubules. The blebbed cells contained normal sarcomeres, sarcoplasmic reticulum, triads and diads. 3. To further establish the validity of the method for ion transport studies, measurements of Na+-K+ pump currents and charge movements are described briefly which demonstrate (i) free access to the cytoplasmic membrane side, (ii) MgATP dependence comparable to reconstituted pump (K(d), 94 μM), (iii) fast, rigorous concentration control and (iv) Na+-K+ pump densities in the range of whole-cell densities. 4. Stimulation of outward Na+Ca2+ exchange current by MgATP attenuated exchange current decay during step increments of cytoplasmic sodium, shifted the secondary activation of outward exchange currently cytoplasmic calcium to lower free calcium concentrations and, particularly in mouse cardiac sarcolemma, induced cytoplasmic calcium-independent current. 5. Upon removal of MgATP the stimulatory effect usually decayed with a t50 (half-time) of about 3 min. However, the reversal took place much more rapidly (t50, 5-20 s) in patches from individual guinea-pig and rabbit myocyte batches. When decay was rapid, secondary activation by cytoplasmic calcium was shifted to higher free cytoplasmic calcium concentrations (K(d), 10-65 μM-free calcium). 6. With repeated applications of MgATP the rate and magnitude of the stimulatory effect progressively decreased. 7. The K(d) for MgATP of the initial rate of stimulation of outward exchange current was 3 mM or greater. When decay was rapid, the steady-state dependence of exchange current on MgATP also had a K(d) of 3 mM or greater. 8. Stimulation of Na+-Ca2+ exchange current by MgATP occurred in the absence of cytoplasmic calcium with 9 mM-EGTA. 9. The stimulatory effect of 2 mM-MgATP was not inhibited by up to 200 μM of the protein kinase inhibitor 1-(5-isoquinoline sulphonyl)-2-methylpiperazine (H7), or by peptide inhibitors of cyclic AMP-dependent protein kinase, protein kinase C and calcium calmodulin-dependent protein kinase II. 10. The stimulatory effect of MgATP was not mimicked by MgATP-γ-S, and it was not reversed by acid phosphatase, alkaline phosphatase or an isolated cardiac protein phosphatase. Further, the effect was not enhanced nor was decay of the effect prolonged by 2 μM of the phosphatase inhibitor, okadaic acid. 11. We conclude that stimulation of Na+-Ca2+ exchange current in excised sarcolemmal patches by MgATP is not a calcium-dependent process and probably does not involve protein kinases.

AB - 1. A giant patch method was used to study the stimulatory effect of cytoplasmic MgATP on outward Na+-Ca2+ exchange current in inside-out cardiac membrane patches (1-10 GΩ seals with 14-24 μm pipette tip diameters) excised from guinea-pig. rabbit and mouse myocytes. 2. To establish the validity of the method with respect to structure, bleb formation was examined with electron microscopy and with confocal fluorescence light microscopy. The blebs, which form as the sarcolemma detaches, excluded intracellular organelles and transverse tubules. The blebbed cells contained normal sarcomeres, sarcoplasmic reticulum, triads and diads. 3. To further establish the validity of the method for ion transport studies, measurements of Na+-K+ pump currents and charge movements are described briefly which demonstrate (i) free access to the cytoplasmic membrane side, (ii) MgATP dependence comparable to reconstituted pump (K(d), 94 μM), (iii) fast, rigorous concentration control and (iv) Na+-K+ pump densities in the range of whole-cell densities. 4. Stimulation of outward Na+Ca2+ exchange current by MgATP attenuated exchange current decay during step increments of cytoplasmic sodium, shifted the secondary activation of outward exchange currently cytoplasmic calcium to lower free calcium concentrations and, particularly in mouse cardiac sarcolemma, induced cytoplasmic calcium-independent current. 5. Upon removal of MgATP the stimulatory effect usually decayed with a t50 (half-time) of about 3 min. However, the reversal took place much more rapidly (t50, 5-20 s) in patches from individual guinea-pig and rabbit myocyte batches. When decay was rapid, secondary activation by cytoplasmic calcium was shifted to higher free cytoplasmic calcium concentrations (K(d), 10-65 μM-free calcium). 6. With repeated applications of MgATP the rate and magnitude of the stimulatory effect progressively decreased. 7. The K(d) for MgATP of the initial rate of stimulation of outward exchange current was 3 mM or greater. When decay was rapid, the steady-state dependence of exchange current on MgATP also had a K(d) of 3 mM or greater. 8. Stimulation of Na+-Ca2+ exchange current by MgATP occurred in the absence of cytoplasmic calcium with 9 mM-EGTA. 9. The stimulatory effect of 2 mM-MgATP was not inhibited by up to 200 μM of the protein kinase inhibitor 1-(5-isoquinoline sulphonyl)-2-methylpiperazine (H7), or by peptide inhibitors of cyclic AMP-dependent protein kinase, protein kinase C and calcium calmodulin-dependent protein kinase II. 10. The stimulatory effect of MgATP was not mimicked by MgATP-γ-S, and it was not reversed by acid phosphatase, alkaline phosphatase or an isolated cardiac protein phosphatase. Further, the effect was not enhanced nor was decay of the effect prolonged by 2 μM of the phosphatase inhibitor, okadaic acid. 11. We conclude that stimulation of Na+-Ca2+ exchange current in excised sarcolemmal patches by MgATP is not a calcium-dependent process and probably does not involve protein kinases.

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