Figure 10.
Relative anion diffusion coefficients determined from time course of coupled Na and MgATP diffusion into murine myocytes (35°C). (A–D) Representative patch clamp records of myocytes opened with pipette solutions containing 130 mM NaCl (A), Na-aspartate (B), Na(Mg)ATP (C), or Na-PEG-COOH (2 kD; D). The pipette also contained 5 mM KCl and 8 mM HEPES titrated to pH 7.0 with KOH. The extracellular solution contained 130 mM NMG-Aspartate, 4 mM MgCl2, and 10 mM HEPES titrated to pH 7.0 with NMG. As indicated, 10 µM vertridine was applied and removed for a few seconds, long enough to achieve a peak outward Na current. The final veratridine application was continued for long enough to induce current decay, indicative of cytoplasmic Na depletion. The time course with which peak veratridine currents approached a maximum was fitted to a single exponential function. (E) Composite results for the rising peak veratridine currents after myocyte opening. The mean time constants for five to nine measurements are given below the respective columns for the four ion pairs examined. (F) Composite results for the decay of currents during continued veratridine application. (G) Simulation of cytoplasmic Na(Mg)ATP accumulation during patch clamp. As noted in Materials and methods, exchange of MgATP takes place threefold faster than overall diffusion through the cytoplasm, while K, Cl, and Na exchange threefold slower across the pipette tip than they diffuse through the cytoplasm. The pipette contains Na(Mg)ATP (120 mM), and its contents are assumed to be constant. DNa, DCl, DK, and DATP are 0.7, 1.2, 1.2, and 0.06 × 10–5 cm2/s, respectively. The extracellular solution contains no Na. A surface membrane Na conductance can be activated and deactivated arbitrarily and was set to generate an outward Na current of ∼2 nA. Upon opening the pipette tip, KCl is lost rapidly from the myocyte. Thereafter, Na(Mg)ATP diffuses slowly into the myocyte in a coupled fashion, limited by the diffusion coefficient of MgATP. When MgATP is the sole anion present in the cytoplasm, activation of the Na conductance results in coupled depletion of Na and MgATP with essentially identical time courses. *P < 0.05, **P < 0.01.

Relative anion diffusion coefficients determined from time course of coupled Na and MgATP diffusion into murine myocytes (35°C). (A–D) Representative patch clamp records of myocytes opened with pipette solutions containing 130 mM NaCl (A), Na-aspartate (B), Na(Mg)ATP (C), or Na-PEG-COOH (2 kD; D). The pipette also contained 5 mM KCl and 8 mM HEPES titrated to pH 7.0 with KOH. The extracellular solution contained 130 mM NMG-Aspartate, 4 mM MgCl2, and 10 mM HEPES titrated to pH 7.0 with NMG. As indicated, 10 µM vertridine was applied and removed for a few seconds, long enough to achieve a peak outward Na current. The final veratridine application was continued for long enough to induce current decay, indicative of cytoplasmic Na depletion. The time course with which peak veratridine currents approached a maximum was fitted to a single exponential function. (E) Composite results for the rising peak veratridine currents after myocyte opening. The mean time constants for five to nine measurements are given below the respective columns for the four ion pairs examined. (F) Composite results for the decay of currents during continued veratridine application. (G) Simulation of cytoplasmic Na(Mg)ATP accumulation during patch clamp. As noted in Materials and methods, exchange of MgATP takes place threefold faster than overall diffusion through the cytoplasm, while K, Cl, and Na exchange threefold slower across the pipette tip than they diffuse through the cytoplasm. The pipette contains Na(Mg)ATP (120 mM), and its contents are assumed to be constant. DNa, DCl, DK, and DATP are 0.7, 1.2, 1.2, and 0.06 × 10–5 cm2/s, respectively. The extracellular solution contains no Na. A surface membrane Na conductance can be activated and deactivated arbitrarily and was set to generate an outward Na current of ∼2 nA. Upon opening the pipette tip, KCl is lost rapidly from the myocyte. Thereafter, Na(Mg)ATP diffuses slowly into the myocyte in a coupled fashion, limited by the diffusion coefficient of MgATP. When MgATP is the sole anion present in the cytoplasm, activation of the Na conductance results in coupled depletion of Na and MgATP with essentially identical time courses. *P < 0.05, **P < 0.01.

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