Figure S4.
Na current kinetics predicted and measured in myocytes that are patch clamped with a cytoplasmic solution containing 40 mM Na and an anion with a variable diffusion coefficient.(A) Predicted results. Na was assumed to have a diffusion coefficient of 1.2 × 10−5 cm2/s, and the diffusion coefficient of the paired anion (X) was assumed to be equal to that of Na (black), 10 times smaller than Na (blue) or 100 times smaller than Na (red). Extracellular Na is initially zero, and a Na conductance is activated that generates 0.4 nA of outward Na current. After 100 s, 100 mM extracellular Na is applied, and after an additional 100 s, the extracellular Na is removed. Cytoplasmic Na depletes and accumulates, respectively, in dependence on anions diffusing into and out of the patch pipette. A 10-fold reduction of the anion diffusion coefficient results in a 5-fold increase of the ion equilibration time constant. When anion diffusion is reduced 100-fold, current decay becomes so slow that it would be difficult to monitor reliably. (B) Results for myocytes superfused continuously with 15 μM veratridine and then additionally with 120, 90, and 40 mM Na, respectively, for 100 s. All cytoplasmic solutions contained 5 mM HEPES, 0.5 mM EGTA, taurine as an osmolyte to give 280 mosmol/liter, and either 40 mM NaCl with 2 mM Na(Mg)ATP (left) or 40 mM Na(Mg)ATP (right). Results for 120 mM extracellular Na (upper panel), employing aspartate as anion, compare current transients obtained after applying veratridine for 100 s with 120 mM Na. Average current decay time constants were distinctly longer in the presence of Na(Mg)ATP in the pipette. Average values during application of Na (τ2) were 15 and 49 s for pipette solutions with Cl versus MgATP, respectively. This 3.3-fold difference in decay rates is recreated in simulations when the diffusion coefficient of MgATP is sevenfold smaller than that of than Cl. In free water, by contrast, MgATP diffuses just 2.6-fold slower than Cl. When experiments were performed with 90 and 40 mM extracellular Na, shown in the middle and lower panels, respectively, current magnitudes were not significantly different in Cl versus MgATP in the pipette. In the presence of cytoplasmic Cl, currents decayed less markedly than with 120 mM extracellular Na. Impressively, no current decay was detectable in the presence of cytoplasmic MgATP. (C) The differences in fractional current decay for Cl- and ATP-containing solutions are quantified. Based on the simulations, these differences require that under these conditions MgATP diffuses >10 times slower than Cl in the myocyte cytoplasm. **P < 0.01, ***P < 0.001.

Na current kinetics predicted and measured in myocytes that are patch clamped with a cytoplasmic solution containing 40 mM Na and an anion with a variable diffusion coefficient. (A) Predicted results. Na was assumed to have a diffusion coefficient of 1.2 × 10−5 cm2/s, and the diffusion coefficient of the paired anion (X) was assumed to be equal to that of Na (black), 10 times smaller than Na (blue) or 100 times smaller than Na (red). Extracellular Na is initially zero, and a Na conductance is activated that generates 0.4 nA of outward Na current. After 100 s, 100 mM extracellular Na is applied, and after an additional 100 s, the extracellular Na is removed. Cytoplasmic Na depletes and accumulates, respectively, in dependence on anions diffusing into and out of the patch pipette. A 10-fold reduction of the anion diffusion coefficient results in a 5-fold increase of the ion equilibration time constant. When anion diffusion is reduced 100-fold, current decay becomes so slow that it would be difficult to monitor reliably. (B) Results for myocytes superfused continuously with 15 μM veratridine and then additionally with 120, 90, and 40 mM Na, respectively, for 100 s. All cytoplasmic solutions contained 5 mM HEPES, 0.5 mM EGTA, taurine as an osmolyte to give 280 mosmol/liter, and either 40 mM NaCl with 2 mM Na(Mg)ATP (left) or 40 mM Na(Mg)ATP (right). Results for 120 mM extracellular Na (upper panel), employing aspartate as anion, compare current transients obtained after applying veratridine for 100 s with 120 mM Na. Average current decay time constants were distinctly longer in the presence of Na(Mg)ATP in the pipette. Average values during application of Na (τ2) were 15 and 49 s for pipette solutions with Cl versus MgATP, respectively. This 3.3-fold difference in decay rates is recreated in simulations when the diffusion coefficient of MgATP is sevenfold smaller than that of than Cl. In free water, by contrast, MgATP diffuses just 2.6-fold slower than Cl. When experiments were performed with 90 and 40 mM extracellular Na, shown in the middle and lower panels, respectively, current magnitudes were not significantly different in Cl versus MgATP in the pipette. In the presence of cytoplasmic Cl, currents decayed less markedly than with 120 mM extracellular Na. Impressively, no current decay was detectable in the presence of cytoplasmic MgATP. (C) The differences in fractional current decay for Cl- and ATP-containing solutions are quantified. Based on the simulations, these differences require that under these conditions MgATP diffuses >10 times slower than Cl in the myocyte cytoplasm. **P < 0.01, ***P < 0.001.

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