Functional analysis of nucleotide turnover between murine myocyte cytoplasm and patch clamp pipette tip (35°C). (A) Pipette perfusion of AMPPNP. A rapid pipette perfusion technique (Hilgemann and Lu, 1998) was used to apply and remove nonhydrolyzable ATP, AMPPNP, from the cytoplasmic side. The pipette contained 4 mM ATP and 80 mM Na throughout. During continuous pump activation with 7 mM K, AMPPNP (2 mM) perfusion inhibits the pump current with a time constant of 25 s. Pumps reactivate with a time constant of 160 s (gray curves) during AMPPNP removal, and the protocol repeats accurately. Gray curves are single exponential functions, fit to results. The red curve in A simulates results with a diffusion coefficient of 3 × 10⁻⁷ cm²/s into and out of a 120 μM long cylinder, open at one end. The dissociation constant of AMPPNP was 5 μM and that of ATP was 50 μM. (B) Pump current changes after myocyte opening in the presence and absence of ATP in the patch pipette. Pump currents were activated repeatedly for just 2 s to ensure that pump activity did not deplete cytoplasmic Na. Pump currents with ATP decreased by 10% over 10 min, while pump currents without ATP decreased by 65%. Without ATP, currents decayed to negligible values in all experiments within 15 min. The final phase of current decay had a time constant of 177 s on average (n = 6; red curve). The time course of decay is simulated (gray curve) with the following assumptions: (1) The initial myocyte ATP concentration is 8 mm, (2) ATP activates Na/K pumps with a K₅₀ of 50 μM, (3) the cytoplasm contains 120 μM of ATP binding sites with a K_d of 50 μM, (4) the diffusion coefficient of free ATP is 8 × 10⁻⁷ cm²/s, (5) the myocyte is 120 μm long, and (6) the pipette access resistance does not limit ATP diffusion out of the myocyte. ***P < 0.001.