Fig. 1. Na⁺, K⁺-pump currents in cortical neurons and its ATP dependence. Identification of the membrane current associated with activities of the Na⁺, K⁺-ATPase. (A) Whole-cell recordings of the Na⁺, K⁺-pump current, I_pump, in cultured cortical neurons. At the holding potential of –60 mV, an inward current was triggered by an acute application of the Na⁺, K⁺-pump inhibitor ouabain (1 mM) or strophanthidin (500 µM), corresponding to the block of a tonic activity of the Na⁺, K⁺-pump. The effect of strophanthidin was reversible, an outward current appeared owing to reactivation of the Na⁺, K⁺-ATPase. (B) The membrane current associated with the Na⁺, K⁺-pump was highly dependent on the presence of intracellular Na⁺ ([Na⁺]_i) and extracellular K⁺ ([K⁺]_o). When Na⁺ was removed from the pipette internal solution or K⁺ was removed from the extracellular solution, little membrane current was observed upon application of 500 µM strophanthidin. (C) The I-V relationship of Na⁺, K⁺-pump current obtained by subtracting I-V curves constructed by membrane current responses to various voltage steps from the –100 mV holding potential in the presence and absence of 1 mM ouabain. The reversal potential of the pump current estimated from the I-V curve is about –133 mV. The relationship showed an outward rectification at depolarized potentials and a decrease of the current at very positive potentials, both are consistent with previous reports of the pump current (De Weer et al., 1988). Voltage pulses were applied every 4 seconds with increments of 20 mV starting at –100 mV. n=10. (D) The ATP-dependence of I_pump was revealed by comparison of the currents recorded with microelectrodes containing either 5 mM ATP or ATP-free internal solution. In the presence of ATP, there was no time-dependent decline of I_pump; without ATP in the internal solution, I_pump was greatly diminished 10 minutes after establishing the whole-cell configuration. n=10 and 8 for ATP and ATP-free groups, respectively. *P<0.05 compared with the current at 1-2 minutes.

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