Na-K pump current (Ipump) is a function of the intracellular Na+ concentration [( Na+]i). We examined the quantitative relationship between Ipump and [Na+]i in isolated guinea pig ventricular myocytes under steady-state conditions. [Na+]i was controlled and "clamped" at several selected concentrations using wide-tipped pipette microelectrodes, and membrane current was measured using the whole cell patch voltage-clamp technique. Ipump generated at a holding potential of -40 mV was determined by measuring the change in steady-state holding current before and during exposure to dihydroouabain (1 mM); Ipump was measured at 11 levels of [Na+]i ranging from 0 to 80 mM (n = 63) with only one measurement per cell and normalized to cell capacitance to account for differences between myocytes in sarcolemmal surface area. Ipump exhibited a nonlinear dependence on [Na+]i; a Hill analysis of the relationship yielded a half-maximal [Na+]i for pump stimulation of 43.2 mM and a Hill coefficient of 1.53. An alternative analysis of the experimental data was performed assuming that occupation of three internal binding sites by Na+ is required for enzyme turnover. Regression analysis gave the best fit when only two different binding affinities (KD) are postulated. The values are KD1 = 1 mM, KD2 = KD3 = 29 mM. From the analysis using the latter model, the level of [Na+]i at which Ipump saturated closely approximated the theoretical saturation level calculated from published estimates of pump turnover rate and density. The maximal sensitivity of the Na-K pump to changes in [Na+]i occurs when internal [Na+] is within the range for the normal resting physiological level.
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