Contribution of electrogenic sodium-potassium ATPase to resting membrane potential of cultured rat skeletal myotubes

The contribution of electrogenic Na⁺-K⁺ ATPase to resting membrane potential (Em) of mature and developing rat skeletal myotubes in culture was determined by examining effects of inhibition of this enzyme on Em. Ouabain, a specific Na⁺-K⁺ ATPase inhibitor, caused resting Em to decrease within 30 s by 5-8 mV and reach a minimum value of about -60 mV after 5 min. The decrease in Em was not accompanied by a decrease in input resistance for up to 15 min after application. Resting Em was found to be dependent on the temperature of the recording medium with maximum values of Em ranging from -85 to -90 mV at a temperature of 35-37°C and minimum values about -60 mV at 10-15°C. Ouabain (1 mM), added to cultures at low temperature (10-15°C) did not further decrease Em but did prevent the increase in Em that occurs with increasing temperature up to 37°C. Resting Em of cultured myotubes was reduced to about -60 mV by reducing the supply of ATP either with 2,4 dinitrophenol (DNP), which inhibits oxidative phosphorylation or with fluorodinitrobenzene (FDNB), which inhibits creatine phosphokinase. Neither of these compounds, when added to cultures in the presence of ouabain, reduced resting Em to a value lower than that obtained with ouabain alone. The developmental increase in Em which occurs after fusion appeared to be due to an increase in activity of Na⁺-K⁺ ATPase; at any given age, Em was higher at 35 °C than at 21 °C, and addition of ouabain caused Em to fall to a value of -60 to -65 mV, regardless of age. The appearance of the electrogenic component of Em was found to be dependent to some extent on initial plating density of the cells.