Energetics of Na+-dependent amino acid co-transport in Ehrlich ascites tumor cells.

The energy available from the Na+ electrochemical potential gradient (delta mu Na) has been evaluated in Ehrlich ascites tumor cells during accumulation of 2-aminoisobutyric acid. Cells were incubated in media of varying [Na+] (25-154 mM) in the presence of 0.25 mM 2-aminoisobutyric acid to establish maximum steady-state accumulation of the amino acid. Membrane potential (Vm) and intracellular Na+ activity (aNa) were estimated using standard electrophysiological techniques. In physiological saline ([Na+] = 154 mM) aNa is 4.4 +/- 0.6 mM, giving an apparent Na+ activity coefficient (gamma app) in the cytoplasm of 0.17 +/- 0.02. Vm under these conditions is -20.8 +/- 2.1 mV. From these values, delta mu Na = 9.9 +/- 0.8 kJ/mol. Concomitant determinations of 2-aminoisobutyric acid (AIB) accumulation show an energy requirement (delta mu AIB) of 8.5 +/- 0.5 kJ/mol. Stepwise reductions in extracellular [Na+] give parallel reductions in aNa, Vm and 2-aminoisobutyric acid accumulation. However, under all conditions tested the energy available from the Na+ electrochemical potential gradient exceeds that needed to drive 2-aminoisobutyric acid uptake. The effects of 2-aminoisobutyric acid on Vm have also been determined. Addition of AIB (10 mM) to steady-state cells leads to membrane depolarization (resting Vm = -22.1 +/- 1.3 mV; plus AIB Vm = -16.2 +/- 1.2 mV) within 1 min. Subsequent repolarization of the membrane to resting levels occurs within 10 min. The repolarization phase is blocked in the presence of ouabain (2 mM). The results establish that the energy available from the Na+ gradient is sufficient to serve as a source for 2-aminoisobutyric acid accumulation.