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

The energy available from the Na⁺ electrochemical potential gradient (Δμ_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 (V_m) and intracellular Na⁺ activity (a_Na) were estimated using standard electrophysiological techniques. In physiological saline ([Na⁺] = 154 mM) a_Na is 4.4 ± 0.6 mM, giving an apparent Na⁺ activity coefficient (γ_app) in the cytoplasm of 0.17 ± 0.02. V_m under these conditions is -20.8 ± 2.1 mV. From these values, Δμ_Na = 9.9 ± 0.8 kJ/mol. Concomitant determinations of 2-aminoisobutyric acid (AIB) accumulation show an energy requirement (Δμ_AIB) of 8.5 ± 0.5 kJ/mol. Stepwise reductions in extracellular [Na⁺] give parallel reductions in a_Na, V_m 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 V_m have also been determined. Addition of AIB (10 mM) to steady-state cells leads to membrane depolarization (resting V_m = -22.1 ± 1.3 mV; plus AIB V_m = -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.