Membrane potential and amino acid transport in a mutant chinese hamster ovary cell line

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Abstract
The bioenergetics of amino acid transport system A was studied in two Chinese hamster ovary (CHO) cell lines, the parent line CHO‐PEOT/1 and CHY‐1, a mutant of the former exhibiting a low activity of the same transport system. The steady‐state transmembrane distribution ratio of the cationic amino acid L‐arginine (RARG) was employed as an indicator of membrane potential (Δψ). Evidence for the reliability of RARG to measure Δψ can be summarized as follows: (1) L‐arginine transmembrane distribution increased under conditions of cell hyperpolarization and decreased under conditions of cell depolarization; (2) L‐arginine distribution conformed closely to that expected for a probe of Δψ in conditions in which Δψ depends largely on the transmembrane potassium gradient; and (3) the value of Δψ obtained through a valinomycin null point experiment (−72.7 mV) was very similar to the value calculated from L‐arginine distribution using the Nernst equation (−73.4 mV). The transmembrane gradient of sodium electrochemical potential (Δ\documentclass{article}\pagestyle{empty}$ \tilde\mu $ Na), the driving force for the operation of system A, was slightly higher in the mutant cell line CHY‐1. In the same line, the intracellular level of the specific system A substrate MeAlB at steady state was also higher. Studies of the rheogenicity of system A in the two lines indicated that the depolarization associated with the entry of substrates of system A was proportional to the amount of amino acid taken up by the cells. Kinetic analysis showed that the low activity of system A in the mutant cell line was referrable to a decrease in transport Vmax. It is concluded that neither a decrease in energy available for the operation of system A nor a decreased efficiency of coupling of the system to Δψ is responsible for the defect observed in the mutant line.