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Membrane potential and amino acid transport in a mutant chinese hamster ovary cell line
Author(s) -
Rotoli Bianca Maria,
Bussolati Ovidio,
Dall'asta Valeria,
Gazzola Gian Carlo
Publication year - 1991
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.1041460312
Subject(s) - chinese hamster ovary cell , valinomycin , membrane potential , depolarization , electrochemical gradient , chemistry , amino acid , arginine , biophysics , nernst equation , hyperpolarization (physics) , steady state (chemistry) , transmembrane domain , biochemistry , membrane , stereochemistry , biology , receptor , electrode , nuclear magnetic resonance spectroscopy
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 (R ARG ) was employed as an indicator of membrane potential (Δψ). Evidence for the reliability of R ARG 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}\begin{document}$ \tilde\mu $\end{document} 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.