Neutral amino acid transport in surface membrane vesicles isolated from mouse fibroblasts: Intrinsic and extrinsic models of regulation

Abstract

AbstractMembrane transport carrier function, its regulation and coupling to metabolism, can be selectively investigated dissociated from metabolism and in the presence of a defined electrochemical ion gradient driving force, using the single internal compartment system provided by vesiculated surface membranes. Vesicles isolated from nontransformed and Simian virus 40‐transformed mouse fibroblast cultures catalyzed carrier‐mediated transport of several neutral amino acids into an osmotically‐sensitive intravesicular space without detectable metabolic conversion of substrate.When a Na+ gradient, external Na+ > internal Na+, was artifically imposed across vesicle membranes, accumulation of several neutral amino acids achieved apparent intravesicular concentrations 6‐ to 9‐fold above their external concentrations. Na+‐stimulated alanine transport activity accompanied plasma membrane material during subcellular fractionation procedures. Competitive interactions among several neutral amino acids for Na+‐stimulated transport into vesicles and inactivation studies indicated that at least 3 separate transport systems with specificity properties previously defined for neutral amino acid transport in Ehrlich ascites cells were functional in vesicles from mouse fibroblasts: the A system, the L system and a glycine transport system. The pH profiles and apparent Km values for alanine and 2‐aminoisobutyric acid transport into vesicles were those expected of components of the corresponding cellular uptake system.Several observations indicated that both a Na+ chemical concentration gradient and an electrical membrane potential contribute to the total driving force for active amino acid transport via the A system and the glycine system. Both the initial rate and quasi‐steady‐state of accumulation were stimulated as a function of increasing concentrations of Na+ applied as a gradient (external > internal) across the membrane. This stimulation was independent of endogenous Na+, K+‐ATPase activity in vesicles and was diminished by monensin or by preincubation of vesicles with Na+. The apparent Km for transport of alanine and 2‐aminoisobutyric acid was decreased as a function of Na+ concentration. Similarly, in the presence of a standard initial Na+ gradient, quasi‐steady‐state alanine accumulation in vesicles increased as a function of increasing magnitudes of interior‐negative membrane potential imposed across the membrane by means of K+ diffusion potentials (internal > external) in the presence of valinomycin; the magnitude of this electrical component was estimated by the apparent distributions of the freely permeant lipophilic cation triphenylme thylphosphonium ion. Alanine transport stimulation by charge asymmetry required Na+ and was blocked by the further addition of either nigericin or external K+. As a corollary, Na+‐stimulated alanine transport was associated with an apparent depolarization, detectable as an increased labeled thiocyanate accumulation. Permeant anions stimulated Na+‐coupled active transport of these amino acids but did not affect Na+‐independent transport. Translocation of K+, H+, or anions did not appear to be directly involved in this transport mechanism. These characteristics support an electrogenic mechanism in which amino acid translocation is coupled t o an electrochemical Na+ gradient by formation of a positively charged complex, stoichiometry unspecified, of Na+, amino acid, and membrane component.Functional changes expressed in isolated membranes were observed t o accompany a change in cellular proliferative state or viral transformation. Vesicles from Simian virus 40‐transformed cells exhibited an increased Vmax of Na+‐stimulated 2‐aminoisobutyric acid transport, as well as an increased capacity for steady‐state accumulation of amino acids in response t o a standard Na+ gradient, relative t o vesicles from nontransformed cells. Density‐inhibition of nontransformed cells was associated with a marked decrease in these parameters assayed in vesicles. Several possibilities for regulatory interactions involving gradient‐coupled transport systems are discussed.