Mechanism of proline transport in Escherichia coli K12. III. Inhibition of membrane potential-driven proline transport by syn-coupled ions and evidence for symmetrical transition states of the 2H+/proline symport carrier.
Specific inhibition of 2H+/proline symport by syn-coupled ions (Na+, Li+, and H+) was investigated using cytoplasmic membrane vesicles prepared from the proline carrier-overproducing strain MinS/ pLC4 -45 of Escherichia coli K12. The 2H+/proline symport driven by the membrane potential generated via respiration with 20 mM ascorbate/Tris, 0.1 mM phenazine methosulfate was specifically inhibited by Na+. The inhibition by Na+ was described by a fully noncompetitive mechanism, and the apparent Ki for Na+ was 15 mM. A linear correlation between the apparent Vmax and the apparent Kd was observed. Li+ stimulated the transport activity 2-fold at 10 mM and inhibited it at concentrations above 50 mM. H+ caused fully noncompetitive inhibition of 2H+/proline symport, and its apparent Ki was 0.6 microM. These results indicate that the concentrations of Na+ and H+ strictly and independently regulate the amount of the active C state carrier responsible for 2H+/proline symport driven by the membrane potential by inhibiting the transition from the C* state carrier which exhibits Na+- and H+-dependent binding of proline and is predominant in nonenergized conditions.
D026921:Amino Acid Transport Systems, Neutral; D000596:Amino Acids / Q000378:metabolism; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006859:Hydrogen / Q000378:metabolism; D008094:Lithium / Q000378:metabolism; D008433:Mathematics; D008564:Membrane Potentials; D008954:Models, Biological; D009992:Osmium / Q000378:metabolism; D011392:Proline / Q000378:metabolism*; D027981:Symporters
D026921:Amino Acid Transport Systems, Neutral; D000596:Amino Acids; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D027981:Symporters; C041832:proline transport protein; C060609:PutP protein, E coli; D009992:Osmium; D006859:Hydrogen; D011392:Proline; D008094:Lithium