A model for the kinetic mechanism of sodium-coupled L-alanine transport in LLC-PK1 cells
Wilson, J.J.; Randles, J.; Kimmich, G.A.
American Journal of Physiology 270(1 Pt 1): C49-C56
1996
ISSN/ISBN: 0002-9513 PMID: 8772429 Document Number: 464846
The kinetics of sodium-dependent L-alanine transport were characterized in ATP-depleted LLC-PK-1 cells, which allows experimental imposition of an interior negative diffusion potential across the plasma membrane. Under these conditions a wide range of sodium concentrations can be studied without altering the membrane potential. When Na+ is the variable substrate, the apparent maximal velocity (V-max) for transport changes nearly fourfold for the five different alanine concentrations studied (0.05-2.0 mM). In contrast, at five different sodium concentrations, ranging from 10 to 135 mM, the apparent V-max with variable alanine remains nearly constant at 5.3 +- 1.2 nmol cntdot min-1 cntdot mg cell protein-1. The ratio of the two primary kinetic parameters, Michaelis constant (K-m)/V-max, varies markedly no matter which solute is treated as the variable substrate. These data are consistent with a simultaneous ordered transport mechanism in which sodium binds before alanine to the transport protein at the extracellular surface of the membrane. Alanine-dependent 22Na+ influx is more than five times faster if unlabeled intracellular sodium is present than in its absence. Sodium-dependent influx of (14C)alanine is more rapid than net alanine flux only if unlabeled Na+ and alanine are both present intracellularly. These results indicate that the cotransporter can function more rapidly in an exchange mode than when it catalyzes net solute uptake and that Na+ is the first solute to be released at the intracellular side of the membrane. A model is presented that can be used for further quantitative analysis of the kinetic and functional properties of the cotransport system.