A model for the kinetic mechanism of sodium-coupled l-alanine transport in llc-pk1 cells. Wilson, Jennifer J., Joan Randles, and George A. Kimmich. Department of Biochemistry, University of Rochester Medical Center, Rochester, New York 14642
APStracts 2:0260C, 1995.
The kinetics of sodium-dependent L-alanine transport were characterized in ATP-depleted LLC-PK1 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 Vmax for transport changes nearly four fold for five different alanine concentrations studied (0.05-2.0 mM). In contrast, at five different sodium concentrations, ranging from 10-135 mM, the apparent Vmax with variable alanine remains nearly constant at 5.31.2 nmoles x min-1 x mg cell protein-1. The ratio of the two primary kinetic parameters (Km/Vmax) 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 prior to 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 co-transport system. 

Received 20 December 1994; accepted in final form 10 July 1995.
APS Manuscript Number C725-4.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 18 July 1995.