AJP - Cell Physiology, Vol 270, Issue 1 C49-C56, Copyright © 1996 by American Physiological Society
A model for the kinetic mechanism of sodium-coupled L-alanine transport in LLC-PK1 cells
J. J. Wilson, J. Randles and G. A. Kimmich
Department of Biophysics, University of Rochester Medical Center, New York 14642, USA.
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 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.min-1.mg cell protein-1. The ratio of
the two primary kinetic parameters, Michaelis constant (Km)/V max, varies
markedly no matter which solute is treated as the variable illustrate.
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.
