A model for the kinetic mechanism of sodium-coupled L-alanine transport in LLC-PK1 cells.
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.
Wilson
J J
JJ
Department of Biophysics, University of Rochester Medical Center, New York 14642, USA.
Randles
J
J
Kimmich
G A
GA
D026905:Amino Acid Transport Systems; D026921:Amino Acid Transport Systems, Neutral; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D018374:LLC-PK1 Cells / Q000378:metabolism*; D008954:Models, Biological; D013552:Swine; D027981:Symporters
D026905:Amino Acid Transport Systems; D026921:Amino Acid Transport Systems, Neutral; D002352:Carrier Proteins; D027981:Symporters; C052168:sodium-alanine cotransporter