Determination of transport parameters of permeant substrates of the vesicular amine transporter

Biological transport of moderately permeant compounds is obscured by diffusion of the compounds back across the membrane, so characterization of the transport of such compounds requires correction for permeability. A relatively simple method for determining kinetic parameters for moderately permeant compounds is presented here. After evaluating a compound's apparent permeability coefficient, its steady-state uptake is measured as a function of concentration. By comparing the concentration dependence of uptake measured both in the presence and in the absence of a complete inhibitor of the transporter, K(m) and Vmax for transport of that substrate may be calculated. When used to analyze transport of tyramine and hydroxyephedrine by the vesicular amine transporter, this method yields results consistent with other methods and with values for analogous impermeant substrates. In bovine adrenal chromaffin vesicles, tyramine and (-)erythro-hydroxyephedrine have apparent permeability coefficients of 4.7 +/- 1.0 x 10(-9) and 1.1 +/- 0.4 x 10(-8) cm/s, respectively. Values for K(m) are 15 +/- 9 and 34 +/- 14 microM and for Vmax are 1.3 +/- 0.2 and 1.4 +/- 0.9 nmol/min.mg of membrane protein, respectively.