Experimental analysis of ion/solute cotransport by substrate binding and facilitated diffusion.

ABSTRACT

An ion/solute cotransporter can be studied in the absence of a transmembrane gradient of the electrochemical potential of the ion. Inspection of the appropriate equations discloses that basic parameters of the cotransport cycle can be obtained by measuring cosubstrate binding and the initial-velocity kinetics of four modes of facilitated diffusion as a function of the concentration of the cotransported ion. The following information can be derived estimates of the affinities of both cosubstrates, the number of binary intermediates participating in cotransport (equivalent to determining the order of cosubstrate binding and release), and the rate constants for the reorientation of the binding sites during cotransport. In general, both maximal velocities and half-saturation constants for the facilitated diffusion of one cosubstrate depend upon the concentration of the other. In some cases, the maximal velocities of influx and efflux do not increase monotonically with the concentration of the ion but pass through a maximum and decrease. If direct binding studies are not possible, affinities of the cosubstrates can be estimated from data for equilibrium exchange or countertransport. Also, an approximate description of the time course of the transient accumulation (overshoot) during countertransport is derived. Under certain circumstances, the height of the overshoot is proportional to the concentration of the cotransported ion. Thus, countertransport can be employed as a simple test to establish if a solute is cotransported with a particular ion. This treatment allows many effects noted in galactoside countertransport in Escherichia coli to be explained.