Rotating disk electrode voltammetry applied to the kinetics of uptake and efflux in wild-type and mutant catecholamine transporters

Rotating disk electrode voltammetry has shown to be a simple, fast, and ine xpensive method for studying substrate kinetics at wild-type and mutant cat echolamine transporters. The transport of the substrates dopamine (DA) and meta- and para-tyramine (m-TYR, p-TYR) are examined at the human norepineph rine transporter (hNET) expressed stably in LLC cells. Following uptake of DA, monitored at 450 mV (vs. Ag/AgCl), efflux is induced by the uptake of a second substrate. The timecourse of induced efflux of DA is found to be de pendent on the structure of the compound used to initiate efflux as well as the concentration of both the preloaded substrate and efflux inducing agen t. A double electrode configuration for the electrochemical cell, including one rotating and one stationary working electrode, is described. A potenti al of 650 mV is applied across the stationary electrode to obtain the timec ourse of single-hydroxyl substrates after correction for any catechol subst rate oxidation. This dual electrode set-up is useful in that it allows the kinetics of two substrates interacting at the transporter to be monitored s imultaneously. The kinetics of the above mentioned substrates are examined at wild-type and mutant hNETs using this two electrode configuration.