STABLE EXPRESSION OF BIOGENIC-AMINE TRANSPORTERS REVEALS DIFFERENCES IN INHIBITOR SENSITIVITY, KINETICS, AND ION DEPENDENCE

We have constructed stable cell lines expressing transporters for dopa mine (DA), norepinephrine (NE), and serotonin (5-HT) by transfection w ith cloned cDNAs. The parental LLC-PK1 cell does not express any of th ese neurotransmitter transporters. Therefore, monoamine transport acti vities in each of these cell lines are due to the transfected DNA only , allowing comparison in the same background. Drug inhibition profiles for each cell line are distinct and as expected for each transporter. LLC-NET and LLC-DAT cells transported both NE and DA and both cell ty pes exhibited a lower K-M for DA transport than for NE transport. Anal ysis of V-max data for LLC-NET cells suggests that substrate is bound to the NE transporter during the rate-limiting step(s) in transport, T he cocaine analog 2-beta carbomethoxy-3 beta-(4-[I-125]iodophenyl)trop ane binds to each cell type, and is displaced by transport substrate i n each case. Binding and transport measurements on parallel cell cultu res allowed estimation of turnover numbers for norepinephrine, dopamin e, and serotonin transporters. All three transporters require external Na+ and Cl-. The Na+ concentration dependence suggests that a single Na+ ion is involved in transport catalyzed by norepinephrine and serot onin transporters while more than one Na+ ion participate in transport mediated by the dopamine transporter.