Structural domains of chimeric dopamine-noradrenaline human transporters involved in the Na(+)- and Cl(-)-dependence of dopamine transport

Catecholamine transporters constitute the biological targets for several important drugs, including antidepressants, cocaine, and related compounds. Some information exists about discrete domains of these transporters that are involved in substrate translocation and uptake blockade, but delineation of domains mediating the ionic dependence of the transport remains to be defined. In the present study, human neuronal transporters for dopamine and noradrenaline (hDAT and hNET) and a series of six functional chimeras were transiently expressed in LLC-PK1 cells. Substitution of Cl(-) by isethionate reveals that cassette IV (i.e., the region of the transporter encompassing transmembrane domain 9 through the COOH terminal) plays an important role in the Cl(-)- dependence of the uptake. Substitutions of Na(+) and NaCl by Tris(+) and sucrose, respectively, demonstrate that three different segments scattered across the transporter are involved in the Na(+)- dependence of the transport activity: cassette I (i.e., the region from the amino terminus through the first two transmembrane domains), cassette IV, and junction between transmembrane domains 3 to 5 and 6 to 8. Results of the present work also suggest that the use of Tris(+) as a substitute for Na(+) results in a biased estimate of the Hill number value for hDAT. This study provides useful clues for identifying specific residues involved in the uptake function of the Catecholamine transporters.