Involvement of the NH2 terminal domain of catecholamine transporters in the Na+ and Cl--dependence of a [H-3]-dopamine uptake

1 The ionic dependence of the [H-3]-dopamine uptake was studied in transfec ted cells expressing the human neuronal transporter fur dopamine (hDAT) or noradrenaline (hNET), and chimeric transporters resulting from the symmetri cal exchange of the region from the NH2 terminal through the first two tran smembrane domains (cassette I). Chimera A is formed by hDAT comprising cass ette I from hNET, whereas chimera B corresponds to the reverse construct. 2 The appearance or the intensity of a Cl -independent component of transpo rt was linked to the presence of the COOH terminal part of hNET in both mon oclonal and polyclonal Ltk cells (Cl- substituted by isethionate and NO3-, respectively), and in transiently transfected COS-7 cells. 3 Cassette I was also involved in the Cl- -dependence because the transport activity of polyclonal Ltk cells expressing A was partly Cl--independent a nd because Ltk(-) cells expressing transporters containing cassette I of hD AT displayed higher K-mCl- values than cells expressing the reverse constru cts. 4 In monoclonal Ltk(-) cell lines, K-mNa+ values and biphasic vs monophasic dependence upon Na+ concentrations differentiate transporters containing c assette I of hNET from those containing cassette I of hDAT. Tn COS-7 cells, the exchange of cassette I produced a significant change in Hill number va lues. 5 In Na+-dependence studies, exchange of the COOH terminal part significant ly modified Hill number values in both Ltk and COS-7 cells. 6 Hill number values close to two were found for hNET and hDAT when sucrose was used as substitute for NaCl. 7 The NH2 terminal part of the transporters bears some of the differences i n the Na+ and Cl--dependence of the uptake that are observed between hDAT a nd hNET. Present results also support a role of the: COOH terminal part in the ionic dependence. British Journal of Pharmacology (2001).