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Involvement of the NH 2 terminal domain of catecholamine transporters in the Na + and Cl − ‐dependence of a [ 3 H]‐dopamine uptake
Author(s) -
Syringas Maria,
Janin François,
Giros Bruno,
Costentin Jean,
Bonnet JeanJacques
Publication year - 2001
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1038/sj.bjp.0704097
Subject(s) - transporter , chimera (genetics) , transfection , dopamine plasma membrane transport proteins , atp binding cassette transporter , biophysics , dopamine transporter , chemistry , biology , biochemistry , gene
The ionic dependence of the [ 3 H]‐dopamine uptake was studied in transfected cells expressing the human neuronal transporter for dopamine (hDAT) or noradrenaline (hNET), and chimeric transporters resulting from the symmetrical exchange of the region from the NH 2 terminal through the first two transmembrane domains (cassette I). Chimera A is formed by hDAT comprising cassette I from hNET, whereas chimera B corresponds to the reverse construct. The appearance or the intensity of a Cl − ‐independent component of transport was linked to the presence of the COOH terminal part of hNET in both monoclonal and polyclonal Ltk − cells (Cl − substituted by isethionate and NO 3 − , respectively), and in transiently transfected COS‐7 cells. Cassette I was also involved in the Cl − ‐dependence because the transport activity of polyclonal Ltk − cells expressing A was partly Cl − ‐independent and because Ltk − cells expressing transporters containing cassette I of hDAT displayed higher K mCl − values than cells expressing the reverse constructs. In monoclonal Ltk − cell lines, K mNa + values and biphasic vs monophasic dependence upon Na + concentrations differentiate transporters containing cassette I of hNET from those containing cassette I of hDAT. In COS‐7 cells, the exchange of cassette I produced a significant change in Hill number values. In Na + ‐dependence studies, exchange of the COOH terminal part significantly modified Hill number values in both Ltk − and COS‐7 cells. Hill number values close to two were found for hNET and hDAT when sucrose was used as substitute for NaCl. The NH 2 terminal part of the transporters bears some of the differences in the Na + and Cl − ‐dependence of the uptake that are observed between hDAT and hNET. Present results also support a role of the COOH terminal part in the ionic dependence.British Journal of Pharmacology (2001) 133 , 387–394; doi: 10.1038/sj.bjp.0704097