Involvement of the NH2 terminal domain of catecholamine transporters in the Na(2+) and Cl(-)-dependence of a [3H]-dopamine uptake.
The ionic dependence of the [3H]-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 NH2 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 NO3(-), 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 NH2 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.
Syringas
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M
UMR C.N.R.S. 6036, IFRMP 23, U.F.R. de Médecine & Pharmacie, 22 Boulevard Gambetta, 76000 Rouen, France.
Janin
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F
Giros
B
B
Costentin
J
J
Bonnet
J J
JJ
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002712:Chlorides / Q000494:pharmacology*; D002522:Chlorocebus aethiops; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins; D006801:Humans; D007700:Kinetics; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D050484:Norepinephrine Plasma Membrane Transport Proteins; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012964:Sodium / Q000494:pharmacology*; D012965:Sodium Chloride / Q000494:pharmacology; D013395:Sucrose / Q000494:pharmacology; D027981:Symporters; D014162:Transfection
D002352:Carrier Proteins; D002712:Chlorides; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D050484:Norepinephrine Plasma Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; C493227:SLC6A2 protein, human; D027981:Symporters; D012965:Sodium Chloride; D013395:Sucrose; D012964:Sodium; D004298:Dopamine