chi-Conopeptide MrIA partially overlaps desipramine and cocaine binding sites on the human norepinephrine transporter.
The interactions of chi-conopeptide MrIA with the human norepinephrine transporter (hNET) were investigated by determining the effects of hNET point mutations on the inhibitory potency of MrIA. The mutants were produced by site-directed mutagenesis and expressed in COS-7 cells. The potency of MrIA was greater for inhibition of uptake by hNET of [3H]norepinephrine (Ki 1.89 microM) than [3H]dopamine (Ki 4.33 microM), and the human dopamine transporter and serotonin transporter were not inhibited by MrIA (to 7 microM). Of 18 mutations where hNET amino acid residues were exchanged with those of the human dopamine transporter, MrIA had increased potency for inhibition of [3H]norepinephrine uptake for three mutations (in predicted extracellular loops 3 and 4 and transmembrane domain (TMD) 8) and decreased potency for one mutation (in TMD6 and intracellular loop (IL) 3). Of the 12 additional mutations in TMDs 2, 4, 5, and 11 and IL1, three mutations (in TMD2 and IL1) had reduced MrIA inhibitory potency. All of the other mutations tested had no influence on MrIA potency. A comparison of the results with previous data for desipramine and cocaine inhibition of norepinephrine uptake by the mutant hNETs reveals that MrIA binding to hNET occurs at a site that is distinct from but overlaps with the binding sites for tricyclic antidepressants and cocaine.
T10 T11 T12 T13 T14 T15 T16 T17 T18 T19 T1 T20 T21 T22 T23 T24 T25 T26 T27 T2 T3 T4 T5 T6 T7 T8 T9
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norepinephrine dopamine [3H]norepinephrine desipramine cocaine norepinephrine hNETs hNET hNET hNET desipramine human dopamine transporter serotonin transporter human norepinephrine transporter hNET human dopamine transporter hNET desipramine and cocaine binding sites human norepinephrine transporter norepinephrine tricyclic cocaine [3H]norepinephrine [3H]dopamine dopamine serotonin amino acid