Extracellular loop 3 of the noradrenaline transporter contributes to substrate and inhibitor selectivity

Lynagh, Timothy, Khamu, Tina S. and Bryan-Lluka, Lesley J. (2014) Extracellular loop 3 of the noradrenaline transporter contributes to substrate and inhibitor selectivity. Naunyn-Schmiedebergs Archives of Pharmacology, 387 1: 95-107. doi:10.1007/s00210-013-0923-7

Author Lynagh, Timothy
Khamu, Tina S.
Bryan-Lluka, Lesley J.
Title Extracellular loop 3 of the noradrenaline transporter contributes to substrate and inhibitor selectivity
Journal name Naunyn-Schmiedebergs Archives of Pharmacology   Check publisher's open access policy
ISSN 0028-1298
Publication date 2014-01-01
Year available 2013
Sub-type Article (original research)
DOI 10.1007/s00210-013-0923-7
Volume 387
Issue 1
Start page 95
End page 107
Total pages 13
Place of publication Heidelberg, Germany
Publisher Springer
Language eng
Abstract The human noradrenaline transporter (NET) and 5-hydroxytryptamine (5-HT) transporter (SERT) are inhibited by antidepressants and psychoactive drugs such as cocaine. Both substrates and inhibitors bind in the transmembrane core of the protein, but molecular divergence at the binding site is not sufficient to account for the NET-selective and SERT-selective inhibition of the antidepressants, desipramine and citalopram, respectively. We considered that the poorly conserved third extracellular loop may contribute to these differences. We substituted single amino acid residues of the third extracellular loop in NET for equivalents from SERT, transiently transfected COS-7 cells with WT NET, 13 mutant NETs and WT SERT, and measured [ 3H]noradrenaline uptake, [3H]nisoxetine binding and [ 3H]5-HT uptake. Mutants F299W, Y300Q, R301K and K303L, at the C-terminal end of EL3, all showed significantly decreased [3H] nisoxetine binding, indicative of a reduced cell surface expression. Most mutants differed little, if at all, from WT NET regarding [3H] noradrenaline uptake; however, the I297P mutant showed no significant uptake activity despite intact cell surface expression, and the A293F mutant showed a significantly slower transporter turnover than WT NET in addition to [ 3H]5-HT uptake that was significantly greater than that of WT NET. The A293F mutation also decreased desipramine potency and increased the inhibition of [3H]noradrenaline uptake by citalopram compared to WT NET. These results suggest that the third extracellular loop allosterically regulates the ability of the transmembrane domains to transport substrates and bind inhibitors and thus contributes to the selectivity of substrates and antidepressants for NET and SERT.
Keyword Noradrenaline
Extracellular loop
Transmembrane domain
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ
Additional Notes Published online: 1 October 2013.

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2014 Collection
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