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

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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.

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Acknowledgments

This work was supported by a research grant from the Australian Research Council. TSK was supported by a University of Queensland Postgraduate Research Award. The authors thank Dr Brett A. Cromer for expert advice on interpreting the data.

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  1. Timothy Lynagh

    Present address: Neurophysiology and Neurosensory Systems, Technical University of Darmstadt, Schnittspahnstraße 3, 64287, Darmstadt, Germany

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  1. School of Biomedical Sciences, The University of Queensland, Brisbane, Australia

    Timothy Lynagh, Tina S. Khamu & Lesley J. Bryan-Lluka

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Correspondence to Timothy Lynagh.

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Lynagh, T., Khamu, T.S. & Bryan-Lluka, L.J. Extracellular loop 3 of the noradrenaline transporter contributes to substrate and inhibitor selectivity. Naunyn-Schmiedeberg's Arch Pharmacol 387, 95–107 (2014). https://doi.org/10.1007/s00210-013-0923-7

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