Lithium Stabilizes the Inward Facing Conformation of Human Serotonin Transporter (hSERT)

Abstract

According to the alternate access model of transport, a transporter such as hSERT adopts two major conformations: the outward facing conformation and the inward facing conformation. For a functional transporter the distribution of these states in equilibrium (with and without substrate) is unknown. Furthermore it is unclear how this distribution is affected by various parameters such as the temperature, the external and internal ion composition, the membrane potential etc. Here we tested the effect of various external cations such as sodium, lithium and NMDG on the conformational equilibrium. In this study we probed the conformational equilibrium using three different techniques. First we utilized intramolecular FRET measurements. This was done utilizing a construct of hSERT that had been genetically modified to contain a CFT at the N-terminal end and a YFP at the C-terminal end (CSERTY). CSERTY was heterologously expressed in HEK cells and FRET changes upon administration of different external ions were correlated with the data we obtained from binding of a radio-labeled inhibitor of SERT (second technique) as well as with the data we obtained by testing the accessibility of cysteines introduced by site directed mutagenesis to MTS reagents (third technique). Binding was conducted in membranes of HEK cells that stably expressed hSERT and for the accessibility studies we employed hSERT expressed in Xenopus laevis oocytes. In accordance with previous studies we find that external sodium supports the outward facing conformation. However for high external lithium concentrations we have evidence that the prevalent conformation is inward facing.