Movements of N‐terminus of LeuT during the Substrate Transport Cycle

Abstract

LeuT from Aquifex aeolicus aeolicus is a bacterial homologue to mammalian solute carrier family 6 (SLC6) of neurotransmitter transporters. SLC6 transporters are of great pharmacological interests because of their crucial role in neurotransmitter clearance. These proteins are also targets of many clinically relevant drugs, including antidepressant drugs and drugs of abuse such as amphetamines. The amino terminus of SLC6 transporters plays an important regulatory role for function.High resolution X‐ray crystal structures of LeuT are available in different conformations. However, these structures are not able to fully explain the dynamic nature of LeuT function during the substrate transport cycle. The aim of this project is to look at the movements of the N terminus of LeuT in the proteoliposomes using spectroscopic based technique called lanthanide based resonance energy transfer (LRET).We developed a LeuT variant in which we inserted a Lanthanide Binding Tag (LBT) at position 335, positioned at the end of extracellular loop 4. Three LeuT mutants (K4C, H7C and A9C) were created in the background of the LeuT‐LBT construct. Using LRET based distance measurements we determined the distance between the LBT bound lanthanide and the fluorophore TMR attached to the cysteines in the N‐terminal of LeuT. The results did not show the expected increase in distance towards the N‐terminus, as the distance to H7C was shorter than to A9C. In contrast, iodate quenching experiments of TMR fluorescence showed no difference in accessibility to KI for all three residues.Intramolecular distances determined by LRET suggest that the fluorophore attached to H7 could be oriented towards the inner vestibule, while the KI based accessibility experiments displays a contrasting picture. In order to elucidate the dynamics of the N‐terminus and support one of the two findings, I am further planning to stabilize the transporter in an outward open conformation using tryptophan, and as an alternative strategy, stabilize LeuT‐LBT in the inward‐facing and an outward‐facing conformation using the R5A or the R30A mutants.Support or Funding InformationThis work is jointly supported by the Austrian Science Fund (FWF) Austria and Higher Education Commission (HEC) PakistanThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.