Transporters in Motion: Combining Computational Approaches and LRET-Measurements

Abstract

Synaptic transmission is regulated by the coordinated action of neurotransmitter release and reuptake. Specialized secondary-active neurotransmitter transporter proteins mediate the presynaptic reuptake of the respective neurotransmitters. Thereby, transporters re-accumulate the neurotransmitter in an economical fashion from the synaptic cleft into the presynaptic specialization by using the pre-established sodium gradient as a driving force. Two major classes can be distinguished: The During the last decade, crystal structures of archeal homologues of mammalian transporters have been published in various states. These structures serve as starting templates to study the structure function relationship in the mammalian counterparts. Hence, we combine molecular dynamics simulations and homology modeling with Lanthanide resonance energy transfer (LRET) measurements to explore substrate translocation in bacterial and mammalian transporters in a triangulated manner. We use the leucine/alanine transporter from Aquifex aeolicus (LeuTAa), the aspartate transporter from Pyrococcus horikoshi (GltPh) and the mammalian excitatory amino acid transporter 3 (EAAT3) and complement our key findings using biochemical studies. Using this multi-facetted approach allows us to ascertain the molecular movements of neurotransmitter transporters and their bacterial homologues and compare our results to the results obtained in crystallization experiments.