Transport dynamics in a glutamate transporter homologue

Glutamate transporters are integral membrane proteins that catalyze neurotransmitter uptake from the synaptic cleft into the cytoplasm of glial cells and neurons(1). Their mechanism involves transitions between extracellular- (outward-) and intracellular- (inward-) facing conformations, whereby substrate binding sites become accessible to the opposite sides of the membrane(2). This process has been proposed to entail trans-membrane movements of three discrete transport domains within a trimeric scaffold(3). Using single-molecule fluorescence resonance energy transfer (smFRET) imaging(4), we have directly observed large-scale transport domain movements in a bacterial homologue of glutamate transporters for the first time. We find that individual transport domains alternate between periods of quiescence and periods of rapid transitions, reminiscent of bursting patterns first recorded in single ion channels using patch-clamp methods(5,6). We suggest that the switch to the dynamic mode in glutamate transporters is due to separation of the transport domain from the trimeric scaffold, which precedes domain movements across the bilayer. This spontaneous dislodging of the substrate-loaded transport domain is approximately 100-fold slower than subsequent trans-membrane movements and may be rate determining in the transport cycle.