Dual and Direction-Selective Mechanisms of Phosphate Transport by the Vesicular Glutamate Transporter

Vesicular glutamate transporters (VGLUTs) fill synaptic vesicles with glutamate and are thus essential for glutamatergic neurotransmission. However, VGLUTs were originally discovered as members of a transporter subfamily specific for inorganic phosphate (P_i). It is still unclear how VGLUTs accommodate glutamate transport coupled to an electrochemical proton gradient ΔμH⁺ with inversely directed P_i transport coupled to the Na⁺ gradient and the membrane potential. Using both functional reconstitution and heterologous expression, we show that VGLUT transports glutamate and P_i using a single substrate binding site but different coupling to cation gradients. When facing the cytoplasm, both ions are transported into synaptic vesicles in a ΔμH⁺-dependent fashion, with glutamate preferred over P_i. When facing the extracellular space, P_i is transported in a Na⁺-coupled manner, with glutamate competing for binding but at lower affinity. We conclude that VGLUTs have dual functions in both vesicle transmitter loading and P_i homeostasis within glutamatergic neurons.