Transport Mechanisms of the Vesicular Glutamate Transporter 1

Abstract

In neuronal signal transmission, vesicular neurotransmitter transporters play a key role in quantal size modification. They are filling synaptic vesicles (SVs) with neurotransmitter, fuelled by an electrochemical gradient (ΔµH+) across the membrane which is generated by the vacuolar ATPase (V-ATPase). Understanding the regulation of SV filling will resolve fundamental questions regarding synaptic function. In particular, questions addressing the vesicular glutamate transporter (VGLUT), which is filling SVs with the excitatory neurotransmitter L-glutamate, such as the mechanism and ionic coupling, remain unanswered. For instance, it is not fully understood which mechanism underlies the biphasic dependence of glutamate uptake on Cl-. Furthermore, there are inconsistent reports on a Cl- conductance by VGLUT1. Recently, a regulatory mode exchanging luminal H+ for cytosolic K+ which is affecting glutamate uptake by changing ΔµH+ has been proposed. In this study, particularly the transport of Cl- by VGLUT1 and a putative K+/H+ exchange mechanism involved in the regulation of VGLUT1 were investigated in order to unravel the ionic coupling of glutamate transport. These questions were addressed in a minimal system with recombinant mouse VGLUT1 and a proton pump from bacillus thermophilus (TF0F1) in artificial membranes, which was established in this work. In reconstituted liposomes containing VGLUT1 and TF0F1, VGLUT1 did not exhibit any Cl- conductance in contrast to previous reports. Additionally, high luminal Cl- concentrations did not enhance glutamate uptake, conflicting with recent studies. This finding further supported a lack of Cl- conductance in VGLUT1. By studying SVs, a K+/H+ exchange mechanism was observed supporting previous studies. Remarkably, preliminary observations in the present work suggest that VGLUT is mediating K+/H+ exchange in SVs. Furthermore, a “hybrid” system of SVs fused with proteoliposomes containing TF0F1 and a fusion mediating machinery was established. The unique benefit of “fused SVs” is that SV contents can be efficiently manipulated, which hitherto has been a challenging task. Preliminary measurements indicate that SVs and TF0F1 maintain their activity after fusion. Fused SVs provide a unique opportunity to study the putative stimulatory effect of high luminal Cl- on glutamate uptake.