Vesicular glutamate transporters are H+-anion exchangers that operate at variable stoichiometry.

Vesicular glutamate transporters accumulate glutamate in synaptic vesicles, where they also function as a major Cl- efflux pathway. Here we combine heterologous expression and cellular electrophysiology with mathematical modeling to understand the mechanisms underlying this dual function of rat VGLUT1. When glutamate is the main cytoplasmic anion, VGLUT1 functions as H+-glutamate exchanger, with a transport rate of around 600 s-1 at -160 mV. Transport of other large anions, including aspartate, is not stoichiometrically coupled to H+ transport, and Cl- permeates VGLUT1 through an aqueous anion channel with unitary transport rates of 1.5 × 105 s-1 at -160 mV. Mathematical modeling reveals that H+ coupling is sufficient for selective glutamate accumulation in model vesicles and that VGLUT Cl- channel function increases the transport efficiency by accelerating glutamate accumulation and reducing ATP-driven H+ transport. In summary, we provide evidence that VGLUT1 functions as H+-glutamate exchanger that is partially or fully uncoupled by other anions.

An amino-terminal point mutation increases EAAT2 anion currents without affecting glutamate transport rates.

Excitatory amino acid transporters (EAATs) are prototypical dual function proteins that function as coupled glutamate/Na+/H+/K+ transporters and as anion-selective channels. Both transport functions are intimately intertwined at the structural level: Secondary active glutamate transport is based on elevator-like movements of the mobile transport domain across the membrane, and the lateral movement of this domain results in anion channel opening. This particular anion channel gating mechanism predicts the existence of mutant transporters with changed anion channel properties, but without alteration in glutamate transport. We here report that the L46P mutation in the human EAAT2 transporter fulfills this prediction. L46 is a pore-forming residue of the EAAT2 anion channels at the cytoplasmic entrance into the ion conduction pathway. In whole-cell patch clamp recordings, we observed larger macroscopic anion current amplitudes for L46P than for WT EAAT2. Rapid l-glutamate application under forward transport conditions demonstrated that L46P does not reduce the transport rate of individual transporters. In contrast, changes in selectivity made gluconate permeant in L46P EAAT2, and nonstationary noise analysis revealed slightly increased unitary current amplitudes in mutant EAAT2 anion channels. We used unitary current amplitudes and individual transport rates to quantify absolute open probabilities of EAAT2 anion channels from ratios of anion currents by glutamate uptake currents. This analysis revealed up to 7-fold increased absolute open probability of L46P EAAT2 anion channels. Our results reveal an important determinant of the diameter of EAAT2 anion pore and demonstrate the existence of anion channel gating processes outside the EAAT uptake cycle.