Franzen, A. | bioRxiv Science

bioRxiv2026

Evolutionary optimization of allosteric activation by Cl- and Cl- conduction in vesicular glutamate transporters

Vesicular glutamate transporters harness proton gradients to load glutamate into synaptic vesicles, while mediating luminal chloride efflux through a channel-like conductance. We combined heterologous expression in mammalian cells, whole-cell patch-clamp recordings and mathematical modeling to functionally characterize the Drosophila melanogaster vesicular glutamate transporter DVGLUT and to compare it to a rat counterpart, rVGLUT1. As in mammalian VGLUTs, DVGLUT glutamate transport is coupled to proton exchange, in a 1:1 stoichiometry. In both, luminal Cl- is necessary as allosteric activator, however, the allosteric affinity is higher in fly than in rat transporters. DVGLUT anion channels exhibit lower unitary currents, but higher anion channel open probabilities, resulting in larger Cl- currents for the fly transporter in the presence of cytoplasmic glutamate. The higher allosteric affinity together with the enhanced anion channel activity may serve as evolutionary adaptation of VGLUTs to lower ion concentrations in Drosophila, illustrating the impact of these particular features for synaptic vesicle filling.

neuroscience↗

bioRxiv2020

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

Excitatory amino acid transporters (EAATs) are prototypic 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 fulfils 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. Moreover, changes in selectivity made gluconate permeant in L46P EAAT2. Non-stationary noise analysis revealed unchanged unitary current amplitudes in mutant EAAT2 anion channels. Rapid L-glutamate application under forward transport conditions demonstrated that L46P does not reduce the transport rate of individual transporters. Since individual transport rates and unitary anion current amplitudes are unaffected, absolute open probabilities of EAAT2 anion channels were quantified as ratio of anion currents by glutamate uptake currents. We found up to sevenfold 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.

physiology↗