Neurotransmitter depletion by bafilomycin is promoted by vesicle turnover

Abstract

Accumulation of neurotransmitter into synaptic vesicles is powered by the vacuolar proton ATPase. We show here that, in brain slices, application of the H(+)-ATPase inhibitors bafilomycin or concanamycin does not efficiently deplete glutamatergic vesicles of transmitter unless vesicle turnover is increased. Simulations of vesicle energetics suggest either that bafilomycin and concanamycin act on the H(+)-ATPase from inside the vesicle, or that the vesicle membrane potential is maintained after the H(+)-ATPase is inhibited.

Figure 1. Effect of veratridine on glutamatergic vesicles depletion by bafilomycin.

A, Synaptic currents evoked at -33mV in CA1 pyramidal neurons by increasing strength of stimulation (at 20, 40, 60, 80 and 100V) of the Schaffer collaterals (in the presence of 100 μM picrotoxin to block GABA_A receptors), in control (Ctl) solution or after soaking slices in 4 μM bafilomycin (Baf) alone. B, Mean EPSCs from experiments as in A (10 control cells, 10 interleaved bafilomycin cells). C, When veratridine (10 μM) was added to the pre-soaking solution for 5 min, EPSCs were almost abolished in bafilomycin-treated cells. D, Mean data from experiments as in C (10 control cells, 10 interleaved bafilomycin cells).

Figure 2. Effects of veratridine and elevated [K⁺] solution on glutamatergic vesicle depletion by concanamycin.

A, Soaking slices in 2μM concanamycin alone had no significant effect on the amplitude of the glutamatergic EPSCs evoked in CA1 pyramidal neurons at -33mV by stimulating the Schaffer collaterals with increasing intensity (4 control cells (Ctl), 4 interleaved concanamycin cells (Conca)). B, When 10 μM veratridine was added to the soaking solution for 5 min, significantly smaller EPSCs were recorded in concanamycin-treated cells than control cells (13 control cells, 10 interleaved concanamycin cells). C, When slices were exposed to an elevated [K⁺] solution for 5 min at the beginning of the soaking period, significantly smaller EPSCs were recorded in concanamycin-treated cells than in control cells (5 control cells, 5 interleaved concanamycin cells).

Figure 3. Simulation of the effect of blocking the vesicular ATPase on the vesicle.

A Membrane potential, B [H⁺], and C glutamate accumulation. At time zero, exocytosis is assumed to equibibrate the vesicle contents with the extracellular space. The vesicle membrane potential is initially negative because vesicular [glutamate] is lower than that in the cytoplasm. The ATPase energises the vesicle for 100 sec and is then blocked (“inhibit ATPase”). Simulations are shown for a vesicle with conductance (g) to glutamate only, to glutamate and H⁺, or to glutamate and Cl^-.