Neurotransmitter corelease: mechanism and physiological role

Abstract

Neurotransmitter identity is a defining feature of all neurons because it constrains the type of information they convey, but many neurons release multiple transmitters. Although the physiological role for corelease has remained poorly understood, the vesicular uptake of one transmitter can regulate filling with the other by influencing expression of the H(+) electrochemical driving force. In addition, the sorting of vesicular neurotransmitter transporters and other synaptic vesicle proteins into different vesicle pools suggests the potential for distinct modes of release. Corelease thus serves multiple roles in synaptic transmission.

Figure 1. Vesicular neurotransmitter transporters depend differentially on the chemical and electrical components of the H⁺ electrochemical gradient

The vacuolar-type H⁺-ATPase generates the H⁺ electrochemical gradient (Δμ_H⁺) required for transport of all classical neurotransmitters into synaptic vesicles. However, different vesicular neurotransmitter transporters rely to differing extents on the two components of Δμ_H⁺, the chemical gradient (ΔpH) and the electrical gradient (Δψ). The vesicular accumulation of monoamines and ACh (left) involves the exchange of protonated cytosolic transmitter for two lumenal H⁺. The resulting movement of more H⁺ than charge dictates a greater dependence on ΔpH than Δψ for both VAChT and VMAT. Vesicular glutamate transport (right) may not involve H⁺ translocation. In the absence of Δψ, however, disruption of ΔpH inhibits uptake, suggesting that the transport of anionic glutamate involves exchange for nH⁺, resulting in the movement of n + 1 charge and hence greater dependence on Δψ than ΔpH. Transport of the neutral zwitterion GABA (and glycine) involves the movement of an equal number of H⁺ and charge, consistent with the similar dependence of VGAT on ΔpH and Δψ. These differences suggest that vesicles storing monoamines or ACh may have mechanisms to favor the accumulation of ΔpH at the expense of Δψ, whereas those storing glutamate may promote a larger Δψ. The extent to which vesicles differ in their expression of these two components remains unknown, but intracellular chloride carriers such as the synaptic vesicle-associated ClC-3 promote vesicle acidification by dissipating the positive Δψ developed by the vacuolar H⁺ pump, thereby disinhibiting the pump to make larger ΔpH. The VGLUTs can also contribute to formation of ΔpH because as an anion, glutamate entry similarly dissipates Δψ to promote ΔpH. Interestingly, a Cl⁻ conductance associated with the VGLUTs may also promote acidification by Cl⁻.

Figure 2. Glutamate flux produces larger and more stable changes in vesicular ΔpH than chloride

Changes in ΔpH of isolated synaptic vesicles were monitored using acridine orange (5 μM) in 140 mM choline gluconate, 10 mM K⁺ gluconate, 10 mM HEPES, pH 7.4. Acidification was triggered by the sequential addition of 1 mM ATP and 2 mM Cl⁻ followed by either 14 mM Cl⁻ (top left) or 4 mM glutamate (top right); more Cl⁻ is required to produce an equivalent initial change in ΔpH. The traces in black indicate vesicles without any further addition. At the arrow, the K⁺ ionophore valinomycin (50 nM, gray), the proton pump inhibitor bafilomycin (250 nM, dark blue/red) or both (light blue/pink) were added. The rate of alkalinization immediately after bafilomycin addition (dark blue/red) is much faster in the vesicles acidified with Cl⁻, indicating that vesicles acidified with glutamate maintain a more stable ΔpH. Although increased buffering may contribute to the stabilization of ΔpH by glutamate, valinomycin accelerates the bafilomycin-induced collapse in ΔpH across membranes acidified with glutamate (pink) but not with Cl⁻ (light blue), indicating an important role for negative Δψ in the stability of ΔpH in glutamate-acidified vesicles. We hypothesize that the negative Δψ developing upon H⁺ efflux impedes further dissipation of ΔpH. In the case of vesicles acidified with Cl⁻, anion efflux through a channel (bottom left) would shunt the developing negative Δψ, allowing the continued efflux of H⁺ and rapid collapse of ΔpH. In the case of vesicles acidified with glutamate, a H⁺ : anion exchange mechanism (bottom right) would impede anion efflux because it would be coupled to the uphill movement of H⁺ into acidic vesicles. Since glutamate efflux is disfavored, H⁺ efflux is slow and ΔpH more stable. Thus, the differences in mechanism of anion flux (channel versus H⁺ exchange) confer differences in the stability of ΔpH. Glutamate thus serves to ‘lock’ H⁺, and hence cationic transmitters such as ACh and monoamines, inside secretory vesicles.