Neurotransmitter release through the V0 sector of V-ATPase.

Neurotransmitter release occurs at specialized areas of the nerve terminal membrane, the active zones, where clusters of synaptic vesicles, the neurotransmitter-storing organelles, are observed (Couteaux and PeÂcot-Dechavassine 1974; Harlow et al. 2001). In resting conditions, a population of synaptic vesicles is docked to the active zone membrane, close to voltage-gated calcium channels (Robitaille et al. 1990), within microdomains where, upon stimulation, cytosolic calcium reaches transiently a very high concentration (Llinas et al. 1992). In spite of the high specialization of the active zone structure and high speed of synaptic transmission, proteins involved in docking and fusion of synaptic vesicles are similar to those operating for much slower membrane fusions, from yeast to neurones (Wickner and Haas 2000). In this respect, the role of SNARE complexes for docking synaptic vesicles at the active zones has been well documented (Rothman 1994; Jahn and SuÈdhof 1999). A detailed genetic and pharmacological dissection of yeast homotypic vacuole fusion revealed the existence, after vacuole docking by trans-SNARE complex formation, of a Ca/calmodulin reaction preceeding the ®nal microcystininhibited step of membrane fusion (Wickner and Haas 2000). Recently, Peters et al. (2001) showed that it was the proteolipids of the membrane sector (V0) of V-ATPase which bind to calmodulin and initiate the ®nal step of membrane fusion. The vacuolar-type H-ATPase is indeed composed of a proteolipid membrane sector (V0) and a catalytic sector (V1). The association between V0 and V1 is reversible and participates in the regulation of proton pumping (Nelson and Harvey 1999). Reconstituted V0 proteolipids form a pore that opens in the presence of calcium and calmodulin. During the fusion of two yeast vacuoles, a V0 trans-complex is formed by the apposition of two proteolipid rings, brought into close contact by the SNARE proteins. The V0 trans-complex may therefore form a proteolipid channel spanning the two interacting membranes at the fusion site (Peters et al. 2001). We would like to discuss the relevance of this model for neurotransmitter release. Synaptosomal membranes were shown to contain a proteolipid oligomer that supported a calcium-dependent release of acetylcholine (ACh) when reconstituted in arti®cial membranes (IsraeÈl et al. 1986; see Fig. 1). This oligomer (mediatophore) turned out to be made of the proteolipid c subunit of V-ATPase (Birman et al. 1990). When cells were transfected for this proteolipid, they acquired a Ca-dependent ACh release mechanism that displayed quantal properties (Falk-Vairant et al. 1996; see Fig. 2). Such reconstitution experiments, using liposomes, transfected cells or Xenopus oocytes (Cavalli et al. 1993), showed that a single proteolipid ring not only opens upon calcium action but is suf®cient to let ACh out down its concentration gradient. This was con®rmed by Peters et al. (2001) who measured the release of choline through reconstituted yeast V-ATPase proteolipids, release that required Ca and, in this case, calmodulin. In synapses, the neurotransmitter is pre-concentrated in synaptic vesicles. This process depends on the proton gradient generated by the V-ATPase, and is blocked by N-N 0-dicyclohexylcarbodiimide (DCCD). In contrast, the ef ̄ux of ACh from already loaded synaptic vesicles is not affected by DCCD (Dolezal et al. 1993). This illustrates that ACh and protons follow different routes. Protons bind to a glutamic residue facing the exterior of the proteolipid ring (Harrison et al. 2000) and are translocated during the ATPdriven rotation of this ring (see Nelson and Harvey 1999 for a review on V-ATPases). ACh is most probably released through a pore found in the middle of the proteolipid oligomer by Jones et al. (1995).