The intermediate resolution model which we have generated bares a striking resemblance to other known channel structures, in particular the gap junction and mechanosensitive channels, both of which have similar conductances to the Syp1 channel (). The overall dimensions of these channels are quite similar as well. Our Syp1 structure has an outer diameter of ~70 Å, with an inner diameter of ~30 Å; these are the similar dimensions of connexin (Unger et al., 1999
) and very close to the dimensions of MscS (80 Å diameter, 25 Å pore) (Bass et al., 2002
). The apparently loose intersubunit intramembrane interactions suggest how Syp1 may interact with Syb and function as a mechanosensitive channel where the SNARE complex formation drives disassembly of the Syp1/Syb multimer leading to channel opening and neurotransmitter release (). Recent work has shown that the fusion pore involved in neurotransmitter release has a conductance of ~.375 nS and that the potential pore diameter based on this conductance should be in the order of >2.3 nm (He et al., 2006
). These numbers correlate very well with the three dimensional structure we have generated.
Comparison of the synaptophysin I complex with other known channel structures
Model for Synaptophysin I (Syp1) complex involvement in vesicle fusion
There are two observed mechanisms of vesicle fusion and recycling at the pre-synaptic active zone: full fusion and transient fusion (also known as Kiss-and-Run) (Sudhof, 2000
). In the full fusion model, SVs are docked at the pre-synaptic membrane by a potential interaction of Syb on the vesicle surface and syntaxin (Syx) on the plasma membrane ( step 1). These proteins are thought to potentially be held together by SNAP25 and complexin (Tang et al., 2006
). Upon depolarization of the neuronal membrane and influx of Ca2+
, Synaptotagmin I (SytI) binds to the SNARE complex and its Ca2+
binding C2 domains form a tight electrostatic interaction with the plasma membrane. A subsequent coiled coil is formed by the SNARE complex (Syx, Syb, and SNAP-25) thus drawing in the SV to fully fuse with the plasma membrane ( step 2 & 3) (Hui et al., 2006
). During transient fusion the SV is also drawn in close to the plasma membrane upon depolarization and Ca2+
influx. However, the SV never fully fuses with the plasma membrane, only a transient fusion pore is formed for neurotransmitter release and the intact SV is eventually released back from the membrane and re-primed for another fusion event ( step 2* & 3*). Currently, the molecular mechanism of transient fusion is not known. Multiple proteins have been proposed as contributors and/or regulators of transient fusion, including Syp1, however, the roles of these proteins has not been confirmed (Fernandez-Alfonso and Ryan, 2004
; Harata et al., 2006
; Wang et al., 2006
; Wang et al., 2003
Based on our structure and published biochemical data on Syp1 and other SV proteins including SytI, Syb, Syx, and synaptotagmin IV (SytIV), we have derived the following model for a possible role of Syp1 in pore formation during full and transient SV fusion. Syb is known to bind to multiple SV proteins exclusive of SNARE complex formation including Syp1 and SytIV [17,26]. SytIV has been implicated in transient fusion (Wang et al., 2003
), although to what extent is unclear. Increased expression of SytIV leads to an increase in kiss-and-run events, and removal of SytIV leads to a disability in memory function associated with neurotransmitter release (Ferguson et al., 2000
) and a loss of SVs (Dean C., Arthur C., Bhalla A., Liu H., Chang P., Stowell M., Jackson M. B., & Chapman E. R. Activity dependent modulation of synaptic vesicle composition regulates synaptic plasticity. submitted) SytIV has also been shown to compete with SytI for SNARE binding (Machado et al., 2004
). We propose that the interaction of SytIV with the Syb/Syp1 complex can regulate the choice between kiss-and-run and full fusion. In our model, Syb and Syp1 are bound in a multimeric complex with SytIV prior to the SV being docked at the active zone ( step 1). Upon membrane depolarization, SytIV binds weakly with the pre-synaptic membrane, Syb is inhibited by Syp1 from entering the coiled-coil SNARE complex and the Syb/Syp1 complex remains stable forming a fusion pore potentially with Syx, another complex on the plasma membrane which has been shown to be active in neurotransmitter release ( step 2*) (Han et al., 2004
). The SytIV/plasma membrane interaction is not as strong as the SytI/plasma membrane interaction and no full SNARE complex is formed. The Syx/Syp1 fusion pore dissociates, potentially upon interaction with dynamin and the SV does not go through full fusion ( step 3*). In this model the exclusion of SytI from the binding machinery inhibits the vesicle from undergoing complete exocytosis. In the full-fusion model, the strong interaction of SytI with the plasma membrane allows the formation of the complete SNARE complex and this strong interaction acts to dissociate Syb from the Syp1 complex thus allowing the Syp1 complex to destabilize and full vesicle fusion to occur ( step 3).
The four transmembrane-helix architecture which makes up the MARVEL domain in Syp1 is seen in a number of other protein families including the myelin and lymphocyte protein (MAL), synaptogyrin and occluding (Sanchez-Pulido et al., 2002
). The involvement of all of these MARVEL domain proteins with membrane contact and interaction is strong evidence for Syp1’s role in vesicle membrane interaction at the plasma membrane. The structure of Syp1 described here provides the first three dimensional data for a MARVEL domain protein and suggests how Syp1 may form a fusion pore complex which regulates synaptic vesicle exocytosis. While further studies are required to fully elucidate the function and mechanism of SypI, as well as other MARVEL domain proteins, our results provide a structural foundation for understanding these important and ubiquitous proteins.