The C Terminus of SNAP25 Is Essential for Ca2+-dependent Binding of Synaptotagmin to SNARE Complexes

Abstract

Regulated neurotransmitter secretion is a specialized version of a general membrane fusion mechanism in which exocytotic fusion is strictly Ca 2ϩ -regulated. Studies of this process have yielded insights into universal mechanisms for intracellular membrane fusion and the identity of core components of the fusion machinery (1-3). VAMP, 1 syntaxin, and SNAP25 are the neural protein substrates for clostridial neurotoxins, a family of highly selective proteases that potently inhibits neurosecretion (4, 5). These proteins are soluble N-ethylmaleimidesensitive factor attachment protein receptors (SNAREs) that mediate the membrane association of N-ethylmaleimide-sensitive factor, a protein required for membrane fusion (6). The SNARE proteins are capable of assembling into extremely stable heterotrimeric complexes (7-9) that consist of a four-helix bundle in parallel alignment (10 -13). A current hypothesis suggests that the formation of SNARE complexes in trans across apposing membranes promotes intimate bilayer interactions and provides the energy to drive membrane fusion (10,(13)(14)(15)(16) (1,18,22,23). The precise role of synaptotagmin and its Ca 2ϩ -dependent interactions with phospholipids or syntaxin in regulated neurosecretion remains to be determined.Studies of regulated exocytosis in membrane preparations from neuroendocrine cells demonstrated that SNAREs are required for a late Ca 2ϩ -dependent step that occurs after vesicle docking and ATP-dependent priming and immediately before fusion (28,29). Tetanus toxin and botulinum neurotoxin (BoNT) B, C1, and E completely inhibited the Ca 2ϩ -dependent triggering of exocytosis, which implied that VAMP, syntaxin, and SNAP25 participate in steps close to or at fusion. Paradoxically BoNT A, which like BoNT E cleaves SNAP25, was only partially inhibitory for triggered fusion despite efficient proteolysis of SNAP25 (28). Because these toxins cleave SNAP25 at distinct C-terminal sites (Arg 180 -Ile 181 for BoNT E and Gln 197 -Arg 198 for BoNT A; see Refs. 30 and 31), it was inferred that the domain within the C terminus of SNAP25 between the toxin cleavage sites plays a distinct role in Ca 2ϩ -dependent membrane fusion events (28). In the present study, this domain of SNAP25 is revealed to be essential for Ca 2ϩ -dependent interactions with synaptotagmin. This finding clarifies classical observations that BoNT A inhibition of neurosecretion is partially reversed by elevating neuronal Ca 2ϩ levels (32). Moreover, the results suggest that an important role for the C terminus of SNAP25 in regulated exocytosis is to mediate Ca 2ϩ -dependent interactions between synaptotagmin and SNARE protein complexes. EXPERIMENTAL PROCEDURES Assays for Ca2ϩ -activated Exocytosis-PC12 cells grown in Dulbecco's modified culture medium supplemented with 5% horse serum and * This work was supported in part by National Institutes of Health Research Grants DK25861 and DK40428 (to T. F. J. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This ar...

Botulinum toxin A, brain and pain

“…This was proposed to result from higher transient increase in calcium concentration upon depolarization in GABA-ergic neurons, in comparison to excitatory neurons (Grumelli et al, 2010). High concentrations of intracellular Ca 2+, in turn, have the ability to overcome the cleaved SNAP-25-mediated paralysis (Gerona et al, 2000;Grumelli et al, 2010;Lawrence et al, 2002).…”

The C2b Domain of Synaptotagmin Is a Ca2+–Sensing Module Essential for Exocytosis

“…This complex is composed of the target SNAREs (t-SNAREs) syntaxin and SNAP-25 and the vesicle SNARE synaptobrevin (Söllner et al 1993); assembly of the complex is necessary (Littleton et al 1998; Chen et al 1999) and may be sufficient (Weber et al 1998) for exocytotic membrane fusion. Synaptotagmin forms direct contacts with both t-SNAREs at all stages of the SNARE complex assembly (data not shown; Chapman et al 1995; Schiavo et al 1997; Davis et al 1999; Gerona et al 2000) and can bind SNAREs and interact with membranes at the same time (Davis et al 1999). These interactions suggest that synaptotagmin may regulate SNARE-mediated membrane fusion by modulating the conformation and/or assembly of SNARE complexes.…”

“…The C2A domain is connected to C2B by a short tether and the tandem C2 domains cooperate to form complexes with components of the soluble N -ethyl maleimide-sensitive factor attachment protein receptor (SNARE) complex (Chapman et al 1995, Chapman et al 1996; Davis et al 1999; Gerona et al 2000). This complex is composed of the target SNAREs (t-SNAREs) syntaxin and SNAP-25 and the vesicle SNARE synaptobrevin (Söllner et al 1993); assembly of the complex is necessary (Littleton et al 1998; Chen et al 1999) and may be sufficient (Weber et al 1998) for exocytotic membrane fusion.…”

Synaptotagmin-1 Utilizes Membrane Bending and SNARE Binding to Drive Fusion Pore Expansion

“…Ca 2ϩ binding to Syt-1 promotes the binding of tandem C2 domains to syntaxin-1 and SNAP25 as well as to SNARE protein complexes (Davis et al, 1999;Gerona et al, 2000;Earles et al, 2001;. Ca 2ϩ -dependent Syt-1-SNARE interactions are mediated in part by acidic residues in the SNAP25 C-terminus binding to basic residues in Syt-1 C2A loop 2 .…”

“…Ca 2ϩ binding imparts a positive electrostatic potential to the C2 domain surface that enables the binding of Syt-1 to its effectors by neutralizing acidic residues in the Ca 2ϩ -binding loops . The two major effectors for Syt-1 are acidic residues on the surface of SNARE complexes and anionic phospholipids in the plasma membrane.Ca 2ϩ binding to Syt-1 promotes the binding of tandem C2 domains to syntaxin-1 and SNAP25 as well as to SNARE protein complexes (Davis et al, 1999;Gerona et al, 2000;Earles et al, 2001; SNARE interactions are mediated in part by acidic residues in the SNAP25 C-terminus binding to basic residues in Syt-1 C2A loop 2 . Syt-1 mutations that eliminate Ca 2ϩ -dependent SNARE binding abrogate Ca 2ϩ -triggered dense-core vesicle exocytosis in PC12 cells , whereas mutations that enhance SNARE binding facilitate Ca 2ϩ -triggered synaptic vesicle exocytosis in neurons (Pang et al, 2006).…”