Interaction of the synprint site of N-type Ca ²⁺ channels with the C2B domain of synaptotagmin I

Abstract

N-type Ca 2؉ channels mediate Ca 2؉ inf lux, which initiates fast exocytosis of neurotransmitters at synapses, and they interact directly with the SNARE proteins syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) through a synaptic protein interaction (synprint) site in the intracellular loop connecting domains II and III of their ␣ 1B subunits. Introduction of peptides containing the synprint site into presynaptic neurons reversibly inhibits synaptic transmission, confirming the importance of interactions with this site in synaptic transmission. Here we report a direct interaction of the synprint peptide from N-type Ca 2؉ channels with synaptotagmin I, an important Ca 2؉ sensor for exocytosis, as measured by an affinity-chromatography binding assay and a solid-phase immunoassay. This interaction is mediated by the second C2 domain (C2B) of synaptotagmin I, but is not regulated by Ca 2؉ . Using both immobilized recombinant proteins and native presynaptic membrane proteins, we found that the synprint peptide and synaptotagmin competitively interact with syntaxin. This interaction is Ca 2؉ -dependent because of the Ca 2؉ dependence of the interactions between syntaxin and these two proteins. These results provide a molecular basis for a physical link between Ca 2؉ channels and synaptotagmin, and suggest that N-type Ca 2؉ channels may undergo a complex series of Ca 2؉ -dependent interactions with multiple presynaptic proteins during neurotransmission.During synaptic transmission, exocytosis of synaptic vesicles in neurons is initiated by the rapid influx of Ca 2ϩ through voltage-gated Ca 2ϩ channels within 200 sec. Major progress has been made toward the understanding of the molecular mechanisms that underlie Ca 2ϩ -dependent exocytosis by identifying proteins that are involved in this process and analyzing their interactions (1, 2). Vesicle docking and fusion are mediated by a core complex of proteins including the synaptic vesicle protein VAMP͞synaptobrevin (3) and the plasmalemmal proteins syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) (4-11). The synaptic vesicle protein p65 or synaptotagmin (12) binds Ca 2ϩ (13,14) and interacts with syntaxin in a Ca 2ϩ -dependent manner (15,16). It is thought to serve as a Ca 2ϩ sensor for fast, Ca 2ϩ -dependent neurotransmitter release (17)(18)(19)(20)(21)(22).N-type Ca 2ϩ channels are localized in nerve terminals (23-25) and participate in neurotransmitter release in central and peripheral synapses (26-28). They are coimmunoprecipitated and copurified with proteins of the synaptic core complex in immunochemical studies (4,5,29), and they are closely associated with sites of transmitter release in physiological experiments (30). The ␣ 1B subunits of N-type Ca 2ϩ channels (31, 32) interact with the synaptic core complex through a synaptic protein interaction (synprint) site in the intracellular loop connecting domains II and III (33, 34) in a Ca 2ϩ -dependent manner with maximum binding in the range of 10-30 M (35). Peptides containing th...

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

“…This was achieved by analyzing the effects of the synprint peptide on secretion. Synprint corresponds to the II-III loop of N-type Ca 2+ channels and binds to t-SNAREs and to synaptotagmin (Kim and Catterall 1997; Sheng et al 1997). A critical point is that synprint binds to synaptotagmin in a Ca 2+ -independent manner and can block Ca 2+ -triggered synaptotagmin-clustering (Chapman et al 1998).…”

“…Soluble synaptotagmin fragments were prepared by thrombin cleavage of GST fusion proteins using thrombin (Chapman et al 1996). His 6 -synprint (Sheng et al 1997) was prepared as previously described (Chapman et al 1998). …”

“…This question was prompted by the finding that mutations within the C2B domain of Drosophila synaptotagmin impair excitation-secretion coupling (DiAntonio and Schwarz 1994; Littleton et al 1993, Littleton et al 1994). A number of C2B–effector interactions have been identified including the following: AP-2 (Zhang et al 1994; Jorgensen et al 1995), SV2 (Schivell et al 1996), β-SNAP (Schiavo et al 1995), Ca 2+ channels (Kim and Catterall 1997; Sheng et al 1997), inositol polyphosphates (Fukuda et al 1995) and, finally, homo-oligomerization (Chapman et al 1996; Sugita et al 1996). Of these interactions, only oligomerization was promoted by Ca 2+ .…”

Old proteins, developing roles: The regulation of calcium channels by synaptic proteins

“…Interestingly, this formation can also occur independent of calcium binding. 89 In addition to binding the SNARE complex, the synaptotagmin I C2B and C2A domains bind the synprint motifs in N-type, 76,90,91 P/Q-type 92 and in an analogous II-III linker motif in R-type channels 53,93,94 which places the calcium binding domains intricately close to the source of calcium influx, helping to account for the speed of exocytosis.…”

“…As syntaxin 1A and synaptotagmin I compete for synprint binding, the functional effect of this mutation may be due to impairment of synaptotagmin's ability to reverse syntaxin 1A inhibition of the channel. The physical association of synaptotagmin I to the II-III linker is a requirement for N-type channel mediated exocytosis 90 although it is yet unknown if this interaction alone has any regulatory effect on the channel. Synaptotagmin I may thus be able to exert a modulatory effect on calcium channel function both directly and through modulating interactions with syntaxin 1A and SNAP-25.…”