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. 2004 Dec 21;101(51):17855-60.
doi: 10.1073/pnas.0408200101. Epub 2004 Dec 10.

Vesicular reuptake inhibition by a synaptotagmin I C2B domain antibody at the squid giant synapse

Rodolfo R Llinás  1 , Mutsuyuki Sugimori, Kimberly A Moran, Jorge E Moreira, Mitsunori Fukuda
Affiliations

Vesicular reuptake inhibition by a synaptotagmin I C2B domain antibody at the squid giant synapse

Rodolfo R Llinás et al. Proc Natl Acad Sci U S A. .

Abstract

Synaptotagmin (Syt) I, a ubiquitous synaptic vesicle protein, comprises a transmembrane region and two C2 domains. The C2 domains, which have been shown to be essential for both synaptic vesicle exocytosis and endocytosis, are also seen as the Ca(2+) sensors in synaptic vesicular release. In a previous study, we reported that a polyclonal antibody raised against the squid (Loligo pealei) Syt I C2B domain, while inhibiting vesicular endocytosis, was synaptic release neutral at the squid giant synapse. Recent reports concerning the C2B requirements for synaptic release prompted us to readdress the role of C2B in squid giant synapse function. Presynaptic injection of another anti-Syt I-C2B antibody (using recombinant whole C2B domain expressed in mammalian cell culture as an antigen) into the presynaptic terminal reproduced our previous results, i.e., reduction of vesicular endocytosis without affecting synaptic release. This set of results addresses the issue of the geometrical arrangement of the Ca(2+) sensor, allowing the C2B domain antibody to restrict Ca(2+)-dependent C2B self-oligomerization without modifying the Ca(2+)-dependent release process.

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Figures

Fig. 1.
Fig. 1.
Characterization of the anti-mSyt I-C2B antibody. (A) Immunoblots showing the specificity of the anti-mSyt I-C2B with the total homogenates of COS-7 cells expressing T7-Syt I-cyto, T7-Syt I-C2A, or T7-Syt I-C2B. Recombinant T7-tagged Syts were subjected to SDS/12.5% PAGE and transferred to a poly(vinylidene difluoride) membrane. The blots were first probed with the anti-mSyt I-C2B antibody (0.5 μg/ml; Left). The same blots were stripped and reprobed with HRP-conjugated anti-T7 tag antibody (1/10,000 dilution) to ensure loading of the same amounts of T7-Syt proteins (Right). Note that the anti-mSyt I-C2B antibody specifically recognized the C2B domain (lane 3), but not the C2A domain (lane 2), even after prolonged exposure to x-ray film (data not shown). (B) Effect of the anti-mSyt I-C2B antibody on Ca2+-dependent oligomerization of the C2B domain. Purified T7-mSyt I-C2B domain (Bottom) was coupled with the anti-T7 tag antibody-conjugated agarose and incubated with FLAG-Syt cytoplasmic domain in the presence of 2 mM EGTA (lane 1) or 1 mM Ca2+ (lanes 2 and 3) (30, 31). After the beads were washed extensively, proteins bound to the beads were analyzed by SDS/12.5% PAGE, followed by immunoblotting with HRP-conjugated anti-FLAG tag antibody (Middle). Input means 1/80 volume of the reaction mixture (Top). Note that the anti-mSyt I-C2B antibody (10 μg) did not inhibit Ca2+-depenent oligomerization of the C2B domain (Lane 3, Middle); instead, it promoted oligomerization. The positions of the molecular mass markers (× 10-3) are shown on the left. (C) Schematic representation of the three distinct ligand-binding sites of the C2B domain (5, 25).
Fig. 2.
Fig. 2.
Effect of intracellular C2B IgG injection in transmitter availability. (A and B) Upper traces, simultaneous presynaptic (black) and postsynaptic (red) recordings after repetitive presynaptic stimulation at 200 Hz. Lower traces are as above at higher sweep speed. (C) Control, the stimulus trains are repeated until all post spikes fail (upper trace). Trains are repeated every 5 sec until all spikes fail at the first stimulus train after 5-sec pause (lower trace). (D) After 15 min, stimulus train demonstrates recovery from transmitter depletion. (E) The same as in C but after IgG injection. (F) Postsynaptic potentials shown at higher gain and sweep speed to show detail time course. (G) Transmission after 15-min rest interval, demonstrating further reduction of transmitter release. (H) The same as in G but at a higher sweep speed. (I) Detailed comparison of the last synaptic potential in F (upper trace) and H to show amplitude reduction but no change in synaptic time course as the smaller potential is enlarged (red) to the amplitude of the potential in F (black).
Fig. 3.
Fig. 3.
IgG injection reduces transmitter release and spontaneous synaptic noise without modifying inward Ca2+ current. (A-C) Reduction of transmitter release (B) after a presynaptic voltage-clamp pulse (C) at different times after IgG injection. Note the lack of change in ICa (A). (D) Postsynaptic noise measurements at different times after IgG injection. Control before injection, extracellular noise level with the postsynaptic electrode outside the post axon is shown.
Fig. 4.
Fig. 4.
Presynaptic localization of fluorescent labeled anti-mSyt I-C2B IgG by using two-photon laser-scanning microscopy. (Upper) Low-magnification image showing membrane distribution of the fluorescent label. (Lower and Inset) En phase imaging of the presynaptic membrane, demonstrating the membrane bound distribution of label. Note the distribution corresponds to the size and geometrical distribution of the synaptic active zones in the terminal.
Fig. 5.
Fig. 5.
Presynaptic injection of anti-mSyt I-C2B results in decreased number of CCVs. Ultrastructure of the giant squid synapse identifying the pre- and postsynaptic structures. (A and B) Electron micrographs from cross sections of nonstimulated, noninjected control synapse. (A) A low-magnification image displaying several active zones with large clusters. (B) Higher-magnification image illustrating the large number of vesicles and number of CCVs (arrows). (C and D) Electron micrographs from cross sections of stimulated, noninjected control synapse to illustrate the effect of stimulation on vesicular number and morphological properties. (C) Low-magnification image showing a decreased number of vesicles during stimulation. (D) An increase in CCVs displayed in higher magnification (arrows). (E and F) Electron micrographs from cross sections of stimulated, anti-mSyt I-C2B-injected synapse. (E) Low-magnification images, demonstrating the significant decreases in vesicle number. (F) Display of low number of CCVs in anti-mSyt I-C2B-injected terminals. Arrows identify clathrin-coated vesicles in B, D, and F.
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