Synaptotagmin I, an integral synaptic vesicle protein, is the putative calcium sensor for fast, synchronous transmission. In vitro, synaptotagmin shows calcium-dependent self-oligomerization and binding to both phospholipids and SNARE (soluble NEM-sensitive factor attachment protein receptor) proteins. Each of these interactions has been postulated to be crucial to synaptotagmin’s role as the calcium sensor, but direct evidence is sparse. We have tested the importance of self-oligomerization to the calcium-sensing mechanism by recording from neurons that express only oligomerization-deficient synaptotagmin.

Hippocampal cell cultures were prepared from synaptotagmin I knock-out mice at embryonic day 18, in accordance with USA and institutional guidelines. After 9-16 days, neurons were infected with Semliki Forest Virus constructs (Invitrogen) containing genes for a synaptotagmin mutant and enhanced green fluorescent protein as a reporter. Whole-cell voltage-clamp recordings were made 8-24 h later from autaptic neurons grown on microislands.

Two different synaptotagmin mutants were used to test the importance of self-oligomerization. One contains an asparagine (N) residue in position 311 instead of a tyrosine (Y). Biochemical studies with the Y311N mutant show a complete lack of calcium-dependent self-oligomerization, while binding to SNARE proteins and phospholipids is normal (Littleton et al. 2001).

In the second mutant, lysines in positions 326 and 327 are mutated to alanines. This K326, 327A mutant also lacks calcium-dependent self-oligomerization in vitro, while binding to SNARE proteins is normal (Earles et al. 2001).

If self-oligomerization is essential for synaptotagmin to work, then these mutants should not be able to support fast synaptic transmission. When neurons were transfected with either mutant, however, they showed clear EPSCs, as did wild-type transfected controls. Thus our results do not support the proposal that self-oligomerization, as determined in biochemical studies, is essential for synaptotagmin’s role as the calcium sensor for neurotransmission.

This work was supported by the Howard Hughes Medical Institute (C.F.S.) and Merck and Chapman Fellowships (C.R.B.).