Identification and characterization of a novel C2B splice variant of synaptotagmin I.

We have identified an alternatively spliced form of synaptotagmin I in Aplysia neurons. This isoform, synaptotagmin I C2B-beta, is generated by alternative exon usage in the C2B domain leading to nine amino acid changes in the C2B sequence from the previously characterized synaptotagmin I, now designated as synaptotagmin I C2B-alpha. Quantitative reverse transcriptase-polymerase chain reaction demonstrated that approximately 25% of mRNA encoding synaptotagmin I contained the C2B-beta exon in the nervous system. Synaptotagmin I C2B-beta showed greater resistance to digestion by chymotrypsin in the absence of calcium than did synaptotagmin I C2B-alpha, although both isoforms required the same amount of calcium to resist chymotrypsin digestion. The source of these changes in C2B properties was mapped to a single amino acid (threonine 358). We have also cloned SNAP 25 in Aplysia and show that it binds synaptotagmin I C2B-beta with a higher affinity than synaptotagmin I C2B-alpha. These results suggest that this splicing alters biochemical properties of the C2B domain, affecting a number of its important known interactions.