No abstract

ABSTRACT

Synaptotagmins I and II are inositol high polyphosphate series (inositol 1,3,4,5-tetrakisphosphate (IP 4 ), inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate) binding proteins, which are thought to be essential for Ca 2؉ -regulated exocytosis of neurosecretory vesicles. In this study, we analyzed the inositol high polyphosphate series binding site in the C2B domain by site-directed mutagenesis and compared the IP 4 binding properties of the C2B domains of multiple synaptotagmins (II-IV). The IP 4 binding domain of synaptotagmin II is characterized by a cluster of highly conserved, positively charged amino acids (321 GKRLKKKKTTVKKK 324). Among these, three lysine residues, at positions 327, 328, and 332 in the middle of the C2B domain, which is not conserved in the C2A domain, were found to be essential for IP 4 binding in synaptotagmin II. When these lysine residues were altered to glutamine, the IP 4 binding ability was completely abolished. The primary structures of the IP 4 binding sites are highly conserved among synaptotagmins I through IV. However, synaptotagmin III did not show significant binding ability, which may be due to steric hindrance by the C-terminal flanking region. These functional diversities of C2B domains suggest that not all synaptotagmins function as inositol high polyphosphate sensors at the synaptic vesicle.Synaptotagmin is an integral membrane protein of secretory vesicles, abundant in neural and some endocrine cells (1). The structure of synaptotagmin is characterized by two copies of highly conserved repeats homologous to the C2 regulatory region of protein kinase C in the cytoplasmic domain (2). The importance of the C2 domain of synaptotagmin in Ca 2ϩ -regulated exocytosis is shown by several studies. A recombinant protein of the C2A domain inhibited Ca 2ϩ -regulated exocytosis of a large dense core vesicle in PC12 cells (3). Injection of basic peptides from the central region of both C2 domains into the squid giant presynaptic terminal also inhibited neurotransmitter release (4). The studies of mutants of Caenorhabditis elegans (5), Drosophila (6, 7), and the knock-out mouse (8) also indicated that synaptotagmin plays a key role in Ca 2ϩ -regulated exocytosis.In our previous study, we characterized synaptotagmin II as an IP 4 1 or rather an inositol-high polyphosphate series (IP 4 , inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate, referred to as IHPS) binding protein (9). Deletion analysis of synaptotagmin II clearly showed that about 30 amino acids of the central region of the C2B domain were essential for IHPS binding (10). This binding domain includes a sequence corresponding to the squid Pep20 peptide, which is essential for neurotransmitter release (4), suggesting that IHPS has some effect on neurotransmitter release. Our recent study of the squid giant presynapse supported this suggestion. Microinjection of the members of IHPS into the squid giant presynaptic terminal is shown to block synaptic transmission withou...