Inositol 1,3,4,5-tetrakisphosphate binding activities of neuronal and non-neuronal synaptotagmins: Identification of conserved amino acid substitutions that abolish inositol 1,3,4,5-tetrakisphosphate binding to synaptotagmins III, V, and X

Synaptotagmins I and II are essential for Ca²⁺-regulated exocytosis of synaptic vesicles from neurons, probably serving as Ca²⁺ sensors. This Ca²⁺-sensing function is thought to be disrupted by binding of an inositol 1,3,4,5-tetrakisphosphate (IP₄) to the C2B domain of synaptotagmin I or II (Fukuda, M., Moreira, J. E., Lewis, F. M. T., Sugimori, M., Niinobe, M., Mikoshiba, K., and Llinas, R. (1995) Proc. Natl. Acad. Sci. U.S. A. 92, 10708-10712). Recently, several synaptotagmin isoforms, expressed outside the nervous system, have been identified in rats and proposed to be involved in constitutive vesicle traffic. To test whether the inositol high polyphosphates also regulate constitutive vesicle traffic by binding to the non-neuronal synaptotagmins, we examined the IP₄ binding properties of the recombinant C2 domains of both neuronal (III, V, X, and XI) and non-neurenal (VI-VIII and IX) synaptotagmins. The C2B domains of synaptotagmins VII-IX and XI had strong IP₄ binding activity, but the C2B domain of synaptotagmin VI showed very weak IP₄ binding activity. In contrast, there was no significant IP₄ binding activity of the C2B domains of synaptetagmins III, V, and X or any of the C2A domains. A phylogenetic tree of the C2 domains of 11 isoforms revealed that synaptotagmins III, V, VI, and X (IP₄-insensitive or very weak IP₄-binding isoforms) belong to the same branch. Based on the sequence comparison between the IP₄-sensitive and -insensitive isoforms, we performed site-directed mutagenesis of synaptotagmin III and identified several amine acid substitutions that abolish IP₄ binding activity. Our data suggest that the inositol high polyphosphates might also regulate constitutive vesicle traffic via binding to the IP₄-sensitive non-neuronal synaptotagmins.