Figure 1.

Expression of exogenous synaptotagmin I in the knock-out neurons. A, Vector NTI software (InfoMax, Frederick, MD) was used to generate structures of C₂A (Shao et al., 1998) and C₂B (Fernandez et al., 2001) domains in the presence of Ca²⁺ (spheres). The linker connecting the C₂ domains was added manually. B, Ca²⁺-binding sites in the C₂B domain of synaptotagmin I, modified from Fernandez et al. (2001). Two Ca²⁺ (Ca1 and Ca2) are coordinated by five Asp residues (D1 through D5) on two loops. C, Expression of synaptotagmin I in neurons from wild-type (WT), heterozygous (Hetero), or knock-out (KO) mice. Cultured hippocampal neurons (2 × 10³ cells) were subjected to immunoblotting. The migration position of synaptotagmin I is indicated by an arrow, and the truncated product weakly expressed in the synaptotagmin mutant is indicated by an asterisk. D, E, Expression of exogenous synaptotagmin I in the knock-out neurons. D, Knock-out neurons (2 × 10⁴ cells) either untransfected (KO) or transfected with either wild-type (Stg I) or mutated synaptotagmin I (D1N-D5N) were analyzed via immunoblotting. Wild-type neurons (WT; 2 × 10³ cells) were loaded as a control. E, Immunoreactivities were measured and normalized to wild-type synaptotagmin I expressed in the knock-out neurons. Data are the mean ± SEM from three experiments.