Direct Interaction of the Rab3 Effector RIM with Ca2+Channels, SNAP-25, and Synaptotagmin

• Published in: The Journal of biological chemistry 2001-08, Vol.276 (35), p.32756-32762
• Main Authors: Coppola, Thierry, Magnin-Lüthi, Sarah, Perret-Menoud, Véronique, Gattesco, Sonia, Schiavo, Giampietro, Regazzi, Romano
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Antigens, Surface - chemistry; Antigens, Surface - metabolism; Binding Sites; Brain - metabolism; Calcium - metabolism; Calcium - pharmacology; Calcium Channels - chemistry; Calcium Channels - metabolism; Calcium-Binding Proteins; Cloning, Molecular; GTP-Binding Proteins; Humans; Kinetics; Membrane Glycoproteins - chemistry; Membrane Glycoproteins - metabolism; Membrane Proteins - chemistry; Membrane Proteins - metabolism; Molecular Sequence Data; Mutagenesis, Site-Directed; Nerve Tissue Proteins - chemistry; Nerve Tissue Proteins - genetics; Nerve Tissue Proteins - metabolism; Protein Isoforms - chemistry; Protein Isoforms - genetics; Protein Isoforms - metabolism; Protein Subunits; rab3 GTP-Binding Proteins - metabolism; Rats; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; RNA, Messenger - genetics; Sequence Alignment; Sequence Homology, Amino Acid; Synaptosomal-Associated Protein 25; Synaptotagmin I; Synaptotagmins; Syntaxin 1; Zinc Fingers
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M100929200

To define the role of the Rab3-interacting molecule RIM in exocytosis we searched for additional binding partners of the protein. We found that the two C2 domains of RIM display properties analogous to those of the C2B domain of synaptotagmin-I. Thus, RIM-C2A and RIM-C2B bind in a Ca2+-independent manner to α1B, the pore-forming subunit of N-type Ca2+ channels (EC50 = ∼20 nm). They also weakly interact with the α1C but not the α1D subunit of L-type Ca2+channels. In addition, the C2 domains of RIM associate with SNAP-25 and synaptotagmin-I. The binding affinities for these two proteins are 203 and 24 nm, respectively, for RIM-C2A and 224 and 16 nm for RIM-C2B. The interactions of the C2 domains of RIM with SNAP-25 and synaptotagmin-I are modulated by Ca2+. Thus, in the presence of Ca2+ (EC50 = ∼75 µm) the interaction with synaptotagmin-I is increased, whereas SNAP-25 binding is reduced. Synaptotagmin-I binding is abolished by mutations in two positively charged amino acids in the C2 domains of RIM and by the addition of inositol polyphosphates. We propose that the Rab3 effector RIM is a scaffold protein that participates through its multiple binding partners in the docking and fusion of secretory vesicles at the release sites.