Direct interaction of the Rab3 effector RIM with Ca2+ channels, SNAP-25, and synaptotagmin

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 C-2 domains of RIM display properties analogous to those of the C2B domain of synaptotagmin-I. Thus, RIM-C(2)A and RIM-C2B bind in a Ca2+-independent manner to alpha 1B, the pore-forming subunit of N-type Ca2+ channels (EC50 = similar to 20 nM). They also weakly interact with the alpha 1C but not the alpha 1D subunit of L-type Ca2+ channels. In addition, the C-2 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-C(2)A and 224 and 16 nM for RIM-C2B. The interactions of the C-2 domains of RIM with SNAP-25 and synaptotagmin-I are modulated by Ca2+. Thus, in the presence of Ca2+ (EC50 = similar to 75 mum) 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 C-2 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.