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

Abstract

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 C 2 B domain of synaptotagmin-I. Thus, RIM-C 2 A and RIM-C 2 B bind in a Ca 2؉ -independent manner to ␣1B, the pore-forming subunit of N-type Ca 2؉ channels (EC 50 ‫؍‬ ϳ20 nM). They also weakly interact with the ␣1C but not the ␣1D subunit of L-type Ca 2؉ 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-C 2 B. The interactions of the C 2 domains of RIM with SNAP-25 and synaptotagmin-I are modulated by Ca 2؉ . Thus, in the presence of Ca 2؉ (EC 50 ‫؍‬ ϳ75 M) 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.Secretion of neurotransmitters, polypeptide hormones, and a variety of other proteins occurs by exocytosis, a multistage process including targeting, docking, and, finally, fusion of secretory vesicles with the plasma membrane. During the last few years, a combination of genetic and biochemical approaches has lead to the identification of several proteins involved in this complex cascade of events. Most of these proteins turned out to be specialized components of the evolutionary conserved machinery that governs intracellular vesicular trafficking in eukaryotic cells (1). Exocytosis was found to necessitate the assembly of a ternary complex between the vesicular SNARE 1 VAMP, associated with the secretory vesicle, and the target SNAREs syntaxin-1 and SNAP-25, localized at the plasma membrane (2). The SNARE complex was initially proposed to ensure the docking of secretory vesicles at the plasma membrane (3). However, it is unlikely that SNARE assembly constitutes the sole determinant for the targeting of secretory vesicles at the release sites because SNARE pairing is rather promiscuous (4, 5), and the localization of syntaxin-1 and SNAP-25 is not restricted to active zones (6). A current hypothesis, supported by biochemical and structural data, proposes that the assembly of the heterotrimeric complex between VAMP, SNAP-25, and syntaxin-1 provides the driving force for membrane fusion (7).In most secretory systems, the exocytotic process is initiated by an increase in the intracellular Ca 2ϩ concentration. In some cells, such as neurons, the elevation of Ca 2ϩ ions is due to opening of voltage-gated calcium channels that are clustered at the release sites, whereas in others, Ca 2ϩ ions are mobilized from intracellular stores. Biochemical and genetic studies indicate...

cAMP/PKA signaling and RIM1α mediate presynaptic LTP in the lateral amygdala

“…RIM1␣, however, interacts directly with Ca 2ϩ -binding proteins such as Munc13-1 and synaptotagmin 1 (19, 20, 32). Moreover, RIM1␣ interacts directly or indirectly with presynaptic VDCCs, thereby regulating the function and/or the physical location of VDCCs in relation to the release site (20,21,33). Thus, RIM1␣ might orchestrate presynaptic signaling complexes and enable compartmentalized signal transduction within presynaptic scaffolds by bringing VDCCs, Ca 2ϩ sensors, and the vesicular fusion machinery into close proximity allowing for activity-dependent and persistent modifications in the vesicular release probability.…”

“…One of the key PKA targets necessary for presynaptic LTP is the active-zone scaffolding protein RIM1␣ (17)(18)(19). RIM1␣ is a multidomain protein that interacts with a number of active-zone proteins involved in neurotransmitter release including Rab3a, Munc13-1, ␣-liprins, synaptotagmin 1, and presynaptic voltage-dependent Ca 2ϩ channels (VDCCs) (19)(20)(21).…”

Old proteins, developing roles: The regulation of calcium channels by synaptic proteins

“…122 The protein comprises an N-terminal zinc-finger motif and C-terminal PDZ and C2 domains. Rim interacts with N-type channels at the synprint motif 123,124 and also weakly with the P/Q-type (Fig. 2).…”

A family of RIM-binding proteins regulated by alternative splicing: Implications for the genesis of synaptic active zones

“…A proline-rich sequence in the linker between the C 2 A and C 2 B domain interacts with the SH3 domain of brain-specific adaptor proteins called RIM-BPs (4) that have been implicated in Ca 2ϩ -channel regulation (10). The C-terminal C 2 B domain binds to ␣-liprins (6), putative adaptor proteins of the active zone that were shown in invertebrates to be essential for regulating active zone size (11)(12)(13), and to synaptotagmin 1, which in turn regulates release (6,14). RIMs thus behave like a molecular scaffold that tethers multiple synaptic proteins.…”