Rab3a interacting molecule (RIM) and the tethering of pre‐synaptic transmitter release site‐associated CaV2.2 calcium channels

Abstract

Biochemical and physiological evidence suggest that pre-synaptic calcium channels are attached to the transmitter release site within the active zone by a molecular tether. A recent study has proposed that 'Rab3a Interacting Molecule' (RIM) serves as the tether for CaV2.1 channels in mouse brain, based in part on biochemical co-immunoprecipitation (co-IP) using a monoclonal antibody, mRIM. We previously argued against this idea for CaV2.2 calcium channel at chick synapses based on experiments using a different anti-RIM antibody, pRIM1,2: while staining for the two proteins co-localized and co-varied at the transmitter release face, consistent with an association, they failed to co-IP from a synaptosome membrane lysate. RIM is, however, a family of proteins and we tested the possibility that the mRIM antibody used in the more recent study identifies a particular channel-tethering variant. We find that co-immunostaining with mRIM and anti-CaV2.2 antibody neither co-localized nor co-varied at the transmitter release face and the two proteins did not co-IP, arguing against a common protein complex and a key CaV2.2 scaffolding role for RIM at the active zone. The differing results might be reconciled, however, in a model where a RIM family member contributes to a protein bridge that anchors the pre-fusion secretory vesicle to the calcium channel protein complex. Keywords: active zone, calcium channel, particle web, presynaptic, RIM1, transmitter release site. RIM proteins are believed to play a scaffolding role in SV docking or priming (Schoch et al. 2002;Koushika et al. 2001;Wang et al. 2000). In vitro binding studies suggest that the C2 domain of RIM fusion protein can bind directly to intracellular domains of the channel as well as other synaptic proteins (Coppola et al. 2001), and it was speculated that such an attachment could contribute to channel scaffolding. We tested this idea using a commercially available anti-RIM antibody, RIM2 [Note: since this antibody cross-reacts with other RIM variants, as shown below, for clarity we now refer to it here as p(oly)RIM1,2]. We found that immunostaining for RIM co-localized and co-varied with staining for CaV2.2 at the pre-synaptic transmitter release face at the chick ciliary ganglion calyx synapse, consistent with at least the idea that the proteins are in the same region, however, a biochemical analysis was negative. Using the pRIM1,2 antibody, we failed to detect RIM and CaV2.2 co-immunoprecipitation (co-IP) from purified synaptosome membrane lysates which are enriched for active zone proteins (Khanna et al. 2006a). A recent study has suggested an alternative mechanism of pre-synaptic calcium channel tethering by RIM. In this model, RIM scaffolds the a1 pore-forming CaV2 family subunit via the intracellular b subunit (Kiyonaka et al. 2007). One observation in favor of such a role in the Kiyonaka paper was the finding that immunoprecipitation (IP) of RIM from mouse brain lysate using a monoclonal anti-RIM antibody, mRIM, co-immunoprecipitates (co-IPs) CaV2.1 ...

Synaptic vesicle tethering and the CaV2.2 distal C-terminal

“…These have been described in detail (Juhaszova et al, 2000; Wong and Stanley, 2010; Gardezi et al, 2013; Wong et al, 2013). Key preparation buffers were: homogenization buffer (HB), 0.32 M sucrose, 10 mM HEPES, 2 mM EDTA, pH 7.4; HEPES lysis buffer, 50 mM HEPES, 2 mM EDTA, pH 7.4; and modified radioimmunoprecipitation assay solubilization buffer (RIPA), 50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.5% Na + deoxycholate, 1 mM EDTA, pH 8.4)…”

“…Our quantitative immunocytochemical analysis [Intensity Correlation Analysis, (Li et al, 2004)] supported the idea that that CaV2.2 and RIM co-vary at presynaptic transmitter release sites (Khanna et al, 2006a). However, the failure to detect a CaV2.2-RIM complex by biochemical analysis suggests that these proteins are parts of two independent, but possibly transiently interacting, complexes (Khanna et al, 2006a; Wong and Stanley, 2010; Wong et al, 2013) and is at odds with the current tether molecular model. …”

“…Several mechanisms of SV tethering by CaVs have been proposed and these fall into two main classes: indirectly via surface membrane docking proteins (Catterall, 1999), or directly via a cytoplasmic link (Wong and Stanley, 2010; Kaeser et al, 2011; Wong et al, 2013). The reduction in transmitter release noted in heterozygote leaner mice, that express a CaV2.1 truncated C-terminal (Kaja et al, 2008), may have been an early hint that this region of presynaptic CaVs plays a role in transmitter release.…”

Physiology and Evolution of Voltage-Gated Calcium Channels in Early Diverging Animal Phyla: Cnidaria, Placozoa, Porifera and Ctenophora

“…The interaction seems to occur via a RIM PDZ motif that binds the Ca v 2 channel C-terminus; in mouse, targeted deletion of the RIM PDZ disrupts proper channel localization and synaptic transmission, and a direct physical interaction was observed between this motif and the channel C-terminus via yeast-two hybrid and NMR spectroscopy assays. However, a similar interaction was not observed in the chick synapse using co-immunoprecipitation (Khanna et al, 2006; Wong and Stanley, 2010), and a separate study found that RIM tethering of Ca v 2 channels requires the Ca v β subunit to serve as an intermediary between the two proteins (Kiyonaka et al, 2007). Recently, evidence has emerged that Ca v 2 channel pre-synaptic scaffolding undergoes a developmental switch in Drosophila , where different mRNA splice isoforms of the vesicle priming protein UNC-13 interact with distinct scaffolding proteins for either microdomain tethering in immature synapses (i.e., with Syd-1 and Liprin-α), or nanodomain tethering in mature synapses (i.e., with Bruchpilot and RIM-associated protein complexes) (Böhme et al, 2016).…”

Regulation of Voltage-Gated Calcium Channels by Synaptic Proteins

“…It was shown that Rim Binding Proteins directly interact with Ca v 2.2 channels (and possibly with Ca v 2.1 channels), suggesting a molecular link between voltage-gated Ca 2+ channels and Rim proteins [ 116 ] . Whereas biochemical studies using synapstosome membranes failed to demonstrate the existence of a Ca v 2.2/Rim complex [ 117 ] , in vitro studies indicate a direct interaction of Rim-1 with the synprint site of Ca v 2.2 channels [ 109 ] as well as with SNAP-25 and synaptotagmin-1 via the C2 domains in a Ca 2+ -dependent manner. At low Ca 2+ concentrations Rim-1 thus preferentially binds SNAP-25 whereas an increase in Ca 2+ concentration (>75 m M) favors its interaction with synaptotagmin-1 [ 109 ] .…”