Abstract
Rab interacting molecules (RIMs) are multi-domain proteins that positively regulate the number of Ca2+ channels at the presynaptic active zone (AZ). Several molecular mechanisms have been demonstrated for RIM-binding to components of the presynaptic Ca2+ channel complex, the key signaling element at the AZ. Here, we report an interaction of the C2B domain of RIM2α and RIM3γ with the C-terminus of the pore-forming α–subunit of CaV1.3 channels (CaV1.3α1), which mediate stimulus-secretion coupling at the ribbon synapses of cochlear inner hair cells (IHCs). Co-expressing full-length RIM2α with a Ca2+ channel complex closely resembling that of IHCs (CaV1.3α1-CaVß2a) in HEK293 cells doubled the Ca2+-current and shifted the voltage-dependence of Ca2+ channel activation by approximately +3 mV. Co-expression of the short RIM isoform RIM3γ increased the CaV1.3α1-CaVß2a-mediated Ca2+-influx in HEK293 cells, but disruption of RIM3γ in mice left Ca2+-influx in IHCs and hearing intact. In conclusion, we propose that RIM2α and RIM3γ directly interact with the C-terminus of the pore-forming subunit of CaV1.3 Ca2+ channels and positively regulate their plasma membrane expression in HEK293 cells.
Highlights
Ca2+-influx through voltage-gated Ca2+ channels triggers the fusion of synaptic vesicles at the presynaptic active zone (AZ)
We studied whether RIM2α and RIM3γ, both expressed at inner hair cells (IHCs) AZs, directly interact with the pore-forming CaV1.3α Ca2+ channel subunit that mediates stimulus-secretion coupling at IHC synapses
Based on co-immunoprecipitation, GST-pulldown assays, fluorescence microscopy of protein co-localization and electrophysiology in HEK293 cells, we indicate that RIM2α and RIM3γ directly bind to the C-terminus of the pore-forming CaV1.3α1 subunit most likely via their C2B domain
Summary
Ca2+-influx through voltage-gated Ca2+ channels triggers the fusion of synaptic vesicles at the presynaptic active zone (AZ). RIM1/2 interact with the pore-forming CaVα1 subunit of CaV2 channels through their central PDZ-domain (CaV2.X (Kaeser et al, 2011)) They have been reported to bind via their C-terminal C2A and C2B domains to the auxiliary β (CaVβ) subunit (Kiyonaka et al, 2007; Gebhart et al, 2010; Gandini et al, 2011) as well as to the ‘‘synaptic protein interaction’’ motif (synprint motif; cytoplasmic linker between domains II and III) of the CaV2.2α1 and CaV1.2α1 subunits, which, was not found for the CaV1.3α1 subunit (Coppola et al, 2001). The extent of this regulation depended on the respective CaVß subunit co-expressed and was least prominent for CaV1.3 in the presence of palmitoylated CaVß2a (Gebhart et al, 2010; Gandini et al, 2011) that we postulate to be the predominant CaVß subunit in inner hair cells (IHCs; Neef et al, 2009)
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