Inter-channel scaffolding of presynaptic CaV2.2 via the C terminal PDZ ligand domain

Abstract

SummaryCalcium entry through CaV2.2 calcium channels clustered at the active zone (AZ) of the presynaptic nerve terminal gates synaptic vesicle (SV) fusion and the discharge of neurotransmitters, but the mechanism of channel scaffolding remains poorly understood. Recent studies have implicated the binding of a PDZ ligand domain (PDZ-LD) at the tip of the channel C terminal to a partner PDZ domain on RIM1/2, a synaptic vesicle-associated protein. To explore CaV2.2 scaffolding, we created intracellular region fusion proteins and used these to test for binding by ‘fishing’ for native CaV2.2 channels from cell lysates. Fusion proteins mimicking the distal half of the channel C terminal (C3strep) reliably captured CaV2.2 from whole brain crude membrane or purified synaptosome membrane lysates, whereas channel I–II loop or the distal half of the II–III loop proteins were negative. This capture could be replicated in a non-synaptic environment using CaV2.2 expressed in a cell line. The distal tip PDZ-LD, DDWC-COOH, was confirmed as the critical binding site by block of pull-down with mimetic peptides. Pull-down experiments using brain crude membrane lysates confirmed that RIM1/2 can bind to the DDWC PDZ-LD. However, robust CaV2.2 capture was observed from synaptosome membrane or in the cell line expression system with little or no RIM1/2 co-capture. Thus, we conclude that CaV2.2 channels can scaffold to each other via an interaction that involves the PDZ-LD by an inter-channel linkage bridged by an unknown protein.

Synaptic vesicle capture by CaV2.2 calcium channels

“…To some extent expression of RIM in these cells varied with batches. Thus, faint protein bands were seen in these experiments ( Figure 5 ) but were almost undetectable in a later batch (Gardezi et al, 2013). …”

“…To test for direct binding to the calcium channel we created an One Strep-tagged C-terminal fusion protein, C3 strep , covering the distal half of the C-terminal to its tip (see Gardezi et al, 2013). SV-PD was carried out by incubating bead-immobilized C3 strep with SVs suspended in detergent-free buffer, using expressed vector as a control.…”

Early Metazoan Origin and Multiple Losses of a Novel Clade of RIM Presynaptic Calcium Channel Scaffolding Protein Homologs

“…The reported conservation of D/E-D/E/H-WC- COOH PDZ ligand motifs on the distal C-termini of Ca V 2 channels from vertebrates ( Kaeser et al. 2011 ; Gardezi et al. 2013 ), fruit flies ( Graf et al.…”

Early metazoan origin and multiple losses of a novel clade of RIM pre-synaptic calcium channel scaffolding protein homologues

“…The reported conservation of D/E-D/E/H-WC- COOH PDZ ligand motifs on the distal C-termini of Ca V 2 channels from vertebrates (Kaeser, et al 2011; Gardezi, et al 2013), fruit flies (Graf, et al 2012) and molluscs (Spafford, et al 2003) is notable given that at the phylum-level protein alignments of orthologous Ca V channel intracellular linkers and N- and C-termini tend to show poor sequence homology (Spafford, et al 2003; Senatore and Spafford 2010; Tyson and Snutch 2013). In addition to binding I-RIMs, the Ca V 2 PDZ ligand motif also mediates interactions with a PDZ domain of the pre-synaptic scaffolding protein Mint, documented in rodents (Maximov, et al 1999), chick (Gardezi, et al 2013) and the gastropod mollusc Lymnaea stagnalis (Spafford, et al 2003). Thus, it seems likely that interactions between Ca V 2 channels and I-RIM/Mint-1 were present in the last common ancestor of the bilaterians.…”