Munc18: A Presynaptic Transmitter Release Site N type (CaV2.2) Calcium Channel Interacting Protein

Abstract

Munc18 is a presynaptic protein that is essential for transmitter release. Recent studies have indicated that this protein is involved in secretory vesicle docking but its binding partners in this role remain a mystery. We demonstrate using the isolated calyx-type presynaptic terminal of the chick ciliary ganglion that staining for Munc18 colocalizes and covaries with that for transmitter release site N type calcium channels (CaV2.2), consistent with elements of a common release site complex. Biochemical analysis demonstrated that the protein coprecipitates with CaV2.2 from lysates of rat or chick brain, including its synaptic, long-splice variant; presynaptic terminal surface membrane proteins, and a cell line coexpressing Munc18 and CaV2.2. Munc18 bound with high affinity to the CaV2.2 II-III intracellular loop, low affinity to the I-II loop but not to other channel intracellular regions. Over-expression of Munc18 in dorsal root ganglion neurons did not affect CaV2.2 current amplitude or fast kinetics but siRNA-knockdown resulted in a negative shift in the steady state inactivation curve, an effect attributed to an indirect action via syntaxin 1. Recombinant Munc18 also coprecipitated strongly with the v-SNARE synaptotagmin, but only weakly with other SNAREs. Thus, the calcium channel may serve as a surface membrane platform anchoring a Munc18-containing bridge to synaptotagmin and the synaptic vesicle.

Regulation of N-type voltage-gated calcium channels (Cav2.2) and transmitter release by collapsin response mediator protein-2 (CRMP-2) in sensory neurons

“…From our studies and those of others, it is clear, however, that this targeting is achieved through interaction of the presynaptic Ca 2+ channel with a growing number of proteins known to be involved in synaptic vesicle exocytosis and endocytosis pathways (Catterall and Few, 2008;Chan et al, 2007;el Far et al, 1995;Evans and Zamponi, 2006;Khanna et al, 2007c;Khanna et al, 2007a;Khanna et al, 2007b;Kisilevsky and Zamponi, 2008;Stanley, 1997;Szabo et al, 2006). For example, syntaxin 1, the cardinal Cav2.2 partner protein (Sheng et al, 1994), is important for the incorporation of Ca 2+ channels into the release site apparatus.…”

“…To test whether there is a change in cell surface Cav2.2 following manipulation of CRMP-2 levels, biotinylation experiments were performed as previously described (Chan et al, 2007). Immunoblotting with Cav2.2 for streptavidin-enriched complexes from biotinylated DRG neurons grown in culture for 7 DIV showed an ~60% increase in cell-surface expression of Cav2.2 in neurons Journal of Cell Science 122 (23) expressing CRMP-2-EGFP compared with EGFP neurons (Fig.…”

“…Immunocytochemistry, confocal microscopy and iterative deconvolution deblurring Immunocytochemistry was performed as described (Chan et al, 2007;Khanna et al, 2006b;Khanna et al, 2007a;Khanna et al, 2007b). Cultured neurons from DRG were imaged on a Nikon Ti swept-field confocal microscope.…”

“…Biotinylation of live DRG neurons grown in culture was performed as described (Chan et al, 2007;Joiner et al, 2001). DRG neurons grown for 5 DIV were transfected with EGFP or CRMP-2-EGFP or treated with scramble or CRMP-2 siRNA for 2 days and then processed for biotinylation experiments.…”

Inhibition of Transmitter Release and Attenuation of Anti-retroviral-associated and Tibial Nerve Injury-related Painful Peripheral Neuropathy by Novel Synthetic Ca2+ Channel Peptides

“…Purification of CRMP2-His and CaV1.2 Ct-dis-and CaV2.2 Ct-dis-GST Fusion Proteins-GST fusion proteins were purified from BL21 (DE3) Escherichia coli bacterial lysates as described previously (10,11), whereas CRMP2-His protein was purified as before (17) with the following modifications (see Fig. 3A).…”