title: 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.
authors.affiliation: Toronto Western Research Institute, University Health Network, Toronto, Ontario, Canada.
authors.forename: Allen W
authors.initials: AW
authors.lastname: Chan
authors.forename: Rajesh
authors.initials: R
authors.lastname: Khanna
authors.forename: Qi
authors.initials: Q
authors.lastname: Li
authors.forename: Elise F
authors.initials: EF
authors.lastname: Stanley
mesh_terms: D017398:Alternative Splicing / Q000502:physiology; D000818:Animals; D001483:Base Sequence; D020864:Calcium Channels, N-Type / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002642:Chick Embryo; D002645:Chickens; D005727:Ganglia, Spinal / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D051938:Munc18 Proteins / Q000235:genetics / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D018377:Neurotransmitter Agents / Q000235:genetics / Q000378:metabolism; D017729:Presynaptic Terminals / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D050600:SNARE Proteins / Q000235:genetics / Q000378:metabolism; D013572:Synaptic Vesicles / Q000235:genetics / Q000378:metabolism*; D050857:Synaptotagmins / Q000235:genetics / Q000378:metabolism; D050827:Syntaxin 1 / Q000235:genetics / Q000378:metabolism
chemical_list: C503279:Cacna1b protein, rat; D020864:Calcium Channels, N-Type; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D018377:Neurotransmitter Agents; D050600:SNARE Proteins; C497939:Stxbp1 protein, rat; D050827:Syntaxin 1; D050857:Synaptotagmins