title: CaV2.2 channel cell surface expression is regulated by the light chain 1 (LC1) of the microtubule-associated protein B (MAP1B) via UBE2L3-mediated ubiquitination and degradation.
abstract: Microtubule-associated protein B is a cytoskeleton protein consisting of heavy and light (LC) chains that play important roles in the regulation of neuronal morphogenesis and function. LC1 is also well known to interact with diverse ionotropic receptors at postsynapse. Much less is known, however, regarding the role of LC1 at presynaptic level where voltage-gated N-type Ca(2+) channels couple membrane depolarization to neurotransmitter release. Here, we investigated whether LC1 interacts with the N-type channels. Co-localization analysis revealed spatial proximity of the two proteins in hippocampal neurons. The interaction between LC1 and the N-type channel was demonstrated using co-immunoprecipitation experiments and in vitro pull-down assays. Detailed biochemical analysis suggested that the interaction occurs through the N-terminal of LC1 and the C-terminal of the pore-forming CaVα1 subunit of the channels. Patch-clamp studies in HEK-293 cells revealed a significant decrease in N-type currents upon LC1 expression, without apparent changes in kinetics. Recordings performed in the presence of MG132 prevented the actions of LC1 suggesting enhanced channel proteasomal degradation. Interestingly, using the yeast two-hybrid system and immunoprecipitation assays in HEK-293 cells, we revealed an interaction between LC1 and the ubiquitin-conjugating enzyme UBE2L3. Furthermore, we found that the LC1/UBE2L3 complex could interact with the N-type channels, suggesting that LC1 may act as a scaffold protein to increase UBE2L3-mediated channel ubiquitination. Together these results revealed a novel functional coupling between LC1 and the N-type channels.
authors.affiliation: Department of Cell Biology, Center for Research and Advanced Studies of the National Polytechnic Institute, (Cinvestav-IPN), Avenida IPN 2508, Colonia Zacatenco, Mexico DF, 07360, Mexico.
authors.forename: María A
authors.initials: MA
authors.lastname: Gandini
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Henríquez
authors.forename: Lizbeth
authors.initials: L
authors.lastname: Grimaldo
authors.forename: Alejandro
authors.initials: A
authors.lastname: Sandoval
authors.forename: Christophe
authors.initials: C
authors.lastname: Altier
authors.forename: Gerald W
authors.initials: GW
authors.lastname: Zamponi
authors.forename: Ricardo
authors.initials: R
authors.lastname: Felix
authors.forename: Christian
authors.initials: C
authors.lastname: González-Billault
mesh_terms: D020864:Calcium Channels, N-Type / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D057809:HEK293 Cells; D006624:Hippocampus / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D059748:Proteolysis; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism*; D054875:Ubiquitination / Q000502:physiology*
chemical_list: C500545:CACNA1B protein, human; D020864:Calcium Channels, N-Type; C475491:MAP1B protein, human; D008869:Microtubule-Associated Proteins; D025801:Ubiquitin; C079437:UBE2L3 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D046988:Proteasome Endopeptidase Complex