title: Homodimerization of the Src homology 3 domain of the calcium channel β-subunit drives dynamin-dependent endocytosis.
abstract: Voltage-dependent calcium channels constitute the main entry pathway for calcium into excitable cells. They are heteromultimers formed by an α(1) pore-forming subunit (Ca(V)α(1)) and accessory subunits. To achieve a precise coordination of calcium signals, the expression and activity of these channels is tightly controlled. The accessory β-subunit (Ca(V)β), a membrane associated guanylate kinase containing one guanylate kinase (β-GK) and one Src homology 3 (β-SH3) domain, has antagonistic effects on calcium currents by regulating different aspects of channel function. Although β-GK binds to a conserved site within the α(1)-pore-forming subunit and facilitates channel opening, β-SH3 binds to dynamin and promotes endocytosis. Here, we investigated the molecular switch underlying the functional duality of this modular protein. We show that β-SH3 homodimerizes through a single disulfide bond. Substitution of the only cysteine residue abolishes dimerization and impairs internalization of L-type Ca(V)1.2 channels expressed in Xenopus oocytes while preserving dynamin binding. Covalent linkage of the β-SH3 dimerization-deficient mutant yields a concatamer that binds to dynamin and restores endocytosis. Moreover, using FRET analysis, we show in living cells that Ca(V)β form oligomers and that this interaction is reduced by Ca(V)α(1). Association of Ca(V)β with a polypeptide encoding the binding motif in Ca(V)α(1) inhibited endocytosis. Together, these findings reveal that β-SH3 dimerization is crucial for endocytosis and suggest that channel activation and internalization are two mutually exclusive functions of Ca(V)β. We propose that a change in the oligomeric state of Ca(V)β is the functional switch between channel activator and channel internalizer.
authors.affiliation: Institut für Neurophysiologie, Medizinische Hochschule Hannover, 30625 Hannover, Germany.
authors.forename: Erick
authors.initials: E
authors.lastname: Miranda-Laferte
authors.forename: Giovanni
authors.initials: G
authors.lastname: Gonzalez-Gutierrez
authors.forename: Silke
authors.initials: S
authors.lastname: Schmidt
authors.forename: Andre
authors.initials: A
authors.lastname: Zeug
authors.forename: Evgeni G
authors.initials: EG
authors.lastname: Ponimaskin
authors.forename: Alan
authors.initials: A
authors.lastname: Neely
authors.forename: Patricia
authors.initials: P
authors.lastname: Hidalgo
mesh_terms: D000818:Animals; D020746:Calcium Channels, L-Type / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D034281:Dynamins / Q000378:metabolism*; D004705:Endocytosis; D008958:Models, Molecular; D009154:Mutation; D016062:Porosity; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D018909:src Homology Domains
chemical_list: D020746:Calcium Channels, L-Type; D004220:Disulfides; D034281:Dynamins