Homodimerization of the Src Homology 3 Domain of the Calcium Channel β-Subunit Drives Dynamin-dependent Endocytosis

Voltage-dependent calcium channels constitute the main entry pathway for calcium into excitable cells. They are heteromultimers formed by an alpha(1) pore-forming subunit (Ca-V alpha(1)) and accessory subunits. To achieve a precise coordination of calcium signals, the expression and activity of these channels is tightly controlled. The accessory beta-subunit (Ca-V beta), a membrane associated guanylate kinase containing one guanylate kinase (beta-GK) and one Src homology 3 (beta-SH3) domain, has antagonistic effects on calcium currents by regulating different aspects of channel function. Although beta-GK binds to a conserved site within the alpha(1)-pore-forming subunit and facilitates channel opening, beta-SH3 binds to dynamin and promotes endocytosis. Here, we investigated the molecular switch underlying the functional duality of this modular protein. We show that beta-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 beta-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 beta form oligomers and that this interaction is reduced by Ca-V alpha(1). Association of Ca-V beta with a polypeptide encoding the binding motif in Ca-V alpha(1) inhibited endocytosis. Together, these findings reveal that beta-SH3 dimerization is crucial for endocytosis and suggest that channel activation and internalization are two mutually exclusive functions of Ca-V beta . We propose that a change in the oligomeric state of Ca-V beta is the functional switch between channel activator and channel internalizer.