title: The Src homology 3 domain of the beta-subunit of voltage-gated calcium channels promotes endocytosis via dynamin interaction.
abstract: High voltage-gated calcium channels enable calcium entry into cells in response to membrane depolarization. Association of the auxiliary beta-subunit to the alpha-interaction-domain in the pore-forming alpha1-subunit is required to form functional channels. The beta-subunit belongs to the membrane-associated guanylate kinase class of scaffolding proteins containing a Src homology 3 and a guanylate kinase domain. Although the latter is responsible for the high affinity binding to the alpha-interaction domain, the functional significance of the Src homology 3 domain remains elusive. Here, we show that injection of isolated beta-subunit Src homology 3 domain into Xenopus laevis oocytes expressing the alpha1-subunit reduces the number of channels in the plasma membrane. This effect is reverted by coexpressing alpha1 with a dominant-negative mutant of dynamin, a GTPase involved in receptor-mediated endocytosis. Full-length beta-subunit also down-regulates voltage-gated calcium channels but only when lacking the alpha-interaction domain. Moreover, isolated Src homology 3 domain and the full-length beta-subunit were found to interact in vitro with dynamin and to internalize the distantly related Shaker potassium channel. These results demonstrate that the beta-subunit regulates the turnover of voltage-gated calcium channels and other proteins in the cell membrane. This effect is mediated by dynamin and depends on the association state of the beta-subunit to the alpha1-pore-forming subunit. Our findings define a novel function for the beta-subunit through its Src homology 3 domain and establish a link between voltage-gated calcium channel activity and the cell endocytic machinery.
authors.affiliation: Centro de Neurociencia de Valparaíso, Universidad de Valparaíso 2349400 Chile.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Gonzalez-Gutierrez
authors.forename: Erick
authors.initials: E
authors.lastname: Miranda-Laferte
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 / Q000502:physiology*; D034281:Dynamins / Q000502:physiology*; D004705:Endocytosis / Q000502:physiology*; D015640:Ion Channel Gating; D008954:Models, Biological; D021122:Protein Subunits; D051381:Rats; D051637:Shaker Superfamily of Potassium Channels / Q000502:physiology; D014982:Xenopus laevis; D018909:src Homology Domains
chemical_list: C491527:Cacnb2 protein, rat; D020746:Calcium Channels, L-Type; D021122:Protein Subunits; D051637:Shaker Superfamily of Potassium Channels; D034281:Dynamins