The α2δ subunits of voltage-gated calcium channels form GPI-anchored proteins, a posttranslational modification essential for function

Abstract

Voltage-gated calcium channels are thought to exist in the plasma membrane as heteromeric proteins, in which the α1 subunit is associated with two auxiliary subunits, the intracellular β subunit and the α2δ subunit; both of these subunits influence the trafficking and properties of CaV1 and CaV2 channels. The α2δ subunits have been described as type I transmembrane proteins, because they have an N-terminal signal peptide and a C-terminal hydrophobic and potentially transmembrane region. However, because they have very short C-terminal cytoplasmic domains, we hypothesized that the α2δ proteins might be associated with the plasma membrane through a glycosylphosphatidylinositol (GPI) anchor attached to δ rather than a transmembrane domain. Here, we provide biochemical, immunocytochemical, and mutational evidence to show that all of the α2δ subunits studied, α2δ-1, α2δ-2, and α2δ-3, show all of the properties expected of GPI-anchored proteins, both when heterologously expressed and in native tissues. They are substrates for prokaryotic phosphatidylinositolphospholipase C (PI-PLC) and trypanosomal GPI-PLC, which release the α2δ proteins from membranes and intact cells and expose a cross-reacting determinant epitope. PI-PLC does not affect control transmembrane or membrane-associated proteins. Furthermore, mutation of the predicted GPI-anchor sites markedly reduced plasma membrane and detergent-resistant membrane localization of α2δ subunits. We also show that GPI anchoring of α2δ subunits is necessary for their function to enhance calcium currents, and PI-PLC treatment only reduces calcium current density when α2δ subunits are coexpressed. In conclusion, this study redefines our understanding of α2δ subunits, both in terms of their role in calciumchannel function and other roles in synaptogenesis.