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

Voltage-gated calcium channels are thought to exist in the plasma membrane as heteromeric proteins, in which the alpha 1 subunit is associated with two auxiliary subunits, the intracellular beta subunit and the alpha(2)delta subunit; both of these subunits influence the trafficking and properties of Ca(V)1 and Ca(V)2 channels. The alpha(2)delta subunits have been described as type 1 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 hypothe- sized that the alpha(2)delta proteins might be associated with the plasma membrane through a glycosylphosphatidylinositol (GPI) anchor attached to delta rather than a transmembrane domain. Here, we provide biochemical, immunocytochemical, and mutational evidence to show that all of the alpha(2)delta subunits studied, alpha(2)delta-1, alpha(2)delta-2, and alpha(2)delta-3, show all of the properties expected of GPI-anchored proteins, both when heterologously expressed and in native tissues. They are substrates for prokaryotic phosphatidylinositol-phospholipase C (PI-PLC) and trypanosomal GPI-PLC, which release the alpha(2)delta 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 alpha(2)delta subunits. We also show that GPI anchoring of alpha(2)delta subunits is necessary for their function to enhance calcium currents, and PI-PLC treatment only reduces calcium current density when alpha(2)delta subunits are coexpressed. In conclusion, this study redefines our understanding of alpha(2)delta subunits, both in terms of their role in calcium-channel function and other roles in synaptogenesis.