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

• Published in: Proceedings of the National Academy of Sciences - PNAS 2010-01, Vol.107 (4), p.1654-1659
• Main Authors: Davies, Anthony, Kadurin, Ivan, Alvarez-Laviada, Anita, Douglas, Leon, Nieto-Rostro, Manuela, Bauer, Claudia S, Pratt, Wendy S, Dolphin, Annette C
• Format: Article
• Language: English
• Subjects: Biochemistry; Biological Sciences; Calcium channels; Cell membranes; COS cells; Epitopes; Focalism; Neurons; Neuroscience; Proteins; Vehicles
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0908735107

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 phosphatidylinositol-phospholipase 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 calcium-channel function and other roles in synaptogenesis.