Voltage-Gated Calcium Channel α2δ Subunits in Lipid Rafts: The Importance of Proteolytic Cleavage Into α2 and δ

The High Voltage-Activated (HVA) subgroup of voltage-gated calcium channels contain an α1 subunit, which forms the selective pore and determines the main functional properties of the channel. The α1 subunit is associated with auxiliary subunits including β and α2δ, which modulate trafficking and functional properties of the channels.There are four known genes encoding α2δ subunits, which are believed to have similar structure. They consist of two peptides: the highly glycosylated α2 which is entirely extracellular is disulfide-bonded to a δ subunit that links the protein into the plasma membrane. The α2 and δ peptides are encoded by a single gene as an uninterrupted α2δ pre-protein, which is further processed post-translationally. We have recently shown that α2δ subunits are glycosyl phophatidyl inositol (GPI)-achored proteins (Davies et al., 2010, PNAS 107:1654-1659), and this is essential for their function, and explains their localization in lipid raft fractions (Davies et al, 2006, J. Neurosci. 26: 8748-8757).We further studied the mechnism and consequences of the proteolytic cleavage of the α2δ subunits (α2δ-1, -2 and -3)and identified the cleavage sites in α2δ-2 and α2δ-3. Western blots from brain tissue revealed only the mature form of the protein strongly associated with lipid rafts. However, transfecting mammalian cells with cDNA for α2δ-1,-2,and -3 resulted in incomplete cleavage (∼ 40-60% processing) most probably due to limitations of the cleavage-mediating protease(s) in heterologous expression systems. The mature form is localized in lipid rafts, suggesting that maturation of the protein might occur in localized membrane domains. Moreover, electrophysiological recordings demonstrated that proteolytic cleavage is key to the function of these subunits.We are currently examining the nature of the protease(s) involved in this proteolytic processing.