Formation of N-type (Cav2.2) voltage-gated calcium channel membrane microdomains: Lipid raft association and...

Voltage-gated calcium channels (Ca(v)s) comprise a pore-forming α₁ with auxiliary α₂δ and β subunits which modulate Ca(v) function and surface expression. Ca(v)α₁ and α₂δ are present in signalling complexes termed lipid rafts but it is unclear whether α₂δ is obligatory for targeting Ca(v)s to rafts or to what extent this influences cell surface organisation of Ca(v)s. Here, we have used imaging, biochemistry and electrophysiology to determine localisation and raft-partitioning of WT and functionally active HA-epitope tagged α₂δ-1 and Ca(v)2.2 subunits expressed in COS-7 cells. We show that α₂δ-1 not only partitions into lipid rafts itself but also mediates raft-partitioning of Ca(v)2.2/β(1b) complexes. Ca(v)α₂δ-1, Ca(v)2.2/β(1b) and Ca(v)2.2/β(1b)/α₂δ-1 complexes are all organised into cell surface clusters although only in the presence of α₂δ-1 do they co-localise with raft markers, caveolin and flotillin. Such clusters persist in the presence of 3-methyl-β-cyclodextrin even though the raft markers disperse. However, clustering is profoundly sensitive to disruption of the actin-based cytoskeleton by cytochalasin-D. We conclude that α₂δ-1, and likely other α₂δ subunits, is necessary and sufficient for targeting Ca(v)s to lipid rafts. However, formation of clusters supporting "hotspots" of Ca(v) activity requires aggregation of macromolecular complexes containing raft components, stabilised by interactions with the cytoskeleton.