RESUMO
Voltage-gated calcium channels (Ca(v)s) comprise a pore-forming α1 with auxiliary α2δ and ß 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/ß(1b) complexes. Ca(v)α2δ-1, Ca(v)2.2/ß(1b) and Ca(v)2.2/ß(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-ß-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.
