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

摘要

Voltage-gated calcium channels (Ca(v)s) comprise a pore-forming alpha with auxiliary alphadelta and beta subunits which modulate Ca(v) function and surface expression. Ca(v)alpha and alphadelta are present in signalling complexes termed lipid rafts but it is unclear whether alphadelta 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 alphadelta-1 and Ca(v)2.2 subunits expressed in COS-7 cells. We show that alphadelta-1 not only partitions into lipid rafts itself but also mediates raft-partitioning of Ca(v)2.2/beta(1b) complexes. Ca(v)alphadelta-1, Ca(v)2.2/beta(1b) and Ca(v)2.2/beta(1b)/alphadelta-1 complexes are all organised into cell surface clusters although only in the presence of alphadelta-1 do they co-localise with raft markers, caveolin and flotillin. Such clusters persist in the presence of 3-methyl-beta-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 alphadelta-1, and likely other alphadelta 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.