Voltage-gated calcium channels (Cav) exist as heteromultimers comprising a pore-forming α1 with accessory β and α2δ subunits which modify channel trafficking and function. We previously showed that α2δ-1 (and likely the other mammalian α2δ isoforms - α2δ-2, 3 and 4) is required for targeting Cavs to lipid rafts, although the mechanism remains unclear. Whilst originally understood to have a classical type I transmembrane (TM) topology, recent evidence suggests the α2δ subunit contains a glycosylphosphatidylinositol (GPI)-anchor that mediates its association with lipid rafts. To test this notion, we have used a strategy based on the expression of chimera, where the reported GPI-anchoring sequences in the gabapentinoid-sensitive α2δ-1 subunit have been substituted with those of a functionally inert Type I TM-spanning protein - PIN-G. Using imaging, electrophysiology and biochemistry, we find that lipid raft association of PIN-α2δ is unaffected by substitution of the GPI motif with the TM domain of PIN-G. Moreover, the presence of the GPI motif alone is not sufficient for raft localisation, suggesting that upstream residues are required. GPI-anchoring is susceptible to phosphatidylinositol-phospholipase C (PI-PLC) cleavage. However, whilst raft localisation of PIN-α2δ is disrupted by PI-PLC treatment, this is assay-dependent and non-specific effects of PI-PLC are observed on the distribution of the endogenous raft marker, caveolin, but not flotillin. Taken together, these data are most consistent with a model where α2δ-1 retains its type I transmembrane topology and its targeting to lipid rafts is governed by sequences upstream of the putative GPI anchor, that promote protein-protein, rather than lipid-lipid interactions. © 2011 Robinson et al.