title: Targeting of voltage-gated calcium channel α2δ-1 subunit to lipid rafts is independent from a GPI-anchoring motif.
abstract: Voltage-gated calcium channels (Ca(v)) exist as heteromultimers comprising a pore-forming α(1) with accessory β and α(2)δ subunits which modify channel trafficking and function. We previously showed that α(2)δ-1 (and likely the other mammalian α(2)δ isoforms--α(2)δ-2, 3 and 4) is required for targeting Ca(v)s to lipid rafts, although the mechanism remains unclear. Whilst originally understood to have a classical type I transmembrane (TM) topology, recent evidence suggests the α(2)δ 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 α(2)δ-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-α(2)δ 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-α(2)δ 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 α(2)δ-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.
authors.affiliation: Faculty of Life Sciences, University of Manchester, Core Technology Facility, Manchester, United Kingdom.
authors.forename: Philip
authors.initials: P
authors.lastname: Robinson
authors.forename: Sarah
authors.initials: S
authors.lastname: Etheridge
authors.forename: Lele
authors.initials: L
authors.lastname: Song
authors.forename: Riddhi
authors.initials: R
authors.lastname: Shah
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Fitzgerald
authors.forename: Owen T
authors.initials: OT
authors.lastname: Jones
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D055592:Biophysical Phenomena; D015220:Calcium Channels / Q000737:chemistry* / Q000378:metabolism*; D020746:Calcium Channels, L-Type; D022461:Caveolins / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D021962:Membrane Microdomains / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D054801:Phosphoinositide Phospholipase C / Q000378:metabolism; D021122:Protein Subunits / Q000378:metabolism*; D021381:Protein Transport; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013395:Sucrose / Q000378:metabolism
chemical_list: C486060:Cacna2d1 protein, rat; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D022461:Caveolins; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D050505:Mutant Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; C106354:flotillins; D013395:Sucrose; D054801:Phosphoinositide Phospholipase C