Targeting of Voltage-Gated Calcium Channel α2δ-1 Subunit to Lipid Rafts Is Independent from a GPI-Anchoring Motif
| Autor: | Philip Robinson, Elizabeth M. Fitzgerald, Riddhi Shah, Lele Song, Sarah L. Etheridge, Owen T. Jones |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2011 |
| Předmět: |
Macromolecular Assemblies
Sucrose Anatomy and Physiology Glycosylphosphatidylinositols Amino Acid Motifs lcsh:Medicine Biochemistry Ion Channels Phosphoinositide Phospholipase C Integrative Physiology Caveolin Molecular Cell Biology lcsh:Science Lipid raft Multidisciplinary Neurochemistry Raft Lipids Transmembrane protein Transport protein Cell biology Electrophysiology Protein Transport Membrane topology lipids (amino acids peptides and proteins) Rabbits Research Article Biotechnology Cell Physiology Calcium Channels L-Type Protein subunit Recombinant Fusion Proteins Molecular Sequence Data Biophysics Neurophysiology Biology Caveolins Biophysical Phenomena Membrane Microdomains Animals Amino Acid Sequence Protein Interactions lcsh:R Proteins Membrane Proteins Rats Transmembrane Proteins Protein Subunits Membrane protein Cellular Neuroscience lcsh:Q Mutant Proteins Calcium Channels Molecular Neuroscience Neuroscience |
| Zdroj: | PLoS ONE PLoS ONE, Vol 6, Iss 6, p e19802 (2011) |
| ISSN: | 1932-6203 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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