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Targeting of Voltage-Gated Calcium Channels to Lipid Rafts: The Role of Auxiliary Alpha2/Delta-1 Subunits.
[Thesis]. Manchester, UK: The University of Manchester; 2011.
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Abstract
This abstract pertains to a thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Life Sciences.Ca2+ entry through voltage-gated calcium channels (CaVs) triggers a range of physiological events, including synaptic neurotransmission and muscular excito-contraction coupling. CaVs are often localised to discrete membrane microdomains and are required to be targeted to such fine structures in order to perform their cellular functions. CaVs are multi-subunit protein complexes that consist of a core, pore-forming α1 subunit and auxiliary β and α2/δ subunits. The α2/δ subunit is required for the optimal cell surface expression and function of CaVs and is itself localised to cholesterol-rich membrane microdomains called lipid rafts. What is unclear is whether the α2/δ subunit is required for whole CaV complexes to be localised to lipid rafts and what effects lipid raft association has on the cell surface distribution and function of CaVs. By a combination of cellular imaging, biochemistry and electrophysiology, this project shows that the auxiliary α2/δ-1 subunit is both necessary and sufficient to target CaV2.2 to lipid rafts in the COS-7 cell heterologous expression system (Robinson et al, 2010). In addition, α2/δ is localised at the cell surface in discrete puncta and co-localises with two endogenous lipid raft resident proteins, caveolin and flotillin-1. While the punctate cell surface distribution of α2/δ is co-incident with that of caveolin and flotillin-1, its distribution is not dependent on cellular cholesterol, but rather the integrity of the actin cytoskeleton. Additional structure-function analysis by employment of the pIN-α2/δ series of deletion and substitution mutants has shown that the association of α2/δ with lipid rafts is bestowed by an extracellular region of the delta peptide, contrary to other evidence supporting the notion that α2/δ may be a GPI-anchored protein. The exact physiological and functional significance of α2/δ and CaV association with lipid rafts remains poorly understood, but the fact that CaVs are enriched within these fine structures provides a potential mechanism for targeting and access to lipid raft associated signalling pathways.Robinson P, Etheridge S, Song L, Armenise P, Jones OT, Fitzgerald EM. (2010)‘Formation of N-type (Cav2.2) voltage-gated calcium channel membrane microdomains:Lipid raft association and clustering’. Cell Calcium. 48(4):183-94.