Targeting of Voltage-Gated Calcium Channels to Lipid Rafts: The Role of Auxiliary Alpha2/Delta-1 Subunits.

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 alpha1 subunit and auxiliary β and alpha2/delta subunits. The alpha2/delta 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 alpha2/delta 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 alpha2/delta-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, alpha2/delta 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 alpha2/delta 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-alpha2/delta series of deletion and substitution mutants has shown that the association of alpha2/delta with lipid rafts is bestowed by an extracellular region of the delta peptide, contrary to other evidence supporting the notion that alpha2/delta may be a GPI-anchored protein. The exact physiological and functional significance of alpha2/delta 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.

Date of Award	31 Dec 2011
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Owen Jones (Supervisor) & Liz Fitzgerald (Supervisor)