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The Journal of Neuroscience, August 23, 2006, 26(34):8748-8757; doi:10.1523/JNEUROSCI.2764-06.2006

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Cellular/Molecular
The Calcium Channel ₂-2 Subunit Partitions with Ca_V2.1 into Lipid Rafts in Cerebellum: Implications for Localization and Function

Anthony Davies,^1 * Leon Douglas,^1 * Jan Hendrich,^1 * Jack Wratten,¹ Alexandra Tran Van Minh,¹ Isabelle Foucault,¹ Dietlind Koch,¹ Wendy S. Pratt,¹ Helen R. Saibil,² and Annette C. Dolphin¹

¹Department of Pharmacology, University College London, London WC1E 6BT, United Kingdom, and ²School of Crystallography, Birkbeck, University of London, London WC1E 7HX, United Kingdom

Correspondence should be addressed to Annette C. Dolphin, Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK. Email: a.dolphin{at}ucl.ac.uk

The accessory ₂ subunits of voltage-gated calcium channels are highly glycosylated transmembrane proteins that interact with calcium channel 1 subunits to enhance calcium currents. We compared the membrane localization and processing of native cerebellar ₂-2 subunits with ₂-2 stably expressed in tsA-201 cells. We identified that ₂-2 is completely concentrated in cholesterol-rich microdomains (lipid rafts) in cerebellum, in which it substantially colocalizes with the calcium channel 1 subunit Ca_V2.1, although Ca_V2.1 is also present in the Triton X-100-soluble fraction. In tsA-201 cells, unlike cerebellum, ₂-2 is not completely proteolytically processed into ₂-2 and -2. However, this processing is more complete in the lipid raft fraction of tsA-201 cells, in which ₂-2 also colocalizes with Ca_V2.1. Cholesterol depletion of intact cells disrupted their lipid rafts and enhanced Ca_V2.1/₂-2/4 currents. Furthermore, ₂-2 coimmunoprecipitates with lipid raft-associated proteins of the stomatin family. The apparent affinity of ₂-2 for its ligand gabapentin is increased markedly in the cholesterol-rich microdomain fractions, in both cerebellum and the stable ₂-2 cell line. In contrast, ₂-2 containing a point mutation (R282A) has a much lower affinity for gabapentin, and this is not enhanced in the lipid raft fraction. This R282A mutant ₂-2 shows reduced functionality in terms of enhancement of Ca_V2.1/4 calcium currents, suggesting that the integrity of the gabapentin binding site may be important for normal functioning of ₂-2. Together, these results indicate that both ₂-2 and Ca_V2.1 are normally associated with cholesterol-rich microdomains, and this influences their functionality.

Key words: calcium; channel; ₂; gabapentin; lipid raft; cerebellum; cholesterol

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Received April 13, 2006; revised July 17, 2006; accepted July 17, 2006.

Correspondence should be addressed to Annette C. Dolphin, Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK. Email: a.dolphin{at}ucl.ac.uk

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