The calcium channel alpha2delta-2 subunit partitions with CaV2.1 into lipid rafts in cerebellum: implications for localization and function.

The accessory alpha2delta subunits of voltage-gated calcium channels are highly glycosylated transmembrane proteins that interact with calcium channel alpha1 subunits to enhance calcium currents. We compared the membrane localization and processing of native cerebellar alpha2delta-2 subunits with alpha2delta-2 stably expressed in tsA-201 cells. We identified that alpha2delta-2 is completely concentrated in cholesterol-rich microdomains (lipid rafts) in cerebellum, in which it substantially colocalizes with the calcium channel alpha1 subunit CaV2.1, although CaV2.1 is also present in the Triton X-100-soluble fraction. In tsA-201 cells, unlike cerebellum, alpha2delta-2 is not completely proteolytically processed into alpha2-2 and delta-2. However, this processing is more complete in the lipid raft fraction of tsA-201 cells, in which alpha2delta-2 also colocalizes with CaV2.1. Cholesterol depletion of intact cells disrupted their lipid rafts and enhanced CaV2.1/alpha2delta-2/beta4 currents. Furthermore, alpha2delta-2 coimmunoprecipitates with lipid raft-associated proteins of the stomatin family. The apparent affinity of alpha2delta-2 for its ligand gabapentin is increased markedly in the cholesterol-rich microdomain fractions, in both cerebellum and the stable alpha2delta-2 cell line. In contrast, alpha2delta-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 alpha2delta-2 shows reduced functionality in terms of enhancement of CaV2.1/beta4 calcium currents, suggesting that the integrity of the gabapentin binding site may be important for normal functioning of alpha2delta-2. Together, these results indicate that both alpha2delta-2 and CaV2.1 are normally associated with cholesterol-rich microdomains, and this influences their functionality.