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.
Davies
Anthony
A
Department of Pharmacology, University College London, London WC1E 6BT, United Kingdom.
Douglas
Leon
L
Hendrich
Jan
J
Wratten
Jack
J
Tran Van Minh
Alexandra
A
Foucault
Isabelle
I
Koch
Dietlind
D
Pratt
Wendy S
WS
Saibil
Helen R
HR
Dolphin
Annette C
AC
D000409:Alanine; D000588:Amines / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001120:Arginine; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D020864:Calcium Channels, N-Type / Q000378:metabolism* / Q000502:physiology; D002460:Cell Line; D002531:Cerebellum / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism; D003509:Cyclohexanecarboxylic Acids / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology; D004553:Electric Conductivity; D000077206:Gabapentin; D047468:Immunoprecipitation; D021962:Membrane Microdomains / Q000378:metabolism*; D051379:Mice; D009154:Mutation / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000378:metabolism; D011689:Purkinje Cells / Q000378:metabolism; D014018:Tissue Distribution; D005680:gamma-Aminobutyric Acid / Q000378:metabolism / Q000494:pharmacology
D000409:Alanine; D000588:Amines; D001120:Arginine; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D020864:Calcium Channels, N-Type; D002784:Cholesterol; D003509:Cyclohexanecarboxylic Acids; D000077206:Gabapentin; D009419:Nerve Tissue Proteins; D005680:gamma-Aminobutyric Acid; C512066:CACNA2D1 protein, mouse