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Functional exofacially tagged N-type calcium channels elucidate the interaction with auxiliary ?2?-1 subunits.

ABSTRACT: CaV1 and CaV2 voltage-gated calcium channels are associated with ? and ?2? accessory subunits. However, examination of cell surface-associated CaV2 channels has been hampered by the lack of antibodies to cell surface-accessible epitopes and of functional exofacially tagged CaV2 channels. Here we report the development of fully functional CaV2.2 constructs containing inserted surface-accessible exofacial tags, which allow visualization of only those channels at the plasma membrane, in both a neuronal cell line and neurons. We first examined the effect of the auxiliary subunits. Although ?2? subunits copurify with CaV2 channels, it has recently been suggested that this interaction is easily disrupted and nonquantitative. We have now tested whether ?2? subunits are associated with these channels at the cell surface. We found that, whereas ?2?-1 is readily observed at the plasma membrane when expressed alone, it appears absent when coexpressed with CaV2.2/?1b, despite our finding that ?2?-1 increases plasma-membrane CaV2.2 expression. However, this was due to occlusion of the antigenic epitope by association with CaV2.2, as revealed by antigen retrieval; thus, our data provide evidence for a tight interaction between ?2?-1 and the ?1 subunit at the plasma membrane. We further show that, although CaV2.2 cell-surface expression is reduced by gabapentin in the presence of wild-type ?2?-1 (but not a gabapentin-insensitive ?2?-1 mutant), the interaction between CaV2.2 and ?2?-1 is not disrupted by gabapentin. Altogether, these results demonstrate that CaV2.2 and ?2?-1 are intimately associated at the plasma membrane and allow us to infer a region of interaction.

SUBMITTER: Cassidy JS

PROVIDER: S-EPMC4066493 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Functional exofacially tagged N-type calcium channels elucidate the interaction with auxiliary α2δ-1 subunits.

Cassidy John S JS Ferron Laurent L Kadurin Ivan I Pratt Wendy S WS Dolphin Annette C AC

Proceedings of the National Academy of Sciences of the United States of America 20140602 24

CaV1 and CaV2 voltage-gated calcium channels are associated with β and α2δ accessory subunits. However, examination of cell surface-associated CaV2 channels has been hampered by the lack of antibodies to cell surface-accessible epitopes and of functional exofacially tagged CaV2 channels. Here we report the development of fully functional CaV2.2 constructs containing inserted surface-accessible exofacial tags, which allow visualization of only those channels at the plasma membrane, in both a neur ...[more]

PMID: 24889613

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Functional exofacially tagged N-type calcium channels elucidate the interaction with auxiliary ?2?-1 subunits.

Publications

Functional exofacially tagged N-type calcium channels elucidate the interaction with auxiliary α2δ-1 subunits.

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