No abstract

The auxiliary α2δ subunits of voltage-gated calcium channels are extracellular membrane-associated proteins, which are post-translationally cleaved into disulfide-linked polypeptides α2 and δ. We now show, using α2δ constructs containing artificial cleavage sites, that this processing is an essential step permitting voltage-dependent activation of plasma membrane N-type (CaV2.2) calcium channels. Indeed, uncleaved α2δ inhibits native calcium currents in mammalian neurons. By inducing acute cell-surface proteolytic cleavage of α2δ, voltage-dependent activation of channels is promoted, independent from the trafficking role of α2δ. Uncleaved α2δ does not support trafficking of CaV2.2 channel complexes into neuronal processes, and inhibits Ca2+ entry into synaptic boutons, and we can reverse this by controlled intracellular proteolytic cleavage. We propose a model whereby uncleaved α2δ subunits maintain immature calcium channels in an inhibited state. Proteolytic processing of α2δ then permits voltage-dependent activation of the channels, acting as a checkpoint allowing trafficking only of mature calcium channel complexes into neuronal processes.DOI: http://dx.doi.org/10.7554/eLife.21143.001

The α2δ proteins are auxiliary subunits of voltage-gated calcium channels, and influence their trafficking and biophysical properties. The α2δ ligand gabapentin interacts with α2δ-1, and inhibits calcium channel trafficking. However, α2-1 has also been proposed to play a synaptogenic role, independent of calcium channel function. In this regard, α2δ-1 was identified as a ligand of thrombospondins, with the interaction involving the thrombospondin synaptogenic domain and the α2δ-1 von-Willebrand-factor domain. Co-immunoprecipitation between α2δ-1 and the synaptogenic domain of thrombospondin-2 was prevented by gabapentin. We therefore examined whether interaction of thrombospondin with α2δ-1 might reciprocally influence 3H-gabapentin binding. We concentrated on thrombospondin-4, because, like α2δ-1, it is upregulated in neuropathic pain models. We found that in membranes from cells co-transfected with α2δ-1 and thrombospondin-4, there was a Mg2+ -dependent reduction in affinity of 3H-gabapentin binding to α2δ-1. This effect was lost for α2δ-1 with mutations in the von-Willebrand-factor-A domain. However, the effect on 3H-gabapentin binding was not reproduced by the synaptogenic EGF-domain of thrombospondin-4. Partial co-immunoprecipitation could be demonstrated between thrombospondin-4 and α2δ-1 when co-transfected, but there was no co-immunoprecipitation with thrombospondin-4-EGF domain. Furthermore, we could not detect any association between these two proteins on the cell-surface, indicating the demonstrated interaction occurs intracellularly.

SummaryA minor α2δ-1 splice-variant, (ΔA+BΔC), with reduced affinity for gabapentin, is differentially upregulated in DRG neurons following ligation. This could influence the efficacy of gabapentin.

Voltage-gated Ca2+ (CaV) channels consist of a pore-forming α1 subunit, which determines the main functional and pharmacological attributes of the channel. The CaV1 and CaV2 channels are associated with auxiliary β- and α2δ-subunits. The molecular mechanisms involved in α2δ subunit trafficking, and the effect of α2δ subunits on trafficking calcium channel complexes remain poorly understood. Here we show that α2δ-1 is a ligand for the Low Density Lipoprotein (LDL) Receptor-related Protein-1 (LRP1), a multifunctional receptor which mediates trafficking of cargoes. This interaction with LRP1 is direct, and is modulated by the LRP chaperone, Receptor-Associated Protein (RAP). LRP1 regulates α2δ binding to gabapentin, and influences calcium channel trafficking and function. Whereas LRP1 alone reduces α2δ-1 trafficking to the cell-surface, the LRP1/RAP combination enhances mature glycosylation, proteolytic processing and cell-surface expression of α2δ-1, and also increase plasma-membrane expression and function of CaV2.2 when co-expressed with α2δ-1. Furthermore RAP alone produced a small increase in cell-surface expression of CaV2.2, α2δ-1 and the associated calcium currents. It is likely to be interacting with an endogenous member of the LDL receptor family to have these effects. Our findings now provide a key insight and new tools to investigate the trafficking of calcium channel α2δ subunits.