Thrombospondin-4 reduces binding affinity of [3H]-gabapentin to calcium-channel α2δ-1-subunit but does not interact with α2δ-1 on the cell-surface when co-expressed

The alpha(2)delta proteins are auxiliary subunits of voltage-gated calcium channels, and influence their trafficking and biophysical properties. The alpha(2)delta ligand gabapentin interacts with alpha(2)delta-1, and inhibits calcium channel trafficking. However, alpha(2)-1 has also been proposed to play a synaptogenic role, independent of calcium channel function. In this regard, alpha(2)delta-1 was identified as a ligand of thrombospondins, with the interaction involving the thrombospondin synaptogenic domain and the alpha(2)delta-1 von-Willebrand-factor domain. Co-immunoprecipitation between alpha(2)delta-1 and the synaptogenic domain of thrombospondin-2 was prevented by gabapentin. We therefore examined whether interaction of thrombospondin with alpha(2)delta-1 might reciprocally influence H-3-gabapentin binding. We concentrated on thrombospondin-4, because, like alpha(2)delta-1, it is upregulated in neuropathic pain models. We found that in membranes from cells co-transfected with alpha(2)delta-1 and thrombospondin-4, there was a Mg2+-dependent reduction in affinity of H-3-gabapentin binding to alpha(2)delta-1. This effect was lost for alpha(2)delta-1 with mutations in the von-Willebrand-factor-A domain. However, the effect on H-3-gabapentin binding was not reproduced by the synaptogenic EGF-domain of thrombospondin-4. Partial co-immunoprecipitation could be demonstrated between thrombospondin-4 and alpha(2)delta-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.