title: Thrombospondin-4 reduces binding affinity of [(3)H]-gabapentin to calcium-channel α2δ-1-subunit but does not interact with α2δ-1 on the cell-surface when co-expressed.
abstract: 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 (3)H-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 Mg(2+) -dependent reduction in affinity of (3)H-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 (3)H-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.
authors.affiliation: Department of Neuroscience, Physiology and Pharmacology, University College London, Gower St., London WC1E 6BT, United Kingdom.
authors.forename: Beatrice
authors.initials: B
authors.lastname: Lana
authors.affiliation: Department of Neuroscience, Physiology and Pharmacology, University College London, Gower St., London WC1E 6BT, United Kingdom.
authors.forename: Karen M
authors.initials: KM
authors.lastname: Page
authors.affiliation: Department of Neuroscience, Physiology and Pharmacology, University College London, Gower St., London WC1E 6BT, United Kingdom.
authors.forename: Ivan
authors.initials: I
authors.lastname: Kadurin
authors.affiliation: Department of Neuroscience, Physiology and Pharmacology, University College London, Gower St., London WC1E 6BT, United Kingdom.
authors.forename: Shuxian
authors.initials: S
authors.lastname: Ho
authors.affiliation: Department of Neuroscience, Physiology and Pharmacology, University College London, Gower St., London WC1E 6BT, United Kingdom.
authors.forename: Manuela
authors.initials: M
authors.lastname: Nieto-Rostro
authors.affiliation: Department of Neuroscience, Physiology and Pharmacology, University College London, Gower St., London WC1E 6BT, United Kingdom.
authors.forename: Annette C
authors.initials: AC
authors.lastname: Dolphin
mesh_terms: D000588:Amines / Q000378:metabolism*; D000700:Analgesics / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D003509:Cyclohexanecarboxylic Acids / Q000378:metabolism*; D000077206:Gabapentin; D047468:Immunoprecipitation; D011485:Protein Binding; D019699:Thrombospondins / Q000378:metabolism*; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
chemical_list: D000588:Amines; D000700:Analgesics; C425903:CACNA2D1 protein, human; D015220:Calcium Channels; D003509:Cyclohexanecarboxylic Acids; D019699:Thrombospondins; C101864:thrombospondin 4; D005680:gamma-Aminobutyric Acid; D000077206:Gabapentin