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.

Beatrice Lana,Ivan Kadurin,Manuela Nieto-Rostro,Karen M Page,Shuxian Ho,Annette C Dolphin

doi:10.1038/srep24531

Beatrice Lana, Ivan Kadurin + Show 4 more

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https://doi.org/10.1038/srep24531

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Journal: Scientific Reports	Publication Date: Apr 14, 2016
Citations: 36	License type: CC BY 4.0

Affiliation: University College London

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 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.

Highlights

Silent synapses, without postsynaptic receptors[14]
In that study α 2δ -1 could be co-immunoprecipitated with a TSP2 synaptogenic region containing its epidermal growth factor (EGF) domains, when they were both co-expressed in HEK-293 cells
In order to examine whether such an interaction could reciprocally affect 3H-gabapentin binding to α 2δ -1, we examined the effect of TSP4 because, like α 2δ -1, it is implicated in neuropathic pain models[18]

Summary

Introduction

Silent synapses, without postsynaptic receptors[14]. TSPs reduce functional postsynaptic AMPA-glutamate receptor accumulation[15]. TSPs 1, 2 and 4 were demonstrated to interact with α 2δ -1 by co-immunoprecipitation from cerebral cortex[16]. We performed radioligand binding experiments to examine whether TSP4 affected 3H-gabapentin binding to α 2δ -1, which might influence the efficacy of this drug. We performed co-immunoprecipitation and immunocytochemical experiments to examine whether α 2δ -1 and TSP4 interacted with each other in this system. Our ligand binding experiments show that co-expression of full length TSP4 modestly reduced the binding affinity for 3H-gabapentin, and only in the presence of Mg2+, whereas the isolated TSP4 EGF domains did not. We were able to demonstrate partial co-immunoprecipitation of α 2δ -1 and full length TSP4, this did not occur for the EGF domains of TSP4. In immunocytochemistry experiments we could not demonstrate co-localisation of α 2δ -1 and TSP4 at the cell surface of transfected cells, both α 2δ -1 and TSP4 could be detected intracellularly and TSP4 was secreted from transfected cells, in the proximity of cells expressing α 2δ -1 on the cell surface

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