Direct, gabapentin-insensitive interaction of a soluble form of the calcium channel subunit α2δ-1 with thrombospondin-4

Abstract

The α2δ‐1 subunit of voltage-gated calcium channels binds to gabapentin and pregabalin, mediating the analgesic action of these drugs against neuropathic pain. Extracellular matrix proteins from the thrombospondin (TSP) family have been identified as ligands of α2δ‐1 in the CNS. This interaction was found to be crucial for excitatory synaptogenesis and neuronal sensitisation which in turn can be inhibited by gabapentin, suggesting a potential role in the pathogenesis of neuropathic pain. Here, we provide information on the biochemical properties of the direct TSP/α2δ-1 interaction using an ELISA-style ligand binding assay. Our data reveal that full-length pentameric TSP-4, but neither TSP-5/COMP of the pentamer-forming subgroup B nor TSP-2 of the trimer-forming subgroup A directly interact with a soluble variant of α2δ-1 (α2δ-1S). Interestingly, this interaction is not inhibited by gabapentin on a molecular level and is not detectable on the surface of HEK293-EBNA cells over-expressing α2δ‐1 protein. These results provide biochemical evidence that supports a specific role of TSP-4 among the TSPs in mediating the binding to neuronal α2δ‐1 and suggest that gabapentin does not directly target TSP/α2δ-1 interaction to alleviate neuropathic pain.

Rab11-dependent recycling of calcium channels is mediated by auxiliary subunit α2δ-1 but not α2δ-3

“…More recently, another study 37 showed a weak interaction between α 2 δ-1 and TSP4, but was unable to demonstrate any interaction between cell-surface expressed α 2 δ-1 and TSP4, concluding that α 2 δ-1 and TSP4 may only interact intracellularly at high concentrations. A further study showed a low affinity interaction between α 2 δ-1 and TSP4, but not other TSPs 38 . However, any interaction of α 2 δ subunits with TSPs is unlikely to be involved in their Rab11-dependent recycling.…”

Analgesia with Gabapentin and Pregabalin May Involve N-Methyl-d-Aspartate Receptors, Neurexins, and Thrombospondins

“…However, two separate studies have failed to show that gabapentin directly disrupts the molecular interaction between thrombospondin and 2-1 proteins in vitro (Lana et al, 2016;El-Awaad et al, 2019), suggesting that the inhibition by gabapentin of thrombospondin actions could be indirect or require the presence of other proteins. Additional studies suggest that signaling proteins, including the scaffolding protein LRP1 (Kadurin et al, 2017) or activation of the small Rho GTPase, Ras-related C3 botulinum toxin substrate 1 (Rac1) are part of the biochemical pathway involved in the synaptogenic action that is inhibited by gabapentinoid drugs (Risher et al, 2018).…”

Rab11-dependent recycling of calcium channels is mediated by auxiliary subunit α2δ-1 but not α2δ-3

“…More recently, another study (Lana et al, 2016) showed a weak interaction between α 2 δ-1 and TSP4, but was unable to demonstrate any interaction between cell-surface expressed α 2 δ-1 and TSP4 nor were they able to demonstrate the importance of the EGF domains of TSP4 in this interaction, concluding that α 2 δ-1 and TSP4 may only interact intracellularly at high concentrations. A further study showed a low affinity interaction between α 2 δ-1 and TSP4, but not other TSPs, which was gabapentin-insensitive and did not involve the EGF domain of TSP4 (El-Awaad et al, 2019). However, any interaction of α 2 δ subunits with TSPs is unlikely to be involved in their Rab11-dependent recycling.…”