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

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

doi:10.1038/srep24531

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Published: 14 April 2016

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

Beatrice Lana¹^na1,
Karen M. Page¹^na1,
Ivan Kadurin¹^na1,
Shuxian Ho¹^na1,
Manuela Nieto-Rostro¹^na1 &
…
Annette C. Dolphin¹^na1

Scientific Reports volume 6, Article number: 24531 (2016) Cite this article

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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.

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Introduction

Ca_V1 and Ca_V2 voltage-gated calcium channels are associated with auxiliary β and α₂δ subunits, which influence both the expression on the plasma membrane and the biophysical properties of the channels (for review see^1,2). Understanding the mechanism of action of the α₂δ-1 subunit is of translational importance, as it is the therapeutic target of the gabapentinoid drugs gabapentin and pregabalin³. These drugs were developed as antiepileptic agents, but also show efficacy in the treatment of neuropathic pain conditions^1,3,4,5. We have found that these drugs reduce calcium currents chronically but not acutely, by inhibiting α₂δ-1 and α₂δ-2 trafficking^6,7,8,9.

We have recently demonstrated that α₂δ-1 and Ca_V2.2 interact both intracellularly and at the plasma membrane, when these proteins are co-expressed⁹. In this and other studies, we found that the von Willebrand Factor-A (VWA) domain of α₂δ subunits is important, both for cell surface expression of α₂δ-1 and for mediating the enhancement by α₂δ-1 of Ca_V2 channel cell surface expression and function^9,10,11. Structural evidence indicates that the region of interaction between α₂δ-1 and Ca_V1.1 involves the VWA domain as well as other regions of α₂δ-1¹². However the VWA domain may also interact with other protein(s) involved in calcium channel trafficking pathways.

The thrombospondins (TSPs) are multi-domain secreted extracellular matrix proteins (Fig. 1A) with diverse functions¹³, one of which is synaptogenesis¹⁴. TSPs are secreted from astrocytes and promote the formation of silent synapses, without postsynaptic receptors¹⁴. TSPs also reduce functional postsynaptic AMPA-glutamate receptor accumulation¹⁵. It was found that postsynaptic expression of α₂δ-1 is required for TSP-induced synaptogenesis in the CNS and this was reported to be independent of the function of α₂δ-1 as a calcium channel subunit¹⁶. Furthermore, TSPs 1, 2 and 4 were demonstrated to interact with α₂δ-1 by co-immunoprecipitation from cerebral cortex¹⁶. The epidermal growth factor (EGF)-like repeats of TSPs were identified to represent their synaptogenic domain and a synaptogenic region of TSP2 containing these EGF repeats was found to interact with full length α₂δ-1 and with its VWA domain, when both were co-expressed in HEK-293 cells¹⁶. In addition, the α₂δ-1 ligand, gabapentin, was observed to inhibit co-immunoprecipitation between the synaptogenic domain of TSP2 and α₂δ-1, when they were co-expressed¹⁶.

In the present study, our aim was to examine whether the gabapentin-sensitive interaction between TSPs and α₂δ-1 demonstrated previously¹⁶ could reciprocally affect ³H-gabapentin binding. We primarily concentrated on TSP4, as, like α₂δ-1^8,17, it is up-regulated in dorsal spinal cord following peripheral sensory nerve injury¹⁸. We therefore performed radioligand binding experiments to examine whether TSP4 affected ³H-gabapentin binding to α₂δ-1, which might influence the efficacy of this drug. We also performed co-immunoprecipitation and immunocytochemical experiments to examine whether α₂δ-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. Furthermore although we were able to demonstrate partial co-immunoprecipitation of α₂δ-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 α₂δ-1 and TSP4 at the cell surface of transfected cells, although both α₂δ-1 and TSP4 could be detected intracellularly and TSP4 was secreted from transfected cells, in the proximity of cells expressing α₂δ-1 on the cell surface.

Results

Effect of TSP4 on ³H-gabapentin binding to α2δ-1

It was demonstrated previously that antibodies to TSP1, 2 and 4 can immunoprecipitate α₂δ-1 from brain¹⁶. In that study α₂δ-1 could also be co-immunoprecipitated with a TSP2 synaptogenic region containing its EGF domains, when they were both co-expressed in HEK-293 cells. It was then shown that this co-immunoprecipitation was reduced in the presence of the α₂δ ligand gabapentin¹⁶. In order to examine whether such an interaction could reciprocally affect ³H-gabapentin binding to α₂δ-1, we examined the effect of TSP4 because, like α₂δ-1, it is implicated in neuropathic pain models¹⁸. We therefore expressed α₂δ-1 and TSP4 together in tsA-201 cells and performed ³H-gabapentin binding assays on membrane preparations. The two main constructs used in these experiments were TSP4_HA (termed TSP4) or its truncated form containing the EGF domains (TSP4-EGF_HA, termed TSP4-EGF) (Fig. 1A). In all preparations used for binding assays, the amount of α₂δ-1 expressed was also examined by western blotting; α₂δ-1 was expressed to a similar extent in the absence and presence of TSP4 (Fig. 1B). Since TSPs were reported to interact with the VWA domain of α₂δ-1¹⁶ and VWA domains bind protein ligands in a divalent cation-dependent manner^19,20, we performed ³H-gabapentin binding in the absence of divalent cations (2 mM EDTA to chelate all divalent cations), or in the presence of 2 mM Mg²⁺ (Fig. 1C,D). We found that the affinity of ³H-gabapentin binding to α₂δ-1 was significantly reduced by the presence of TSP4, but only in the presence of Mg²⁺ and not in the presence of EDTA (Fig. 1C,D). This is shown by the increased K_D value in Mg²⁺ (Fig. 1E). In control experiments we observed no direct binding of ³H-gabapentin to membranes of tsA-201 cells expressing only TSP4 (Fig. 1D).

We then utilised an α₂δ-1 construct containing three point mutations in the metal ion dependent adhesion site (MIDAS) motif of the VWA domain¹¹. Mutation of the MIDAS motif has been reported to prevent divalent cation-mediated interaction of VWA domains with their protein ligands²¹. We found that α₂δ-1 MIDAS^AAA bound ³H-gabapentin with an affinity similar to WT α₂δ-1 (Fig. 1F). However, no effect of TSP4 was observed on the affinity of ³H-gabapentin binding to α₂δ-1 MIDAS^AAA, determined in the presence or absence of Mg²⁺ (Fig. 1F). This supports the possibility that TSP4 can interact with the VWA domain of α₂δ-1 in a Mg²⁺ -dependent manner and that this interaction allosterically reduces the affinity of ³H-gabapentin binding to α₂δ-1, but does not affect the B_max.

The EGF-like domains of TSPs were identified previously as their synaptogenic region and for TSP2 a domain including the EGF repeats was found to be the region interacting with α₂δ-1¹⁶. We therefore examined the ability of an equivalent TSP4-EGF domain construct to affect ³H-gabapentin binding to α₂δ-1. However, we found no effect of this domain on the affinity of ³H-gabapentin binding to α₂δ-1, either in the presence or absence of Mg²⁺ (Fig. 2A,B). In a control experiment, TSP4-EGF alone showed no ³H-gabapentin binding (data not shown).

Examination of the presence of TSP4 with α₂δ-1 in DRM fractions

The α₂δ proteins are strongly concentrated in detergent-resistant membrane (DRM) fractions^22,23,24 (Fig. 3A). For this reason we examined whether TSP4 would co-purify with α₂δ-1 in DRMs, as evidence of an interaction. When co-expressed with α₂δ-1 in tsA-201 cells, only a very small proportion (about 7% from the experiment shown in Fig. 3A) of TSP4 was also observed in DRM fraction 5 (Fig. 3A). This distribution was not affected by DRM preparation in the presence or absence of Mg²⁺, as shown by Western blotting of α₂δ-1 and TSP4 from concentrated DRMs (Fig. 3B). To examine whether the amount of TSP4 that co-localises with α₂δ-1 in DRMs is sufficient to affect the binding of gabapentin to α₂δ-1, ³H-gabapentin binding was determined in these DRMs. The observed K_D for ³H-gabapentin binding in DRMs in Mg²⁺ -containing medium was unchanged by the presence of TSP4 (Fig. 3C) and was similar to that found previously in DRMs²⁵. This result indicates that the amount of TSP4 that is present with α₂δ-1 in the DRMs does not cause a reduction in the binding affinity of gabapentin to α₂δ-1.

We then investigated whether we could demonstrate any evidence for an effect of TSP4 on ³H-gabapentin binding in brain. The binding affinity of ³H-gabapentin is known to increase successively as α₂δ-1 is purified from brain membranes²⁶. We wished to test the hypothesis that an interaction between TSPs and α₂δ-1 might contribute to the lower ³H-gabapentin binding affinity in brain membranes compared to the purified α₂δ-1 protein. To do this, we took advantage of our finding that the reduction of ³H-gabapentin binding affinity in the presence of TSP4 that we observed in tsA-201 cell membranes was dependent on the presence of Mg²⁺ and was not observed in the presence of EDTA.

We therefore examined whether there was a Mg²⁺ -dependent effect on ³H-gabapentin binding affinity in brain, which would be compatible with a Mg²⁺ -dependent interaction between an endogenous binding partner (such as TSP4) and endogenous α₂δ-1. However, in an experiment using adult rat brain tissue, we found that the ³H-gabapentin binding affinity was not altered by the presence of EDTA or MgCl₂, either in crude brain membranes (K_D 217.9 nM and 189.1 nM, respectively; Supplementary Fig. S1A) or in DRMs prepared from brain (K_D 40.9 nM and 52.7 nM, respectively; Supplementary Fig. S1B). As previously observed in tsA-201 cells, the presence of Mg²⁺ reduced the B_max for ³H-gabapentin in brain membranes. This suggests that the ³H-gabapentin binding affinity of α₂δ-1 in brain membranes is not influenced by the interaction of any proteins (including TSPs) binding to the VWA domain of α₂δ-1 in a Mg²⁺ -dependent manner.

Interaction between α2δ-1 and TSP4

In order to examine whether there was an interaction between α₂δ-1 and TSP4 in our system and to understand where in the cell this putative interaction was occurring we also performed co-immunoprecipitation and immunocytochemistry. For co-immunoprecipitation studies, we expressed α₂δ-1 and TSP4_HA together in tsA-201 cells. Full-length TSP4 was well-expressed (Fig. 4A) and could be immunoprecipitated with HA antibody (Fig. 4B). We observed a small amount of co-immunoprecipitation of α₂δ-1 with full-length TSP4, but the proportion of α₂δ-1 that was co-immunoprecipitated with TSP4 was very low, compared to that in the WCL (Fig. 4B) and compared to the amount of TSP4 which was efficiently immunoprecipitated. Co-immunoprecipitation of α₂δ-1 with TSP4 was observed in 8/13 experiments; the amount of co-immunoprecipitation of α₂δ-1 in these experiments is quantified relative to TSP4 immunoprecipitated in Supplementary Table S1. These results indicate that the interaction between α₂δ-1 and full length TSP4 when they are co-expressed is probably weak, in agreement with the small amount of TSP4 co-purifying with α₂δ-1 in DRM fractions.

No specific co-immunoprecipitation of α₂δ-1-MIDAS^AAA was seen with TSP4 (Supplementary Fig. S2A–D, lane 2). Furthermore, we did not observe co-immunoprecipitation of α₂δ-1 with TSP4-EGF_HA (Supplementary Fig. S2, lane 4). These results agree with the lack of effect of TSP4 on ³H-gabapentin binding to α₂δ-1-MIDAS^AAA (Fig. 1F) and the lack of effect of TSP4-EGF domain on ³H-gabapentin binding to α₂δ-1 (Fig. 2A,B).

TSPs have been documented to bind to a number of proteins including the cell surface receptor LRP1^27,28. LRP1 contains four ligand binding domains²⁹, which can each be separately expressed as a minigene fused with the LRP1 transmembrane domain and retain ligand binding activity^30,31. As a control, we immunoprecipitated these constructs using the same immunoprecipitation protocol and found that TSP4-EGF (Fig. 5) and full length TSP4 (Supplementary Fig. S3) co-immunoprecipitated robustly with LRP1 ligand binding domains.

Immunocytochemical investigation of distribution of expressed α₂δ-1 with TSP4 in tsA-201 cells

TSPs are secreted extracellular matrix proteins. In order to determine whether secreted TSP4 was able to bind to α₂δ proteins expressed on the cell surface of transfected cells, mimicking what might occur in native tissues, we first co-transfected α₂δ-1 and TSP4 into tsA-201 cells. TSP4 protein was detected by immunoreactivity to its HA tag. As expected, the two proteins were both found to be present intracellularly in permeabilised cells when they were co-transfected (Fig. 6A, upper row). Similarly, TSP4-EGF and α₂δ-1 could both be detected intracellularly when co-transfected (Fig. 6A, lower row).

In non-permeabilised cells, we observed secreted TSP4 spreading around the base of co-transfected cells, associated with the substrate on the coverslip, whereas α₂δ-1 was expressed on the cell surface (Fig. 6B, lower row). In contrast, at a higher focal plane, there was very little TSP4 in the proximity of the cell surface, where α₂δ-1 was observed (Fig. 6B, upper row), although in the same experiment, but in permeabilising conditions, both were again detected intracellularly (data not shown). TSP4 was also detected in the medium (Fig. 6C), further evidence that it was secreted from transfected tsA-201 cells. In none of these experiments did we observe TSP4 co-localised with α₂δ-1 on the cell surface. Furthermore, fewer cells were observed expressing α₂δ-1 on the cell surface when TSP4 was co-expressed, suggesting α₂δ-1 may have been retained intracellularly by co-expression with TSP4.

In order to mimic the situation in the brain where TSP4 is secreted mainly from astrocytes and α₂δ-1 is on the cell surface of neurons, in the next set of experiments we transfected α₂δ-1 and TSP4 separately and then mixed the two populations of transfected cells (Fig. 7). However, again no co-localisation was observed on the cell surface of closely-apposed cells. When we permeabilised cells and examined the region where two neighboring cells bordered each other, one expressing α₂δ-1 and the other secreting TSP4 (Fig. 7A), there was no co-localisation on the bordering cell surface (right panel). Furthermore in non-permeabilised cells (Fig. 7B), no co-localisation of α₂δ-1 with TSP4 was seen around α₂δ-1-expressing cells (arrow indicates α₂δ-1 on cell surface), despite the clear presence of a neighbouring cell secreting TSP4 (*Fig. 7B, the lower middle panel shows the base of the cells with secreted TSP4).

In order to determine the intracellular compartments in which α₂δ-1 and TSP4 were localised we performed immunocytochemistry experiments using antibodies against markers of the endoplasmic reticulum (ER) and Golgi apparatus. In permeabilised cells, when both TSP4 and α₂δ-1 were transfected together, TSP4 was co-localised in part with both the ER marker PDI and the Golgi marker Golgin-97, but also showed extensive punctate localisation near the plasma membrane, which was not associated with ER or Golgi (Fig. 8A,C). In contrast, α₂δ-1 was extensively co-localised with the ER marker but very little was associated with the Golgi (Fig. 8B,D).

Discussion

The α₂δ proteins were first identified as auxiliary subunits of voltage gated calcium channels, affecting both their trafficking and biophysical properties (for review see¹), but more recently they have also been proposed to play other roles, independent of their involvement in calcium channel function^16,32,33. Nevertheless, since α₂δ proteins traffic calcium channels to the plasma membrane and to presynaptic terminals as well as affecting channel function^9,11, it is difficult to separate their independent roles from their roles as calcium channel auxiliary subunits.

In view of the fact that the gabapentinoid drugs, gabapentin and pregabalin, bind to α₂δ-1 and α₂δ-2 proteins^1,3,4,5, the potential of these proteins to have multiple functions is of translational importance. The affinity of gabapentin binding has been shown to depend on the state of purification of α₂δ-1, being reported to be 92 nM in brain membranes and 9.4 nM in purified protein²⁶, reflecting the possibility that an endogenous small molecule binding partner is lost during purification. In agreement with this we have found here that the binding affinity for ³H-gabapentin is increased in purified DRM fractions from brain.

Gabapentin interacts with a binding site found in both α₂δ-1 and α₂δ-2, involving three arginine residues, upstream of the VWA domain^3,24,34. In agreement with this, we have found here that gabapentin still binds to α₂δ-1 MIDAS^AAA with a similar affinity to that observed for WT α₂δ-1, indicating that VWA domain function is not directly involved in the binding of gabapentin. We also demonstrated a similar result for α₂δ-2¹⁰. It was previously found that gabapentin was able to prevent co-immunoprecipitation between α₂δ-1 and a TSP2 synaptogenic domain when they were co-expressed and that the interaction involved the α₂δ-1 VWA domain¹⁶. Therefore gabapentin could be considered to be an allosteric modulator of TSP binding to α₂δ-1, since gabapentin itself does not bind directly to the VWA domain. Allosteric interactions between two sites are normally reciprocal³⁵ and therefore we hypothesized that the interaction of TSPs with α₂δ-1 might influence ³H-gabapentin binding. Indeed, we found this to be the case; in membrane preparations prepared from co-transfected cells, the presence of TSP4 significantly reduced the affinity for ³H-gabapentin binding to α₂δ-1, although this result was not observed in more purified DRM fractions, where little TSP4 co-purified with α₂δ-1. The effect of TSP4 on ³H-gabapentin binding was only observed in the presence of Mg²⁺, not when divalent cations were chelated. Furthermore, it was absent when using α₂δ-1 MIDAS^AAA, indicating that it is likely to be mediated by the interaction of TSP4 with the α₂δ-1 MIDAS motif, in a Mg²⁺ -dependent manner.

In summary, our studies indicate that if α₂δ-1 and TSP4 are both expressed in the same cells and are therefore present at high concentration intracellularly, then TSP4 can influence the properties of α₂δ-1, in terms of ³H-gabapentin binding affinity. However, this effect was not reproduced by the EGF repeat domain of TSP4, which is comparable to the region of TSP2 previously found to interact with α₂δ-1¹⁶, although the TSP4-EGF construct has four, rather than three EGF repeats and it does not contain a properdin-like domain.

In the co-immunoprecipitation experiments presented here, using C-terminally HA-tagged TSP4, some co-immunoprecipitation with co-expressed full length α₂δ-1 was found, although this was not observed in all experiments and was not reproduced with the TSP4-EGF domain. This suggests that the interaction between α₂δ-1 and TSP4 is probably of low affinity and this is in agreement with our finding that the effect of TSP4 on ³H-gabapentin binding is absent in DRM preparations, where any interaction between α₂δ-1 and TSP4 is not retained, as there is very little co-purification between α₂δ-1 and TSP4. In agreement with this, the affinity of ³H-gabapentin binding to brain membranes and brain DRMs was not affected by chelating divalent cations with EDTA, which would disrupt any interaction between endogenous α₂δ-1 and TSPs, indicating that TSPs do not represent a significant native interaction partner of α₂δ-1 in adult rat brain, despite the presence of TSP4 in this tissue³⁶.

Co-immunoprecipitation was previously demonstrated of the TSP2 synaptogenic domain with α₂δ-1 or its VWA domain when the two proteins were co-expressed in HEK-293 cells, using C-terminal FLAG or other tags on α₂δ-1 for the immunoprecipitation¹⁶. We have previously observed that C-terminal tags were cleaved from α₂δ subunits during their processing and were not associated with cell surface α₂δ subunits²³. This was one of the initial supporting pieces of evidence for glycosylphosphatidylinositol (GPI)-anchoring of the α₂δ proteins²³. Furthermore, the vast majority of transfected α₂δ-1 expressed in tsA-201 cells is not on the cell surface, as also demonstrated in cell surface biotinylation experiments²². Therefore immunoprecipitation of the C-terminal FLAG-tagged α₂δ-1 with co-expressed TSPs¹⁶ is likely to be occurring mainly between intracellular proteins. This is also true for the co-immunoprecipitation studies performed here, since in these experiments the two proteins were co-expressed and we have shown that both are in part associated with the ER compartment, as expected. Furthermore, we have obtained no evidence from the immunocytochemical experiments presented here that TSP4, once secreted, interacts with α₂δ-1 on the cell surface of the same or adjacent cells. It is of course possible that additional factors, not present in the transfected cells, are required for such an interaction. Of interest, it has recently been identified that TSPs interact intracellularly with the ER-resident protein stromal interaction molecule-1³⁷.

α₂δ-1 is upregulated in DRG neurons following peripheral sensory nerve injury^8,38,39 and there is increased trafficking of α₂δ-1 to presynaptic terminals in the dorsal horn of the spinal cord⁸. TSPs are mainly expressed and secreted by astrocytes in the CNS, including the spinal cord^14,18 and TSP4 is secreted by astrocytes in the subventricular zone after brain injury⁴⁰. TSP4 is also up-regulated in dorsal spinal cord following peripheral sensory nerve injury in rats and this up-regulation correlates with the development of neuropathic pain¹⁸. From the ³H-gabapentin binding studies described here, one inference might be that the up-regulation of TSP4 in neuropathic pain conditions could limit the effectiveness of gabapentin, as it reduces the affinity of gabapentin binding to α₂δ-1. However, as described above, in the spinal cord, α₂δ-1 is present mainly on the surface of presynaptic terminals⁸, whereas TSP4 is a secreted protein, produced primarily by non-neuronal cells^14,18. Thus physiologically or pathologically the two proteins would be likely to interact and affect gabapentin binding to α₂δ-1 on the cell surface, primarily of the DRG neurons. However in the present study we have been unable to identify a cell surface interaction between TSP4 and α₂δ-1, although this might occur under conditions not tested here. Interestingly, it has recently been demonstrated that TSP4 is also expressed within DRG neurons and is upregulated in the ganglia following nerve injury⁴¹. Since we have shown previously that gabapentin reduces the trafficking of α₂δ-1 to the cell surface⁷, it is therefore possible that TSP4 will interfere with the ability of gabapentin to bind to α₂δ-1 and reduce calcium channel trafficking, if they are both present in the same cells.

Materials and Methods

Molecular biology

The pcDNA3 TSP4_HA construct was generated by PCR from the human TSP4 cDNA. pcDNA3 TSP4-EGF_HA was then generated (containing only the EGF-like repeats after the signal sequence; residues 286–462) with an HA tag on the C terminus, Fig. 1A. LRP1 minigenes 1–4 were also used⁴² and for immunoprecipitation a triple FLAG tag was inserted in place of the original HA tag between residues 24 and 25 of their coding sequences. The α₂δ constructs used here have been described previously^9,10.

Cell culture and Transfection

The tsA-201 cells were cultured as described previously^10,22. Cells were transfected using FuGENE 6 reagent (Roche) for all biochemical experiments, or PolyJet (SignaGen) for imaging experiments in tsA-201 cells, according to the manufacturers’ instructions.

Membrane and detergent-resistant membrane (DRM) preparation

Cell membrane and DRM fractions were prepared essentially as described previously²². Cultured tsA-201 cells were washed twice in ice-cold PBS and then left in ice-cold PBS containing Complete protease inhibitor cocktail (Roche, concentration according to manufacturer’s instructions) for 5 min. Cells were gently washed off the bottom of the flask and pelleted at 2000 × g for 10 min. Pellets were kept on ice if used immediately, or stored at −20 °C until required. For brain membranes, adult male Sprague Dawley rats (200–250 g) were euthanised by CO₂ inhalation and then decapitated. The whole brain minus cerebellum was homogenised on ice in a buffer containing the following (in mM): 20 HEPES, pH 7.4, 2 EDTA, EDTA-free protease inhibitor cocktail (Roche, concentration according to manufacturer’s instructions).

For membrane preparation, pellets obtained from cell harvesting, resuspended in ice-cold 10 mM Hepes plus protease inhibitors (pH 7.4), or brain homogenate, were mechanically lysed by 10–15 passages through a 23 gauge needle. Cell debris and non-lysed cells were pelleted for 15 min at 1,000 × g. The supernatant obtained was transferred to a 30 ml thick-walled ultracentrifuge tube (Beckman-Coulter). Membranes were pelleted in an ultracentrifuge (Beckman-Coulter) for 90 min at 60,000 × g and resuspended as required. Aliquots were used to determine the protein concentration. For detection of secreted TSP4, medium was collected from transfected cells and concentrated 15-fold using 10 kDa cut-off filter (GE Healthcare).

For DRM preparation, the method used was essentially as described previously²². Briefly, tsA-201 cell pellets or brain homogenate, were resuspended in Mes-buffered saline (MBS: 25 mM Mes, pH 6.5, 150 mM NaCl and EDTA-free protease inhibitor cocktail, containing 1% (v/v) Triton X-100 (Thermo Scientific)) and incubated on ice for 1 h. An equal volume of 90% (w/v) sucrose in MBS was then added to obtain 45% final concentration and overlaid with 35% (w/v) and 5% (w/v) sucrose in MBS to form a discontinuous gradient. The samples were centrifuged at 14,000 × g for 18 h at 4 °C (Beckman SW40 rotor). 1 ml fractions were subsequently harvested from the top to the bottom of the tube. When necessary, protein fractions from the gradient were washed free of sucrose by dilution into 25 volumes of PBS and centrifugation (150,000 × g, for 1 h at 4 °C) to pellet the DRM material. The material was either used immediately or stored at −20 °C until required.

³H-Gabapentin binding assay

³H-gabapentin binding was performed essentially as described previously²⁵. [³H]-gabapentin (ARC, St. Louis, MO), was aliquoted and stored at −80 °C. The specific activity was about 110 Ci/mmol and the concentration 1 mCi/ml. Non-radioactive gabapentin (U.S. Pharmacopea or Pfizer) was stored at 4 °C until use and all the solutions and the reagents were kept on ice during the preparation. Binding of [³H]-gabapentin was performed in a final volume of 250 μl at room temperature for 90 min. 50 μg/tube of membranes or 3 μg/tube of DRMs were incubated with various concentrations of [³H]-gabapentin in 10 mM HEPES/KOH, pH 7.4. Each experiment was carried out in triplicate and the mean value used for subsequent analysis. Concentrations of [³H]-gabapentin higher than 20 nM were achieved by adding non-radioactive gabapentin and correcting the specific binding by the dilution factor (as described previously⁴).

Non-specific binding was determined in the presence of 100-fold or more excess of non-radioactive gabapentin (100 μM). A maximum level of non-specific binding of 25% of total was accepted. Total counts per minute were determined (in duplicate) to give an estimate of the activity of the [³H]-gabapentin. Harvesting of the samples was performed by filtration through GF/B filter papers (Whatman, GE Healthcare) pre-soaked in 0.3% polyethyleneimine solution, in order to reduce non-specific binding to the filter paper. The samples were transferred to the filters by washing the tubes three times with Tris-HCl (100 mM, pH 7.4, Sigma 7–9) using a Brandel Harvester (Brandel). The GF/B filter papers were then cut out and transferred to vials containing 5 ml of scintillant (Liquid scintillant cocktail, Beckman Coulter). Samples were equilibrated for 18 h and then the activity was counted in triplicate in a scintillation counter (Beckman Coulter) for 10 min per sample. Data were fit using the Hill- equation (y = B_max*xⁿ/(K_Dⁿ + xⁿ), where B_max is the total number of binding sites; n, termed n_H in the text, is the Hill coefficient and K_D is the binding affinity. Hill coefficients were usually >1 and <1.5, which is very common for ligand binding studies, for reasons discussed previously⁴³.

Primary Antibodies

The following primary antibodies were used: for α₂δ-1 (unless stated) an anti-α₂δ-1 (mouse monoclonal, Sigma) was used. Where stated, a custom-made rabbit polyclonal antipeptide antibody directed against residues EPFPSAVTIKSWVDK (equivalent to residues 1–15 of mature rat α₂δ-1 following cleavage of the signal sequence). Other antibodies used were: anti-HA (rabbit polyclonal, Santa Cruz or rat monoclonal, Roche), anti-flotillin-1 (mouse monoclonal, BD Biosciences), anti-GAPDH (mouse monoclonal, Ambion), anti-TSP4 antibody (mouse monoclonal #276523, R&D systems), rabbit polyclonal or mouse monoclonal anti-FLAG antibodies (Sigma), mouse Golgin-97 (Molecular Probes) and mouse Protein disulphide isomerase (PDI, Abcam).

Co-immunoprecipitation

The tsA-201 cells were transfected with the α₂δ-1 and TSP4_HA (or non-tagged TSP4 in Fig. 6C) cDNA, or with LRP1 m1-m4_Flag cDNA. Receptor-associated protein (RAP) cDNA was included in all transfections in which LRP1 constructs were used, to ensure their correct folding⁴⁴. Freshly harvested cell pellets were re-suspended in Lysis Buffer (Supplementary Table S2) using first a 200 μl Gilson pipette and then a 23 gauge needle. Samples were then left on ice for 30 min and centrifuged at 14,000 × g for 30 min at 4 °C. The supernatant WCL (1 mg protein) was pre-cleared for 2 h at 4 °C with 20 μl of Protein-G agarose beads (Invitrogen) to reduce the non-specific binding to the agarose beads (except in Figs 4,5 and S3, in which Protein-A/G-PLUS agarose (Santa Cruz) were used without a pre-clearing step). The supernatant was recovered by centrifugation at 400 × g for 30 s. and then incubated overnight with 5 μg/ml of rabbit anti-HA antibody or mouse anti-FLAG antibody at 4 °C. It was then incubated with Protein-G (or A/G-PLUS) beads for 2 h at 4 °C. A series of washes were carried out in order to reduce the non-specific binding to the beads (Supplementary Table S2). Protein bound to the beads was eluted with loading sample buffer at a final concentration of 1×, containing 100 mM dithiothreitol, followed by incubation for 15 min in a water bath at 55 °C and subsequent centrifugation of the protein-G-beads. Samples of the supernatant were analysed by western blot.

Western blotting

This was performed essentially as described previously²². The following secondary antibodies were used for western blot: goat anti-rabbit-coupled to horseradish peroxidase (HRP) and goat anti-mouse coupled to HRP (Biorad).

Immunocytochemistry in tsA-201 cells

The method used is essentially as previously described²². Cells were transfected in low-serum medium. In co-transfection conditions, α₂δ-1 and the stated TSP construct were used in a ratio of 1:1. For tsA-201 cells transfected separately, α₂δ-1 was transfected directly onto poly-lysine coated coverslips and TSP4_HA-expressing cells from a different flask were layered onto α₂δ-1-expressing cells 16–24 h after transfection. Cells were then incubated for a further 16–24 h before being fixed.

Cells were fixed with 4% paraformaldehyde in TBS for 5 min at room temperature and then washed twice with TBS. Either no permeabilisation step was used, or cells were permeabilised for 15 min with 0.02% Triton X-100. The primary antibodies used were: mouse anti α₂δ-1 (1:100) with rabbit anti-HA (1:500) for experiments where α₂δ-1 and TSP4_HA were co-expressed; rabbit anti α₂δ-1 (1:100) with mouse anti-Golgin-97 (1:500) or mouse anti-PDI (1:100) antibodies. Primary antibodies were incubated overnight at 4 °C, followed by the secondary antibodies, fluorescein isothiocyanate (FITC)-conjugated anti-mouse (1:500, Sigma) or anti-rabbit Alexa Fluor 594 (1:500, Invitrogen), for 1 h at room temperature. DAPI (4′,6-diamidine-2′-phenylindole dihydrochloride) was also used to visualise the nuclei. Cells were mounted in Vectashield (Vector laboratories, Burlingame, CA) to reduce photobleaching. Cells were examined on a confocal laser scanning microscope (Zeiss LSM780, except in Fig. 6A,B, taken with an LSM510), using a ×63 (1.4 NA) oil-immersion objective. Confocal optical sections were 1 μm. Photomultiplier settings were kept constant in each experiment and all images were scanned sequentially. Image processing was performed using ImageJ. Data illustrated are representative of images taken of at least 10 cells for non-permeabilised conditions and >100 cells for permeabilised conditions, from 3 independent experiments, unless otherwise stated.

Additional Information

How to cite this article: Lana, B. et al. 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. Sci. Rep. 6, 24531; doi: 10.1038/srep24531 (2016).

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Acknowledgements

This work was supported by a Wellcome Trust senior Investigator award (098360/Z/12/Z) and Medical Research Council (UK) (grants G0801756 and G0901758) to ACD. BL was a PhD student supported in part by a small grant from Pfizer. SH was a UCL Pharmacology MSci student and also supported by a Lister Institute vacation award. We thank Dr Guojon Bu (Mayo Clinic, USA) for the LRP minigene constructs and RAP cDNA and Prof Olga Stenina for TSP4 cDNAs. We thank Lucien West for participating in initial construction of truncated TSPs used in this study.

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Lana Beatrice and Page Karen M. contributed equally to this work.

Authors and Affiliations

Department of Neuroscience, Physiology and Pharmacology, University College London, Gower St., London, WC1E 6BT, United Kingdom
Beatrice Lana, Karen M. Page, Ivan Kadurin, Shuxian Ho, Manuela Nieto-Rostro & Annette C. Dolphin

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

B.L. performed ligand binding studies. B.L. and I.K. performed co-immunoprecipitation and western blotting studies. K.M.P. and M.N.-R. performed immunocytochemistry experiments, with input from S.H. A.C.D. wrote the paper, aided by all the authors.

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Lana, B., Page, K., Kadurin, I. et al. 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. Sci Rep 6, 24531 (2016). https://doi.org/10.1038/srep24531

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Received: 11 January 2016
Accepted: 30 March 2016
Published: 14 April 2016
DOI: https://doi.org/10.1038/srep24531

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