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The inhibition of functional expression of calcium channels by prion protein demonstrates competition with α2δ for GPI-anchoring pathways.

Alvarez-Laviada A, Kadurin I, Senatore A, Chiesa R, Dolphin AC - Biochem. J. (2014)

Bottom Line: In the present study we examined whether there was an effect of PrP on calcium currents.We have shown that when PrP is co-expressed with calcium channels formed from CaV2.1/β and α2δ-1 or α2δ-2, there is a consistent decrease in calcium current density.We now find that PrP does not inhibit CaV2.1/β currents formed with α2δ-1ΔC, rather than α2δ-1.

View Article: PubMed Central - PubMed

Affiliation: *Department of Neuroscience, Physiology and Pharmacology, University College London, London WC1E 6BT, U.K.

ABSTRACT
It has been shown recently that PrP (prion protein) and the calcium channel auxiliary α2δ subunits interact in neurons and expression systems [Senatore, Colleoni, Verderio, Restelli, Morini, Condliffe, Bertani, Mantovani, Canovi, Micotti, Forloni, Dolphin, Matteoli, Gobbi and Chiesa (2012) Neuron 74, 300-313]. In the present study we examined whether there was an effect of PrP on calcium currents. We have shown that when PrP is co-expressed with calcium channels formed from CaV2.1/β and α2δ-1 or α2δ-2, there is a consistent decrease in calcium current density. This reduction was absent when a PrP construct was used lacking its GPI (glycosylphosphatidylinositol) anchor. We have reported previously that α2δ subunits are able to form GPI-anchored proteins [Davies, Kadurin, Alvarez-Laviada, Douglas, Nieto-Rostro, Bauer, Pratt and Dolphin (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 1654-1659] and show further evidence in the present paper. We have characterized recently a C-terminally truncated α2δ-1 construct, α2δ-1ΔC, and found that, despite loss of its membrane anchor, it still shows a partial ability to increase calcium currents [Kadurin, Alvarez-Laviada, Ng, Walker-Gray, D'Arco, Fadel, Pratt and Dolphin (2012) J. Biol. Chem. 1287, 33554-33566]. We now find that PrP does not inhibit CaV2.1/β currents formed with α2δ-1ΔC, rather than α2δ-1. It is possible that PrP and α2δ-1 compete for GPI-anchor intermediates or trafficking pathways, or that interaction between PrP and α2δ-1 requires association in cholesterol-rich membrane microdomains. Our additional finding that CaV2.1/β1b/α2δ-1 currents were inhibited by GPI-GFP, but not cytosolic GFP, indicates that competition for limited GPI-anchor intermediates or trafficking pathways may be involved in PrP suppression of α2δ subunit function.

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Effect of GPI–GFP on CaV2.1/β1b/α2δ-1 calcium channel currents(A) I–V relationships for IBa recorded from tsA-201 cells expressing CaV2.1/β1b/α2δ-1 with GFP (■; n=9), GPI–GFP (▲; n=9), or GFP and ΔGPI–PrP (△; n=6). The broken line indicates the level of IBa observed for CaV2.1/β1b/α2δ-1 plus WT PrP from Figure 3(B). ***P<0.001 between the peak IBa when GPI–GFP was co-expressed compared with when GFP was co-expressed (Student's t test). (B) Western blot of α2δ-1 (upper panels) co-expressed (1:1) with GFP–GPI (lane 1), GFP (lane 2) or empty vector (lane 3; con). GFP expression is shown in the lower panel. All three lanes are from the same blots from which irrelevant lanes have been excised and the molecular mass is given on the right-hand side in kDa.
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Figure 5: Effect of GPI–GFP on CaV2.1/β1b/α2δ-1 calcium channel currents(A) I–V relationships for IBa recorded from tsA-201 cells expressing CaV2.1/β1b/α2δ-1 with GFP (■; n=9), GPI–GFP (▲; n=9), or GFP and ΔGPI–PrP (△; n=6). The broken line indicates the level of IBa observed for CaV2.1/β1b/α2δ-1 plus WT PrP from Figure 3(B). ***P<0.001 between the peak IBa when GPI–GFP was co-expressed compared with when GFP was co-expressed (Student's t test). (B) Western blot of α2δ-1 (upper panels) co-expressed (1:1) with GFP–GPI (lane 1), GFP (lane 2) or empty vector (lane 3; con). GFP expression is shown in the lower panel. All three lanes are from the same blots from which irrelevant lanes have been excised and the molecular mass is given on the right-hand side in kDa.

Mentions: This result suggests either that association of the α2δ and PrP proteins in the plasma membrane, and potentially in the same cholesterol-rich membrane microdomains, facilitated by their GPI anchoring, is essential for the inhibition of calcium currents observed or that there is competition between α2δ-1 and PrP for the GPI-anchor intermediates, resulting in less maturation of α2δ subunit in the presence of PrP. We therefore examined whether the effect of PrP might be related to the GPI anchor and tested the effect of an unrelated protein engineered to contain a GPI anchor (GFP–GPI). We found that co-expression of GFP–GPI markedly reduced calcium channel currents formed by CaV2.1/β1b/α2δ-1 compared with the co-expression of cytosolic GFP, and in this experiment ∆GPI–PrP again had no effect (Figure 5A). This result is in agreement with the hypothesis that overexpression of a GPI-anchored protein can produce this inhibition. We then compared the level of α2δ-1 expression in the presence of co-expressed GFP–GPI and cytosolic GFP (Figure 5B). In all cases substantial α2δ-1 was co-expressed.


The inhibition of functional expression of calcium channels by prion protein demonstrates competition with α2δ for GPI-anchoring pathways.

Alvarez-Laviada A, Kadurin I, Senatore A, Chiesa R, Dolphin AC - Biochem. J. (2014)

Effect of GPI–GFP on CaV2.1/β1b/α2δ-1 calcium channel currents(A) I–V relationships for IBa recorded from tsA-201 cells expressing CaV2.1/β1b/α2δ-1 with GFP (■; n=9), GPI–GFP (▲; n=9), or GFP and ΔGPI–PrP (△; n=6). The broken line indicates the level of IBa observed for CaV2.1/β1b/α2δ-1 plus WT PrP from Figure 3(B). ***P<0.001 between the peak IBa when GPI–GFP was co-expressed compared with when GFP was co-expressed (Student's t test). (B) Western blot of α2δ-1 (upper panels) co-expressed (1:1) with GFP–GPI (lane 1), GFP (lane 2) or empty vector (lane 3; con). GFP expression is shown in the lower panel. All three lanes are from the same blots from which irrelevant lanes have been excised and the molecular mass is given on the right-hand side in kDa.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3924758&req=5

Figure 5: Effect of GPI–GFP on CaV2.1/β1b/α2δ-1 calcium channel currents(A) I–V relationships for IBa recorded from tsA-201 cells expressing CaV2.1/β1b/α2δ-1 with GFP (■; n=9), GPI–GFP (▲; n=9), or GFP and ΔGPI–PrP (△; n=6). The broken line indicates the level of IBa observed for CaV2.1/β1b/α2δ-1 plus WT PrP from Figure 3(B). ***P<0.001 between the peak IBa when GPI–GFP was co-expressed compared with when GFP was co-expressed (Student's t test). (B) Western blot of α2δ-1 (upper panels) co-expressed (1:1) with GFP–GPI (lane 1), GFP (lane 2) or empty vector (lane 3; con). GFP expression is shown in the lower panel. All three lanes are from the same blots from which irrelevant lanes have been excised and the molecular mass is given on the right-hand side in kDa.
Mentions: This result suggests either that association of the α2δ and PrP proteins in the plasma membrane, and potentially in the same cholesterol-rich membrane microdomains, facilitated by their GPI anchoring, is essential for the inhibition of calcium currents observed or that there is competition between α2δ-1 and PrP for the GPI-anchor intermediates, resulting in less maturation of α2δ subunit in the presence of PrP. We therefore examined whether the effect of PrP might be related to the GPI anchor and tested the effect of an unrelated protein engineered to contain a GPI anchor (GFP–GPI). We found that co-expression of GFP–GPI markedly reduced calcium channel currents formed by CaV2.1/β1b/α2δ-1 compared with the co-expression of cytosolic GFP, and in this experiment ∆GPI–PrP again had no effect (Figure 5A). This result is in agreement with the hypothesis that overexpression of a GPI-anchored protein can produce this inhibition. We then compared the level of α2δ-1 expression in the presence of co-expressed GFP–GPI and cytosolic GFP (Figure 5B). In all cases substantial α2δ-1 was co-expressed.

Bottom Line: In the present study we examined whether there was an effect of PrP on calcium currents.We have shown that when PrP is co-expressed with calcium channels formed from CaV2.1/β and α2δ-1 or α2δ-2, there is a consistent decrease in calcium current density.We now find that PrP does not inhibit CaV2.1/β currents formed with α2δ-1ΔC, rather than α2δ-1.

View Article: PubMed Central - PubMed

Affiliation: *Department of Neuroscience, Physiology and Pharmacology, University College London, London WC1E 6BT, U.K.

ABSTRACT
It has been shown recently that PrP (prion protein) and the calcium channel auxiliary α2δ subunits interact in neurons and expression systems [Senatore, Colleoni, Verderio, Restelli, Morini, Condliffe, Bertani, Mantovani, Canovi, Micotti, Forloni, Dolphin, Matteoli, Gobbi and Chiesa (2012) Neuron 74, 300-313]. In the present study we examined whether there was an effect of PrP on calcium currents. We have shown that when PrP is co-expressed with calcium channels formed from CaV2.1/β and α2δ-1 or α2δ-2, there is a consistent decrease in calcium current density. This reduction was absent when a PrP construct was used lacking its GPI (glycosylphosphatidylinositol) anchor. We have reported previously that α2δ subunits are able to form GPI-anchored proteins [Davies, Kadurin, Alvarez-Laviada, Douglas, Nieto-Rostro, Bauer, Pratt and Dolphin (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 1654-1659] and show further evidence in the present paper. We have characterized recently a C-terminally truncated α2δ-1 construct, α2δ-1ΔC, and found that, despite loss of its membrane anchor, it still shows a partial ability to increase calcium currents [Kadurin, Alvarez-Laviada, Ng, Walker-Gray, D'Arco, Fadel, Pratt and Dolphin (2012) J. Biol. Chem. 1287, 33554-33566]. We now find that PrP does not inhibit CaV2.1/β currents formed with α2δ-1ΔC, rather than α2δ-1. It is possible that PrP and α2δ-1 compete for GPI-anchor intermediates or trafficking pathways, or that interaction between PrP and α2δ-1 requires association in cholesterol-rich membrane microdomains. Our additional finding that CaV2.1/β1b/α2δ-1 currents were inhibited by GPI-GFP, but not cytosolic GFP, indicates that competition for limited GPI-anchor intermediates or trafficking pathways may be involved in PrP suppression of α2δ subunit function.

Show MeSH
Related in: MedlinePlus