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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 PrP on CaV2.1/β4/α2δ-2 calcium channel currents(A) Current–voltage (I–V) relationships for IBa recorded from tsA-201 cells expressing CaV2.1/β4/α2δ-2 (■; n=25), CaV2.1/β4 alone (○; n=7) and CaV2.1/β4/α2δ-2/WT PrP (△; n=23). The ratio of cDNAs used for transfection for CaV2.1/β4/α2δ-2/WT-PrP was 3:2:2:1, with empty vector used where α2δ or PrP was absent. (B) Examples of families of IBa current traces resulting from step potentials from −100 mV to between −30 and +15 mV in 5 mV increments for CaV2.1/β4/α2δ-2 (top panel), CaV2.1/β4 alone (middle panel) and CaV2.1/β4/α2δ-2/WT PrP (bottom panel). (C) Individual peak IBa currents at +10 mV, expressed as the mean±S.E.M. percentage of the control condition with WT α2δ-2 and CaV2.1/β4/α2δ-2 (■; n=25), CaV2.1/β4 alone (○; n=7) and CaV2.1/β4/α2δ-2/WT PrP (△; n=23). **P<0.01 and ***P<0.001. (D) Voltage-dependence of activation (V50 activation) determined by fitting a modified Boltzmann function to the individual I–V relationships shown in (A) for CaV2.1/β4/α2δ-2 (black bar), CaV2.1/β4 alone (white bar) and CaV2.1/β4/α2δ-2/WT PrP (grey bar). *P<0.05 and **P<0.01. (E) Steady-state inactivation curves for IBa recorded from cells expressing CaV2.1/β4/α2δ-2 (■; n=12), CaV2.1/β4 alone (○; n=4) and CaV2.1/β4/α2δ-2/WT PrP (△; n=8). (F) Voltage-dependence of steady-state inactivation (V50 inactivation) determined by fitting a Boltzmann function to the individual steady-state inactivation relationships for the data shown in (E); CaV2.1/β4/α2δ-2 (black bar), CaV2.1/β4 alone (white bar) and CaV2.1/β4/α2δ-2/WT PrP (grey bar). **P<0.01; NS, not significant. All statistical differences were determined by one-way ANOVA and Dunnett's multiple comparison test.
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Figure 2: Effect of PrP on CaV2.1/β4/α2δ-2 calcium channel currents(A) Current–voltage (I–V) relationships for IBa recorded from tsA-201 cells expressing CaV2.1/β4/α2δ-2 (■; n=25), CaV2.1/β4 alone (○; n=7) and CaV2.1/β4/α2δ-2/WT PrP (△; n=23). The ratio of cDNAs used for transfection for CaV2.1/β4/α2δ-2/WT-PrP was 3:2:2:1, with empty vector used where α2δ or PrP was absent. (B) Examples of families of IBa current traces resulting from step potentials from −100 mV to between −30 and +15 mV in 5 mV increments for CaV2.1/β4/α2δ-2 (top panel), CaV2.1/β4 alone (middle panel) and CaV2.1/β4/α2δ-2/WT PrP (bottom panel). (C) Individual peak IBa currents at +10 mV, expressed as the mean±S.E.M. percentage of the control condition with WT α2δ-2 and CaV2.1/β4/α2δ-2 (■; n=25), CaV2.1/β4 alone (○; n=7) and CaV2.1/β4/α2δ-2/WT PrP (△; n=23). **P<0.01 and ***P<0.001. (D) Voltage-dependence of activation (V50 activation) determined by fitting a modified Boltzmann function to the individual I–V relationships shown in (A) for CaV2.1/β4/α2δ-2 (black bar), CaV2.1/β4 alone (white bar) and CaV2.1/β4/α2δ-2/WT PrP (grey bar). *P<0.05 and **P<0.01. (E) Steady-state inactivation curves for IBa recorded from cells expressing CaV2.1/β4/α2δ-2 (■; n=12), CaV2.1/β4 alone (○; n=4) and CaV2.1/β4/α2δ-2/WT PrP (△; n=8). (F) Voltage-dependence of steady-state inactivation (V50 inactivation) determined by fitting a Boltzmann function to the individual steady-state inactivation relationships for the data shown in (E); CaV2.1/β4/α2δ-2 (black bar), CaV2.1/β4 alone (white bar) and CaV2.1/β4/α2δ-2/WT PrP (grey bar). **P<0.01; NS, not significant. All statistical differences were determined by one-way ANOVA and Dunnett's multiple comparison test.

Mentions: We then examined whether PrP would influence the ability of α2δ to increase calcium channel currents. We expressed CaV2.1/β4 and α2δ-2 to mimic the calcium channel combination present in cerebellar Purkinje cells, either with or without PrP. We found that PrP co-expression produced a moderate, but consistent, reduction in peak calcium channel currents at +10 mV of approximately 34%, although the currents in the presence of PrP and α2δ remained significantly larger than those in the absence of α2δ (Figures 2A–2C). The voltage-dependence of activation of the CaV2.1/β4/α2δ-2 currents in the presence of PrP was also significantly depolarized, compared with in its absence, although not to the same extent as in the absence of α2δ (Figure 2D). Similarly, the voltage-dependence of steady-state inactivation of CaV2.2/β4/α2δ-2 was depolarized significantly in the additional presence of PrP (Figures 2E and 2F). All these effects are indicative of a reduced enhancement by α2δ-2 of CaV2.1/β4 calcium channel currents when PrP 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 PrP on CaV2.1/β4/α2δ-2 calcium channel currents(A) Current–voltage (I–V) relationships for IBa recorded from tsA-201 cells expressing CaV2.1/β4/α2δ-2 (■; n=25), CaV2.1/β4 alone (○; n=7) and CaV2.1/β4/α2δ-2/WT PrP (△; n=23). The ratio of cDNAs used for transfection for CaV2.1/β4/α2δ-2/WT-PrP was 3:2:2:1, with empty vector used where α2δ or PrP was absent. (B) Examples of families of IBa current traces resulting from step potentials from −100 mV to between −30 and +15 mV in 5 mV increments for CaV2.1/β4/α2δ-2 (top panel), CaV2.1/β4 alone (middle panel) and CaV2.1/β4/α2δ-2/WT PrP (bottom panel). (C) Individual peak IBa currents at +10 mV, expressed as the mean±S.E.M. percentage of the control condition with WT α2δ-2 and CaV2.1/β4/α2δ-2 (■; n=25), CaV2.1/β4 alone (○; n=7) and CaV2.1/β4/α2δ-2/WT PrP (△; n=23). **P<0.01 and ***P<0.001. (D) Voltage-dependence of activation (V50 activation) determined by fitting a modified Boltzmann function to the individual I–V relationships shown in (A) for CaV2.1/β4/α2δ-2 (black bar), CaV2.1/β4 alone (white bar) and CaV2.1/β4/α2δ-2/WT PrP (grey bar). *P<0.05 and **P<0.01. (E) Steady-state inactivation curves for IBa recorded from cells expressing CaV2.1/β4/α2δ-2 (■; n=12), CaV2.1/β4 alone (○; n=4) and CaV2.1/β4/α2δ-2/WT PrP (△; n=8). (F) Voltage-dependence of steady-state inactivation (V50 inactivation) determined by fitting a Boltzmann function to the individual steady-state inactivation relationships for the data shown in (E); CaV2.1/β4/α2δ-2 (black bar), CaV2.1/β4 alone (white bar) and CaV2.1/β4/α2δ-2/WT PrP (grey bar). **P<0.01; NS, not significant. All statistical differences were determined by one-way ANOVA and Dunnett's multiple comparison test.
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Figure 2: Effect of PrP on CaV2.1/β4/α2δ-2 calcium channel currents(A) Current–voltage (I–V) relationships for IBa recorded from tsA-201 cells expressing CaV2.1/β4/α2δ-2 (■; n=25), CaV2.1/β4 alone (○; n=7) and CaV2.1/β4/α2δ-2/WT PrP (△; n=23). The ratio of cDNAs used for transfection for CaV2.1/β4/α2δ-2/WT-PrP was 3:2:2:1, with empty vector used where α2δ or PrP was absent. (B) Examples of families of IBa current traces resulting from step potentials from −100 mV to between −30 and +15 mV in 5 mV increments for CaV2.1/β4/α2δ-2 (top panel), CaV2.1/β4 alone (middle panel) and CaV2.1/β4/α2δ-2/WT PrP (bottom panel). (C) Individual peak IBa currents at +10 mV, expressed as the mean±S.E.M. percentage of the control condition with WT α2δ-2 and CaV2.1/β4/α2δ-2 (■; n=25), CaV2.1/β4 alone (○; n=7) and CaV2.1/β4/α2δ-2/WT PrP (△; n=23). **P<0.01 and ***P<0.001. (D) Voltage-dependence of activation (V50 activation) determined by fitting a modified Boltzmann function to the individual I–V relationships shown in (A) for CaV2.1/β4/α2δ-2 (black bar), CaV2.1/β4 alone (white bar) and CaV2.1/β4/α2δ-2/WT PrP (grey bar). *P<0.05 and **P<0.01. (E) Steady-state inactivation curves for IBa recorded from cells expressing CaV2.1/β4/α2δ-2 (■; n=12), CaV2.1/β4 alone (○; n=4) and CaV2.1/β4/α2δ-2/WT PrP (△; n=8). (F) Voltage-dependence of steady-state inactivation (V50 inactivation) determined by fitting a Boltzmann function to the individual steady-state inactivation relationships for the data shown in (E); CaV2.1/β4/α2δ-2 (black bar), CaV2.1/β4 alone (white bar) and CaV2.1/β4/α2δ-2/WT PrP (grey bar). **P<0.01; NS, not significant. All statistical differences were determined by one-way ANOVA and Dunnett's multiple comparison test.
Mentions: We then examined whether PrP would influence the ability of α2δ to increase calcium channel currents. We expressed CaV2.1/β4 and α2δ-2 to mimic the calcium channel combination present in cerebellar Purkinje cells, either with or without PrP. We found that PrP co-expression produced a moderate, but consistent, reduction in peak calcium channel currents at +10 mV of approximately 34%, although the currents in the presence of PrP and α2δ remained significantly larger than those in the absence of α2δ (Figures 2A–2C). The voltage-dependence of activation of the CaV2.1/β4/α2δ-2 currents in the presence of PrP was also significantly depolarized, compared with in its absence, although not to the same extent as in the absence of α2δ (Figure 2D). Similarly, the voltage-dependence of steady-state inactivation of CaV2.2/β4/α2δ-2 was depolarized significantly in the additional presence of PrP (Figures 2E and 2F). All these effects are indicative of a reduced enhancement by α2δ-2 of CaV2.1/β4 calcium channel currents when PrP 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