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Biochem J
2014 Mar 01;4582:365-74. doi: 10.1042/BJ20131405.
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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.
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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.
Figure 1. PI-PLC treatment and phase separation of α2δ-1, α2δ-2 and PrP in mouse cerebella(A) DRM fractions from WT (left-hand panel), PrP Tg(WT) (middle panel) and PrP KO (right-hand panel) mouse cerebella were prepared as described in the Materials and methods section. Aliquots were resolved on 3–8% Tris/acetate (to resolve α2δs) or 4–12% Bis-Tris gels (to resolve PrP in the same samples), and analysed by Western blotting (WB) with relevant antibodies as indicated. The full profile is not shown, but only the fractions of the sucrose gradient corresponding to DRMs identified by the presence of flotillin-1 (fractions 4–7 harvested from the top). The anti-α2δ-1 and anti-α2δ-2 antibodies recognize the α2-1 and α2-2 moieties. (B) Aliquots of concentrated DRM fractions from WT (left-hand panel), PrP Tg(WT) (middle panel) and PrP KO (right-hand panel) cerebella were treated with PNGase F and analysed by Western blotting with the indicated antibodies. (C) DRM fractions analysed in (A) were subjected to PI-PLC treatment and Triton X-114 phase separation (see the Materials and methods section), followed by PNGase F deglycosylation. The proteins remaining in the aqueous (aq) and detergent (det) phase were then resolved on 4–12% Bis-Tris gels and analysed with the indicated antibodies. Lanes 1 and 2 in each panel are from detergent phase fractions, whereas lanes 3 and 4 are from the respective aqueous phase, treated or not with PI-PLC as indicated. (D) The PrP Tg(WT) fractions from (C, middle panel) were also blotted for PrP using the 3F4 antibody. Molecular mass is shown on the right-hand side of the gels in kDa.
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.
Figure 3. Effect of PrP constructs on CaV2.1/β1b/α2δ-1 calcium channel currents(A) Examples of families of IBa current traces resulting from step potentials from −90 mV to between −30 and +10 mV in 5 mV steps for CaV2.1/β1b/α2δ-1 alone (top panel) with WT PrP (middle panel) or with ΔGPI–PrP (bottom panel). The ratio of cDNAs used for the transfection of CaV2.1/β1b/α2δ-1/PrP was 3:2:2:1, with empty vector used where α2δ or PrP was absent. (B) I–V relationships for IBa recorded from tsA-201 cells expressing CaV2.1/β1b/α2δ-1 alone (■; n=28) with WT PrP (▲; n=19) or with ΔGPI–PrP (△; n=13). (C) Individual mean±S.E.M. peak IBa currents at +5 mV for CaV2.1/β1b/α2δ-1 alone (■; n=28) with WT PrP (▲; n=19) or ΔGPI–PrP (△; n=13). **P<0.01. (D) V50 activation for CaV2.1/β1b/α2δ-1 alone (black bar) with WT PrP (white bar) or with ΔGPI–PrP (grey bar). All statistical differences were determined by one-way ANOVA and Dunnett's multiple comparison test. (E and F) CaV2.2/β1b/α2δ-1 was expressed in Xenopus oocytes either alone or together with PrP at the ratios shown. (E) Representative current traces elicited by steps to test potentials between −25 and +10 mV in 5 mV steps from a holding potential of −100 mV for CaV2.2/β1b/α2δ-1/PrP (1:0.25; upper panel) or CaV2.2/β1b/α2δ-1/PrP (1:1; lower panel). The residual voltage clamp transients have been truncated. (F) Peak currents measured at +10 mV for three α2δ-1/PrP ratios plotted as the mean±S.E.M. percentage of the mean control IBa recorded in the absence of PrP in the same experiment. α2δ-1/PrP 1:1 (black bar; n=6), α2δ-1/PrP ratio 1:0.5 (grey bar; n=18), α2δ-1/PrP ratio 1:0.25 (white bar; n=20) and mean normalized control for α2δ-1 in the absence of PrP (hatched bar; n=21). *P=0.016 between the individual conditions and their respective control condition performed in the same experiment as determined by Student's t test.
Figure 4. Comparison of the effect of PrP constructs on calcium channel currents containing α2δ-1 or anchorless α2δ-1(A) I--V relationships for IBa recorded from tsA-201 cells expressing CaV2.1/β1b/α2δ-1 alone (■; n=16) with WT PrP (▲; n=10) or ΔGPI–PrP (◆; n=14) or cells expressing CaV2.1/β1b/α2δ-1ΔC alone (□; n=11) with WT PrP (∆; n=13) or ΔGPI–PrP (◇; n=15). The ratio of cDNAs was the same as in Figure 3(A). (B) Examples of families of IBa current traces resulting from step potentials from −90 mV to between −30 and +10 mV in 5 mV steps for CaV2.1/β1b/α2δ-1 alone (top left-hand panel) or α2δ-1ΔC alone (top right-hand panel) with WT PrP (middle panel) or ΔGPI–PrP (bottom panels). (C) Peak IBa currents (means±S.E.M.) for the data shown in (A) for CaV2.1/β1b with α2δ-1 (solid bars) or with α2δ-1ΔC (hatched bars) either alone (black bars) or with WT PrP (light grey bars), or ΔGPI-PrP (dark grey bars). *P<0.05 as determined by one-way ANOVA and Bonferroni's post-hoc test. (D) Western blot of α2δ-1 (upper panels; 4–12% Bis-Tris gel) co-expressed (1:1) with WT PrP (left-hand lane), ∆GPI–PrP (middle lane) or empty vector (right-hand lane; con). PrP expression is shown in the lower panel (4–12% Bis-Tris gel). The lower expression of ∆GPI–PrP in the cell lysate is because much of it is secreted [53]. (E) Western blot of α2δ-1∆C in medium (upper panels; 3–8% Tris/acetate gel) and cell lysate (lower panels; 4–12% Bis-Tris gel) co-expressed (1:1) with PrP (left-hand panels), ∆GPI–PrP (middle panel) or empty vector (right-hand panels; con). In both (D) and (E), 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.
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.
Altier,
The Cavβ subunit prevents RFP2-mediated ubiquitination and proteasomal degradation of L-type channels.
2011, Pubmed
Altier,
The Cavβ subunit prevents RFP2-mediated ubiquitination and proteasomal degradation of L-type channels.
2011,
Pubmed Arikkath,
Auxiliary subunits: essential components of the voltage-gated calcium channel complex.
2003,
Pubmed Barclay,
Ducky mouse phenotype of epilepsy and ataxia is associated with mutations in the Cacna2d2 gene and decreased calcium channel current in cerebellar Purkinje cells.
2001,
Pubmed
,
Xenbase Berrow,
Properties of cloned rat alpha1A calcium channels transiently expressed in the COS-7 cell line.
1997,
Pubmed Biasini,
Prion protein at the crossroads of physiology and disease.
2012,
Pubmed Bordier,
Phase separation of integral membrane proteins in Triton X-114 solution.
1981,
Pubmed Brandner,
Normal host prion protein necessary for scrapie-induced neurotoxicity.
1996,
Pubmed Bremer,
Axonal prion protein is required for peripheral myelin maintenance.
2010,
Pubmed Brodbeck,
The ducky mutation in Cacna2d2 results in altered Purkinje cell morphology and is associated with the expression of a truncated alpha 2 delta-2 protein with abnormal function.
2002,
Pubmed
,
Xenbase Büeler,
Mice devoid of PrP are resistant to scrapie.
1993,
Pubmed Büeler,
Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein.
1992,
Pubmed Campana,
Characterization of the properties and trafficking of an anchorless form of the prion protein.
2007,
Pubmed Campbell,
Inhibition of the interaction of G protein G(o) with calcium channels by the calcium channel beta-subunit in rat neurones.
1995,
Pubmed Cantí,
Identification of residues in the N terminus of alpha1B critical for inhibition of the voltage-dependent calcium channel by Gbeta gamma.
1999,
Pubmed
,
Xenbase Cantí,
Evidence for two concentration-dependent processes for beta-subunit effects on alpha1B calcium channels.
2001,
Pubmed
,
Xenbase Cantí,
The metal-ion-dependent adhesion site in the Von Willebrand factor-A domain of alpha2delta subunits is key to trafficking voltage-gated Ca2+ channels.
2005,
Pubmed Catterall,
Structure and regulation of voltage-gated Ca2+ channels.
2000,
Pubmed Chiesa,
Neurological illness in transgenic mice expressing a prion protein with an insertional mutation.
1998,
Pubmed Collinge,
Molecular neurology of prion disease.
2005,
Pubmed Collinge,
Prion protein is necessary for normal synaptic function.
1994,
Pubmed Cormack,
FACS-optimized mutants of the green fluorescent protein (GFP).
1996,
Pubmed Davies,
The calcium channel alpha2delta-2 subunit partitions with CaV2.1 into lipid rafts in cerebellum: implications for localization and function.
2006,
Pubmed Davies,
Functional biology of the alpha(2)delta subunits of voltage-gated calcium channels.
2007,
Pubmed Davies,
The alpha2delta subunits of voltage-gated calcium channels form GPI-anchored proteins, a posttranslational modification essential for function.
2010,
Pubmed Dolphin,
Beta subunits of voltage-gated calcium channels.
2003,
Pubmed Elfrink,
Interaction of the cellular prion protein with raft-like lipid membranes.
2007,
Pubmed Fujita,
Structural remodeling of GPI anchors during biosynthesis and after attachment to proteins.
2010,
Pubmed Gurnett,
Dual function of the voltage-dependent Ca2+ channel alpha 2 delta subunit in current stimulation and subunit interaction.
1996,
Pubmed
,
Xenbase Hooper,
Glypican-1 facilitates prion conversion in lipid rafts.
2011,
Pubmed Hoppa,
α2δ expression sets presynaptic calcium channel abundance and release probability.
2012,
Pubmed Jay,
Structural characterization of the dihydropyridine-sensitive calcium channel alpha 2-subunit and the associated delta peptides.
1991,
Pubmed Kadurin,
Calcium currents are enhanced by α2δ-1 lacking its membrane anchor.
2012,
Pubmed Lehmann,
A mutant prion protein displays an aberrant membrane association when expressed in cultured cells.
1995,
Pubmed Leroy,
Interaction via a key tryptophan in the I-II linker of N-type calcium channels is required for beta1 but not for palmitoylated beta2, implicating an additional binding site in the regulation of channel voltage-dependent properties.
2005,
Pubmed Mallucci,
Depleting neuronal PrP in prion infection prevents disease and reverses spongiosis.
2003,
Pubmed Mouillet-Richard,
Signal transduction through prion protein.
2000,
Pubmed Nichols,
Rapid cycling of lipid raft markers between the cell surface and Golgi complex.
2001,
Pubmed Page,
Dominant-negative calcium channel suppression by truncated constructs involves a kinase implicated in the unfolded protein response.
2004,
Pubmed
,
Xenbase Pragnell,
Calcium channel beta-subunit binds to a conserved motif in the I-II cytoplasmic linker of the alpha 1-subunit.
1994,
Pubmed Prusiner,
Prions.
1998,
Pubmed Rutishauser,
The comprehensive native interactome of a fully functional tagged prion protein.
2009,
Pubmed Senatore,
Mutant PrP suppresses glutamatergic neurotransmission in cerebellar granule neurons by impairing membrane delivery of VGCC α(2)δ-1 Subunit.
2012,
Pubmed Stahl,
Scrapie prion protein contains a phosphatidylinositol glycolipid.
1987,
Pubmed Steele,
The prion protein knockout mouse: a phenotype under challenge.
2007,
Pubmed Stewart,
Mutational analysis of topological determinants in prion protein (PrP) and measurement of transmembrane and cytosolic PrP during prion infection.
2003,
Pubmed Stuermer,
Reggie/flotillin and the targeted delivery of cargo.
2011,
Pubmed Taylor,
Glypican-1 mediates both prion protein lipid raft association and disease isoform formation.
2009,
Pubmed Tomlinson,
Functional properties of a neuronal class C L-type calcium channel.
1993,
Pubmed
,
Xenbase Viles,
Copper binding to the prion protein: structural implications of four identical cooperative binding sites.
1999,
Pubmed Waithe,
Beta-subunits promote the expression of Ca(V)2.2 channels by reducing their proteasomal degradation.
2011,
Pubmed Wakamori,
Auxiliary subunits operate as a molecular switch in determining gating behaviour of the unitary N-type Ca2+ channel current in Xenopus oocytes.
1999,
Pubmed
,
Xenbase Walker,
Subunit interaction sites in voltage-dependent Ca2+ channels: role in channel function.
1998,
Pubmed
