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

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.

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 Ca_V2.1/β4/α₂δ-2 calcium channel currents

(A) Current–voltage (I–V) relationships for I_Ba recorded from tsA-201 cells expressing Ca_V2.1/β4/α₂δ-2 (■; n=25), Ca_V2.1/β4 alone (○; n=7) and Ca_V2.1/β4/α₂δ-2/WT PrP (△; n=23). The ratio of cDNAs used for transfection for Ca_V2.1/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 I_Ba current traces resulting from step potentials from −100 mV to between −30 and +15 mV in 5 mV increments for Ca_V2.1/β4/α₂δ-2 (top panel), Ca_V2.1/β4 alone (middle panel) and Ca_V2.1/β4/α₂δ-2/WT PrP (bottom panel). (C) Individual peak I_Ba currents at +10 mV, expressed as the mean±S.E.M. percentage of the control condition with WT α₂δ-2 and Ca_V2.1/β4/α₂δ-2 (■; n=25), Ca_V2.1/β4 alone (○; n=7) and Ca_V2.1/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 Ca_V2.1/β4/α₂δ-2 (black bar), Ca_V2.1/β4 alone (white bar) and Ca_V2.1/β4/α₂δ-2/WT PrP (grey bar). *P<0.05 and **P<0.01. (E) Steady-state inactivation curves for I_Ba recorded from cells expressing Ca_V2.1/β4/α₂δ-2 (■; n=12), Ca_V2.1/β4 alone (○; n=4) and Ca_V2.1/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); Ca_V2.1/β4/α₂δ-2 (black bar), Ca_V2.1/β4 alone (white bar) and Ca_V2.1/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 Ca_V2.1/β1b/α₂δ-1 calcium channel currents

(A) Examples of families of I_Ba current traces resulting from step potentials from −90 mV to between −30 and +10 mV in 5 mV steps for Ca_V2.1/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 Ca_V2.1/β1b/α₂δ-1/PrP was 3:2:2:1, with empty vector used where α₂δ or PrP was absent. (B) I–V relationships for I_Ba recorded from tsA-201 cells expressing Ca_V2.1/β1b/α₂δ-1 alone (■; n=28) with WT PrP (▲; n=19) or with ΔGPI–PrP (△; n=13). (C) Individual mean±S.E.M. peak I_Ba currents at +5 mV for Ca_V2.1/β1b/α₂δ-1 alone (■; n=28) with WT PrP (▲; n=19) or ΔGPI–PrP (△; n=13). **P<0.01. (D) V₅₀ activation for Ca_V2.1/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) Ca_V2.2/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 Ca_V2.2/β1b/α₂δ-1/PrP (1:0.25; upper panel) or Ca_V2.2/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 I_Ba 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 I_Ba recorded from tsA-201 cells expressing Ca_V2.1/β1b/α₂δ-1 alone (■; n=16) with WT PrP (▲; n=10) or ΔGPI–PrP (◆; n=14) or cells expressing Ca_V2.1/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 I_Ba current traces resulting from step potentials from −90 mV to between −30 and +10 mV in 5 mV steps for Ca_V2.1/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 I_Ba currents (means±S.E.M.) for the data shown in (A) for Ca_V2.1/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 Ca_V2.1/β1b/α₂δ-1 calcium channel currents

(A) I–V relationships for I_Ba recorded from tsA-201 cells expressing Ca_V2.1/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 I_Ba observed for Ca_V2.1/β1b/α₂δ-1 plus WT PrP from Figure 3(B). ***P<0.001 between the peak I_Ba 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.