The alpha2delta subunits of voltage-gated calcium channels form GPI-anchored proteins, a posttranslational modification essential for function

Abstract

Voltage-gated calcium channels are thought to exist in the plasma membrane as heteromeric proteins, in which the alpha1 subunit is associated with two auxiliary subunits, the intracellular beta subunit and the alpha(2)delta subunit; both of these subunits influence the trafficking and properties of Ca(V)1 and Ca(V)2 channels. The alpha(2)delta subunits have been described as type I transmembrane proteins, because they have an N-terminal signal peptide and a C-terminal hydrophobic and potentially transmembrane region. However, because they have very short C-terminal cytoplasmic domains, we hypothesized that the alpha(2)delta proteins might be associated with the plasma membrane through a glycosylphosphatidylinositol (GPI) anchor attached to delta rather than a transmembrane domain. Here, we provide biochemical, immunocytochemical, and mutational evidence to show that all of the alpha(2)delta subunits studied, alpha(2)delta-1, alpha(2)delta-2, and alpha(2)delta-3, show all of the properties expected of GPI-anchored proteins, both when heterologously expressed and in native tissues. They are substrates for prokaryotic phosphatidylinositol-phospholipase C (PI-PLC) and trypanosomal GPI-PLC, which release the alpha(2)delta proteins from membranes and intact cells and expose a cross-reacting determinant epitope. PI-PLC does not affect control transmembrane or membrane-associated proteins. Furthermore, mutation of the predicted GPI-anchor sites markedly reduced plasma membrane and detergent-resistant membrane localization of alpha(2)delta subunits. We also show that GPI anchoring of alpha(2)delta subunits is necessary for their function to enhance calcium currents, and PI-PLC treatment only reduces calcium current density when alpha(2)delta subunits are coexpressed. In conclusion, this study redefines our understanding of alpha(2)delta subunits, both in terms of their role in calcium-channel function and other roles in synaptogenesis.

Fig. 1.

Evidence that α₂δ-3 is GPI-anchored. (A) Amino acid sequence of rat α₂δ-3 C terminus showing the peptide used to generate the δ-3 (1035-1049) Ab in red, the two predicted GPI-anchor sites (potential ω amino acids arrowed and underlined in purple), and the C-terminal hydrophobic sequence (green). (B Upper) Immunoblot profile of WT α₂δ-3 in lipid-raft fractions (DRM) from transfected tsA-201 cells using α₂-3 (71–90) Ab. (Lower) Flotillin distribution in the same DRM profile. (C) Immunoblot of heterologously expressed α₂δ-3 in DRM fraction five, before and after incubation with PI-PLC and subsequent deglycosylation with endoglycosidase F (EndoF) as indicated. Deglycosylation was performed, because it enables accurate assessment of the protein MW and generally enhances immunoreactivity of the anti-peptide Abs. The Ab used was anti-δ-3 (1035-1049). The arrows show the upward shift of the δ-3 protein after PI-PLC. Similar results were obtained with PI-PLC from three different commercial sources. (D) Immunoblot profile of hippocampal α₂δ-3 with α₂-3 (71-90) Ab (Top) and flotillin (Bottom) in DRM fractions. (E) Hippocampal α₂δ-3 in DRM fraction five after deglycosylation with EndoF before and after PI-PLC as indicated. (Top) Immunoblot with α₂-3 Ab; (Bottom) immunoblot with δ-3 Ab. (Left) Equal loading of both lanes (25 μg protein); (Right) 8 μg protein in left lane and 20 μg in right lane to better compare the upward mobility shift after PI-PLC treatment, which is indicated by arrows. (F) PI-PLC-treated material from hippocampal DRM fraction five was subjected to phase separation in Triton X-114. This procedure is used to identify GPI-anchored proteins. The intact, amphipathic protein remains in the detergent-rich phase, whereas the PI-PLC-cleaved hydrophilic form is found in the detergent-poor (aqueous) phase (asterisk; see SI Materials and Methods (13). Western blots were performed on the detergent and aqueous phases to identify the presence of the different proteins. (Top) α₂-3. (Middle) Flotillin. (Bottom) δ-3 showing protein in the detergent (left two lanes) and aqueous phases (right two lanes) with and without PI-PLC treatment as indicated. The α₂-3 and δ-3 are indicated by an asterisk in the aqueous phase. Below this is shown a parallel blot that was probed with the CRD Ab, which shows a band at the level of δ-3 in the aqueous phase that was only observed after PI-PLC treatment (arrow). (G Left) Mean I-V relationship for Ca_V2.2/β1b coexpressed with α₂δ-3 in tsA-201 cells and treated for 90 min with vehicle (▪ n = 5) or with PI-PLC (4 U/mL; red • n = 11). (Right) Representative I_Ba currents (steps from −30 mV to +15 mV from a holding potential of −90 mV) are shown for the vehicle-treated control (black traces, Upper) and PI-PLC-treated (red traces, Lower) conditions; 1 mM Ba²⁺ was used as charge carrier. (H) Mean I-V relationship for Ca_V2.2/β1b expressed without α₂δ in tsA-201 cells and treated for 90 min with a vehicle control (▪ n = 11), a control in which the same amount of water was added (□ n = 20), or a control with PI-PLC (4 U/mL; red • n = 28). No significant differences were observed under any of the conditions. Note that 5 mM Ba²⁺ was used as the charge carrier, because the currents were very small in 1 mM Ba²⁺.

Fig. 2.

Evidence that GPI-anchoring of α₂δ-3 is required for cell-surface localization and function. (A Left) Immunoblot profile of GAS-WKW α₂δ-3 (Upper) and CGG-WKW α₂δ-3 (Lower) in DRM fractions from transfected tsA-201 cells using α₂-3 (71–90) Ab. (Right) Bar chart showing the average proportion of the total α₂δ-3 found to be in DRM (sucrose gradient fractions 4–6) for WT α₂δ-3 (black bar), CGG-WKW α₂δ-3 (blue bar), and GAS-WKW α₂δ-3 (red bar) from two independent experiments each. (B) Mean I-V relationship for Ca_V2.2/β1b coexpressed with WT α₂δ-3 (▪ n = 12), with GAS-WKW α₂δ-3 (red • n = 25), or without α₂δ (△ n = 23) in tsA-201 cells. All recordings are in 5 mM Ba²⁺. (C) Peak I_Ba (mean ± SEM) measured at +15 mV was determined from I-V relationships including those in A. Ca_V2.2/β1b is shown without α₂δ (white bar; n = 36), with WT α₂δ-3 (black bar; n = 16), with CGG-WKW α₂δ-3 (blue bar; n = 22), or with GAS-WKW α₂δ-3 (red bar; n = 25). Data were pooled from several experiments and normalized to the respective control (+WT α₂δ-3) in each experiment. The statistical significances of the differences compared to +WT α₂δ-3 were determined by ANOVA and post hoc Bonferroni test. **, P < 0.001. Example I_Ba currents (from −30 mV to +15 mV from a holding potential of −90 mV) are shown above the bars for no α₂δ (gray), WT α₂δ-3 (black), CGG-WKW α₂δ-3 (blue), and GAS-WKW α₂δ-3 (red). (D Upper) Cell-surface biotinylation of α₂δ-3 (WT), CGG-WKW α₂δ-3 and GAS-WKW α₂δ-3. The left three lanes show WCL, and the right six lanes show WT and mutant α₂δ-3 after cell-surface biotinylation, before and after deglycosylation with EndoF as indicated. Open arrow, full-length α₂δ-3; closed arrow, cleaved α₂-3. Lower shows lack of biotinylation of cytoplasmic Akt. (E) Quantification of relative amounts of full-length α₂δ-3 and cleaved α₂-3 for the CGG-WKW α₂δ-3 (blue) and GAS-WKW α₂δ-3 (red) mutants at the cell surface from two experiments, including the data shown in C, that were normalized to the amount in the WCL. (F) Confocal microscopic images showing membrane localization of α₂δ-3 using the δ-3 Ab (Left, red) for WT (Upper) and CGG-WKW α₂δ-3 (Lower) when coexpressed with green fluorescent protein Ca_V2.2 (Middle) and β1b in nonpermeabilized Cos-7 cells. (Right) Merged images show nuclear staining with DAPI (blue). Note that cell-surface staining in nonpermeabilized Cos-7 cells is seen, not as a fine ring but as a wide annulus, because of the flattened geometry (see also Fig. S3A). (Scale bar: 50 μm on merged images.) (G) Quantification of cell-surface immunofluorescence using δ-3 Ab for WT α₂δ-3 (black bars; n = 68), CGG-WKW α₂δ-3 (blue bars; n = 38), and GAS-WKW α₂δ-3 (red bars; n = 38). Statistical significance is P < 0.001 for one-way ANOVA and Tukey’s post hoc tests.

Fig. 3.

Biochemical and functional evidence that α₂δ-2 is anchored by GPI. (A) Amino acid sequence of mouse α₂δ-2 C terminus showing the peptides used to generate the δ-2 (1062-1079), δ-2 (1080-1094), and δ-2 (1133-1147; C-terminus, CT) Abs in blue, red, and pink, respectively. The predicted GPI-anchor site (in purple and underlined with ω indicated by the arrow) and the C-terminal hydrophobic sequence (green) are also shown. (B) Immunoblot of δ-2 from peak DRM fractions prepared from an α₂δ-2 stable cell line (14), which shows the absence of immunoreactivity to the C-terminal δ-2 (1133-1147) Ab (Left; see Fig. S4A for validation of this Ab) but immunoreactivity to other δ-2 Abs (1062-1079; Center) and (1080-1094; Right). (C) Immunoblot of δ-2 from the peak DRM fraction derived from α₂δ-2–expressing tsA-201 cells before and after incubation with PI-PLC and subsequent deglycosylation with EndoF as indicated. The Ab used was δ-2 (1080-1094). The arrows show the upward shift of the δ-2 protein after PI-PLC. (D Top) Immunoblot of α₂-2 from the peak DRM fraction derived from cerebellum before and after incubation with PI-PLC as indicated and subsequent deglycosylation with EndoF. (Middle) Immunoblot of δ-2 from the same fractions. A mixture of all anti–δ-2 Abs was used for this immunoblot. The arrows show the upward shift of the δ-2 protein after PI-PLC. (Bottom) Immunoblot of δ-2 with the δ-2 (1133-1147; CT) Ab alone from the same fractions before PI-PLC treatment, which shows the absence of immunoreactivity. (E) Immunoprecipitated HA-tagged α₂δ-2 treated or not treated with PI-PLC before purification is indicated in panel 1. Silver-stained gel was used for the affinity-purified HA-tagged α₂δ-2; full-length α₂δ-2 is the main species purified by this procedure, possibly because of the greater accessibility of the HA tag. These data also confirm that the PI-PLC used does not have any significant proteolytic activity, because no proteolytic fragments of α₂δ-2 were observed after 3 h of incubation. Panel 2 shows corresponding anti-HA blot; panel 3 shows blot for α₂-2 with α₂-2 (102-117) Ab. Panels 1–3 used 3–8% Tris acetate gel. Panel 4 shows the blot for δ-2 with δ-2 (1080-1094) Ab using a 12% Bis-Tris gel for better resolution of δ-2. Arrows indicate the position of α₂δ-2, and arrowheads show α₂-2 and δ-2. The material shown in panel 1 was then adjusted to equal protein concentration before immunoblotting for CRD. (F) Immunoblot with anti-CRD Ab. Lane 1 shows α₂δ-2 without PI-PLC treatment. Lane 2 shows α₂δ-2 after PI-PLC treatment. Lane 3 shows deglycosylated α₂δ-2 without PI-PLC treatment. Lane 4 shows deglycosylated α₂δ-2 after PI-PLC treatment that showed an ∼170-kDa band corresponding to HA-a₂δ-2 and a 150-kDa band corresponding to deglycosylated HA–α₂δ-2 (arrows). The CRD antibody has a low affinity, and we were unable to examine immunoreactivity against a band corresponding to free δ-2, because very little was purified using the internal HA tag in α₂-2 (see silver-stained gel and δ-2 blot in E). (G) Effect of acute incubation of Cos-7 cells for 1 h with PI-PLC (4 U/mL) on cell-surface localization of transfected 5′NT and α₂δ-2. Representative confocal microscopic images of α₂δ-2 (102-117 Ab; Upper) and 5′NT (Lower) cell-surface expression in transfected nonpermeabilized COS-7 cells (nuclei visualized with DAPI) were treated with either vehicle (con, left) or PI-PLC (right). (Scale bar: 20 μm.) (H) Quantification of immunofluorescence data similar to those shown in G were obtained from epifluorescence images for 5′NT incubated with vehicle (black bar; n = 25) or PI-PLC (Glyko; hatched bar; n = 31) and for α₂δ-2 incubated with vehicle (white bar; n = 30) or PI-PLC (cross-hatched bar; n = 20). The statistical significances of the differences with and without PI-PLC treatment were determined by Student's t test. *, P = 0.0083 for 5′NT; **, P = 0.0019 for α₂δ-2. (I Upper) Mean I-V relationship for Ca_V2.2/β1b coexpressed with α₂δ-2 (▪ n = 12), with α₂δ-2 GAS-WKW (red • n = 24), or without α₂δ (△ n = 18) in tsA-201 cells. All recordings are in 5 mM Ba²⁺. (Lower) Example I_Ba currents (from −30 mV to +15 mV from a holding potential of −90 mV) are shown for no α₂δ (gray), α₂δ-2 (black), and GAS-WKW α₂δ-2 (red).

Fig. 4.

Evidence that GPI anchoring of α₂δ-1 is required for cell-surface localization and function. (A) Amino acid sequence of rat α₂δ-1 C terminus showing the predicted GPI-anchor sites (ω) and the C-terminal hydrophobic sequence (green). (B and C) Lysates from the hippocampus were incubated with vehicle (B) or PI-PLC (C) at 37°C in the presence of protease inhibitors, and then they were subjected to sucrose gradient centrifugation. (Upper) The immunoblot profile of α₂δ-1 in DRMs from hippocampus is shown using the α₂-1 monoclonal Ab. (Lower) Flotillin distribution in same DRM profile. (D) Phase separation of α₂-1 after PI-PLC treatment. (Upper) Peak DRM fraction before phase separation showing input for α₂-1 and flotillin. (Lower) Phase separation after vehicle or PI-PLC treatment and Triton X-114 extraction shows α₂-1 in aqueous phase after PI-PLC (indicated by asterisk). Flotillin was not redistributed into the aqueous phase after PI-PLC treatment. (E Left) Immunolocalization of α₂δ-1 in nonpermeabilized DRG neurons after 2 days in culture after incubation with vehicle (Upper; confocal field contains 12 DRG neurons) or with 4 U/mL PI-PLC for 60 min (Lower; field contains 10 DRG neurons). (Scale bar: 50 μm.) (Right) Quantification of α₂δ-1 immunostaining expressed as number of pixels per cell (log₁₀ scale) in the intensity range 500-3,000 for 18 images from three different coverslips for each condition, each containing between 5 and 12 cells (Upper). Black line, vehicle-treated; red line, 4 U/mL PI-PLC; blue line, 8 U/mL PI-PLC. (Lower) Quantification of immunostaining in four separate experiments after vehicle (black bar; normalized to 100%) relative to PI-PLC (4 U/mL or 8 U/mL; red bar). **, P < 0.001 for Student's t test. (F) Immunoblot of supernatant after PI-PLC treatment of DRGs, as in E, for 4 U/mL and 8 U/mL PI-PLC. The arrow shows the presence of α₂-1. (G) Cartoon showing the structure of α₂δ subunits that is identified here. The GPI anchor consists of ethanolamine (orange), three mannose rings (blue), glucosamine (pink), and inositol (yellow). (H) Proposed scheme for processing α₂δ proteins. The N-terminal signal sequence is in gray, the C-terminal GPI signal sequence is in green, the α₂ sequence is in black, and the δ sequence is in white. The GPI anchor is drawn as in G. The position of the ω amino acid is indicated by a purple arrow in G and H.