Proteolytic maturation of α2δ represents a checkpoint for activation and neuronal trafficking of latent calcium channels
- University College London, United Kingdom
Figures
Figure 1 with 3 supplements
Effect of mutation of α2δ-1 to disrupt the proteolytic cleavage site.
(a) Cartoon of uncleaved pro-α2δ-1 and cleaved α2δ-1, showing the approximate position of inserted HA tag and disulfide bonds between α2 (grey) and δ (blue). (b) Rat α2δ-1 sequence at the identified …
Figure 1—figure supplement 1
Alignment of the proteolytic cleavage site in α2δ-1, showing species conservation.
/ indicates cleavage site previously identified in rabbit α2δ-1 by N-terminal sequencing of delta Jay et al., 1991; De Jongh et al., 1990.
Figure 1—figure supplement 2
α2(V6)δ-1 is localised in DRMs to a similar extent to α2δ-1.
(a) Sucrose gradient fractions showing a comparison of DRM localisation (lanes 2–4) of α2δ-1 in untransfected (UTR) tsA-201 cells (top panels, showing some endogenous α2δ-1), α2δ-1 transfected cells …
Figure 1—figure supplement 3
α2(3C)δ-1 is localised in DRMs to a similar extent to α2δ-1.
(a) Comparison of DRM localisation of α2δ-1-HA (left panels) and α2(3C)δ-1-HA (right panels), isolated from transfected tsA-201 cells. Only peak DRM (4–7) and soluble (11–13) fractions are shown. …
Figure 2 with 3 supplements
Effect of mutation of α2δ-1 cleavage site to an HRV-3C site on cell-surface expression and functional properties of CaV2.2.
(a) Example traces (−30 to +10 mV in 5 mV steps) for CaV2.2/β1b-GFP and no α2δ (black traces), WT α2δ-1 (red traces) or α2(3C)δ-1 (blue traces). Charge carrier 1 mM Ba2+. Scale bars refer to all …
Figure 2—figure supplement 1
Examination of effect of α2(3C)δ-1 on CaV2.2/β1b calcium channel currents in tsA-201 cells.
These experiments were performed using 10 mM Ba2+ to amplify any differences in current amplitude between the two conditions examined. (a) Example traces (−25 to +25 mV steps) for CaV2.2/β1b-GFP and …
Figure 2—figure supplement 2
Examination of effect of α2(V6)δ-1 on CaV2.2/β1b calcium channel currents in tsA-201 cells.
(a) Example traces (−25 to +10 mV in 5 mV steps) for CaV2.2/β1b-GFP and either no α2δ (black traces), α2δ-1 (red traces) or α2(V6)δ-1 (blue traces). The scale bars refer to all traces. Charge …
Figure 2—figure supplement 3
Examination of effect of α2(3C)δ-1 on CaV2.2/β1b cell surface expression in tsA-201 cells.
(a) Immunocytochemical detection of cell-surface expression of CaV2.2-BBS, with β1b, and empty vector (left), WT α2δ-1-HA (middle) or α2(3C)δ-1-HA (right) in tsA-201 cells. Upper panel: CaV2.2-BBS …
Figure 3 with 2 supplements
Effect of proteolytic cleavage of α2δ-1 containing an HRV-3C cleavage site on cell-surface expression and functional properties of CaV2.2.
(a) Cartoon showing intracellular cleavage by 3C-protease (green) of α2(3C)δ-1 (gray/blue) associated with CaV2.2 (cyan). (b) Deglycosylated, cell-surface biotinylated fractions for α2δ-1-HA (lanes …
Figure 3—figure supplement 1
The effect of 3C-protease on α2δ-1 and α2(3C)δ-1 expressed in tsA-201 cells.
(a) Deglycosylated WCL for the experiment shown in Figure 3b; α2δ-1-HA (lanes 1 and 2) and α2(3C)δ-1-HA (lanes 3 and 4), expressed in tsA-201 cells, without (lanes 1 and 3) or with (lanes 2 and 4) …
Figure 3—figure supplement 2
Lack of effect of 3C-protease on CaV2.2/β1b currents expressed in tsA-201 cells.
Top: example traces (−25 to +15 mV steps) for CaV2.2/β1b-GFP and either no protease (black traces), WT 3C-protease (blue traces) or inactive mutant 3C-protease (C147V, cyan traces). The charge …
Figure 4 with 3 supplements
Effect of proteolytic cleavage of α2δ-3 containing an HRV-3C cleavage site on cell-surface expression and functional properties of CaV2.2.
(a) Images showing cell-surface CaV2.2-BBS (upper row, white), and total CaV2.2 (II-III loop Ab, lower row, red), for CaV2.2-BBS/β1b in N2A cells, with empty vector (panel 1) or α2δ-3-HA (panel 2). …
Figure 4—figure supplement 1
Lack of cleavage of α2(V6)δ-3.
The expression of α2δ-3 compared to α2(V6)δ-3. The constructs were expressed transiently in tsA-201 cells; peak lipid raft fractions were taken for western blotting. There was a complete loss of …
Figure 4—figure supplement 2
Effect of 3C-protease on expression and cleavage of α2(3C)δ-3.
Full data from experiment shown in Figure 4d. WT α2δ-3-HA (lanes 1, 2 and 3) and α2(3C)δ-3-HA (lanes 4, 5 and 6) were expressed in tsA-201 cells, either without or with inactive (C147V, lanes 2 and …
Figure 4—figure supplement 3
Lack of effect of purified 3C-protease on expression and cleavage of α2(3C)δ-3.
Incubation of cells expressing α2(3C)δ-3-HA with 3C-protease enzyme did not result in cleavage on the cell surface. Cell surface biotinylated deglycosylated α2(3C)δ-3-HA, from cells incubated …
Figure 5 with 1 supplement
Effect of thrombin on the properties and function of α2δ-3 containing a thrombin proteolytic cleavage site.
(a) Sequence at α2δ-3 cleavage site mutated to a thrombin cleavage site. (b) Cartoon of thrombin cleavage of α2δ-3 on cell-surface. (c) Cell-surface biotinylation (left panel) shows efficient …
Figure 5—figure supplement 1
Controls for cleavage of α2(Th)δ-3 by thrombin.
(a) Deglycosylated cell surface biotinylation for tsA-201 cells expressing WT α2δ-3-HA and incubated either with vehicle or thrombin for 60 min (lanes 1 and 2), showing WT α2δ-3 is not cleaved by …
Figure 6
Proteolytic processing of endogenous α2δ-1 and effect of exogenous expression of α2δ-1 and α2(3C)δ-1 in DRGs.
(a) DRGs, spinal nerves and dorsal roots from rats, 4 days after SNL, were dissected and segmented according to the diagram. X marks site of ligation. Tissue was pooled from 4 rats. It was either …
Figure 7 with 1 supplement
Effect of α2δ-1 and proteolytic cleavage of α2(3C)δ-1 on trafficking of CaV2.2 into hippocampal neurites.
(a) Images showing CaV2.2-HA in permeabilized hippocampal neurons (top, white), when co-transfected with β1b, mCherry (bottom, red) and either no α2δ (left), WT α2δ-1 (middle) or α2(3C)δ-1 (right). …
Figure 7—figure supplement 1
Cartoon showing processing and trafficking of α2δ in neurons.
Cartoon of the effect of proteolytic processing of α2δ (dark gray α2, blue δ) on CaV2.2 trafficking and voltage-dependent activation. The gray channel is one that cannot be activated by …
Figure 8
Effect of proteolytic cleavage of α2(3C)δ-1 on Ca2+ influx in presynaptic terminals of hippocampal neurons.
(a) Fluorescence changes in presynaptic terminals of hippocampal neurons expressing sy-GCaMP6f and VAMP-mOr2 in response to electrical stimulation. White arrows point to transfected boutons. Top …
Additional files
-
Supplementary file 1
Summary of statistics shown for the quantitative data in the Figures.
The statistical data were obtained using Graphpad Prism 5.
- https://doi.org/10.7554/eLife.21143.023
