A potent voltage-gated calcium channel inhibitor engineered from a nanobody targeted to auxiliary CaVβ subunits
- Columbia University, Vagelos College of Physicians and Surgeons, United States
Figures
Figure 1 with 1 supplement
Development of a pan-CaVβ nanobody.
(a) Size-exclusion chromatograph and Coomassie gel (inset) showing purified Cavβ1 from baculovirus-infected HEK293 GnTl- cells. (b) Flow-chart of nanobody generation. (c) Phage ELISA using CaVβ1 as …
Figure 1—figure supplement 1
Nb.F3 binds all four CaVβ subunits in the cytosol of mammalian cells Left, schematic of phorbol ester 12,13-dibutyrate (PdBu) translocation assay.
Right, confocal images of HEK293 cells expressing nb.F3-CFP-C1PKCγ and a YFP-CaVβ before (top) and after (bottom) the addition of 1 µM Pdbu.
Figure 2 with 2 supplements
Nb.F3 is functionally silent on reconstituted CaV2.2 channels.
(a) Schematic of experimental strategy; BBS-α1B-YFP was transfected in HEK293 cells with α2δ, CaVβ and either CFP or nb.F3-P2A-CFP. (b) Exemplar flow cytometry dot plot of cells expressing BBS-α1B-YF…
Figure 2—figure supplement 1
Exemplar flow cytometry data for BBS-α1B with YFP-CaVβ2- CaVβ4.
(a) Exemplar flow cytometry dot plot of cells transfected with BBS-α1B, α2-δ, YFP-CaVβ2 and CFP (left) or nb.F3 (right). (b) Cumulative distribution histogram of data from (a) of Alexa-647 (left) or …
Figure 2—figure supplement 2
Nb.F3 is functionally silent on reconstituted CaV1.2 channels.
(a) Cartoon of experimental strategy. BBS-α1C was transfected in HEK293 cells with each YFP-CaVβ and either CFP or nb.F3-P2A-CFP. (b) Exemplar flow cytometry dot plot of cells transfected with BBS-α1…
Figure 3 with 2 supplements
Functional impact of a chimeric nb.F3-Nedd4L protein (CaV-aβlator) on reconstituted Cav2.2 channels.
(a) Schematic of experimental design; HEK293 cells were transfected with BBS-α1B + CaVβ-YFP + α2δ−1, and either nb.F3, nb.F3-Nedd4L, or nb.F3-Nedd4L[C942S]. (b) Exemplar histograms (left) and …
Figure 3—figure supplement 1
Exemplar flow cytometry data for CaV-aβlation of BBS-α1B with YFP-CaVβ2- CaVβ4.
(a) Exemplar flow cytometry dot plot of cells transfected with BBS-α1B, α2-δ, YFP-CaVβ1 and nb.F3 (left), nb.F3-Nedd4L (middle) or nb.F3-Nedd4L[C942S] (right). (b) Histogram a of YFP (left) or …
Figure 3—figure supplement 2
Functional impact of a chimeric nb.F3-Nedd4L protein (CaV-aβlator) on reconstituted Cav1.2 channels.
(a) Schematic of experimental design. HEK293 cells were transfected with BBS-α1C, YFP-CaVβ, and either nb.F3, nb.F3-Nedd4L or nb.F3-Nedd4L[C942S]. (b) Exemplar flow cytometry dot plot of cells …
Figure 4
CaV-aβlator inhibits distinct reconstituted HVACCs.
(a) Population I-V curves from HEK293 expressing α1C + β1b + α2δ−1 with either nb.F3 (black, Ipeak, 0mV = −48.4 ± 8.4 pA/pF, n = 12) or CaV-aβlator (red, Ipeak, 0mV = −0.93 ± 0.16 pA/pF, n = 8). (b-d…
Figure 5 with 1 supplement
CaV-aβlation of endogenous CaV1.2 in cardiomyocytes.
(a) Confocal images (top) and exemplar traces from whole-cell recordings of uninfected guinea pig cardiomyocytes (left), or infected with adenovirus expressing either CaV-βlator (middle) or …
Figure 5—figure supplement 1
CaV-aβlator does not redistribute α1C to Rab5 early-endosomes or lysosomes, nor decrease total levels of α1C or β2.
(a) Comparison of total α1C levels in uninfected cardiomyocytes (gray, n = 15) and those infected with nb.F3-Nedd4L (red, n = 19). (b). Comparison of total β2 levels in uninfected cardiomyocytes …
Figure 6
CaV-aβlation of HVACCs in DRG neurons and pancreatic β-cells.
(a) Exemplar Fura-2 traces of murine DRG neurons infected with GFP (left), F3-Nedd4L (middle), F3-Nedd4L[C942S] (right), with confocal images in inset. The orange bars represent depolarization with …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (rat) | CACNA1B | NM_147141 | ||
| Gene (rabbit) | CACNA1C | NM_001136522 | ||
| Gene (rat) | CACNB1 | NM_017346 | ||
| Gene (human) | CACNB2 | NM_201590 | ||
| Gene (rat) | CACNB3 | NM_012828.2 | ||
| Gene (rat) | CACNB4 | NM_001105733.1 | ||
| Gene (human) | CACNA2D1 | NM_000722.4 | ||
| Gene (human) | NEDD4L | NM_001144965.2 | ||
| strain, strain background (Escherichia coli) | Rosetta DE3 | Millipore Sigma | ||
| Cell line (Human) | HEK293 | Other | RRID: CVCL_0045 | Laboratory of Dr. Robert Kass |
| Recombinant DNA reagent | nb.F3-CFP-PKCγ | This paper | Made by PCR, see molecular biology and cloning | |
| Recombinant DNA reagent | nb.F3-P2A-CFP | This paper | Made by PCR, see molecular biology and cloning | |
| Recombinant DNA reagent | nb.F3-Nedd4L-P2A-CFP | This paper | Made from pCI HA Nedd4L (Addgene #27000); see molecular biology and cloning | |
| Recombinant DNA reagent | nb.F3-Nedd4L [C942S]-P2A-CFP | This paper | Made by site-directed mutagenesis | |
| Recombinant DNA reagent | BBS-α1B | PMID: 20308247 | ||
| Recombinant DNA reagent | BBS-α1C | PMID: 20308247 | ||
| Antibody | Anti-α1C | Alomone | Cat#: ACC-003 | 1:1000 WB/IF |
| Antibody | Anti-α1C | NeuroMab | Clone: N263/31 | 1:200 IF |
| Antibody | Anti-CaVβ1 | NeuroMab | Clone: N7/18 | 1:500 WB |
| Antibody | Anti-CaVβ2 | Alomone | Cat#: ACC-105 | 1:200 |
| Antibody | Anti-Rab5 | Cell Signaling Technology | Cat#: 3547 | 1:200 |
| Antibody | Anti-Rab7 | Cell Signaling Technology | Cat#: 9367 | 1:200 |
| Antibody | Anti-LAMP1 | Developmental Studies Hybridoma Bank at the University of Iowa | RRID: AB_528127 | 1:100 |
| Antibody | Anti-RyR | Thermo Fisher Scientific | Cat#: MA3-916 | 1:1000 |
| Antibody | Anti-actin | Sigma | Cat#: A5060 | 1:1000 |
| Antibody | Anti-ubiquitin, VU-1 | LifeSensors | Cat#: VU101 | 1:500 |
| Antibody | RFP-trap agarose beads | Chromotek | Cat#: rta-20 | |
| Antibody | Anti-FLAG affinity gel | Sigma-Aldrich | Cat#: A2220 | |
| Peptide, recombinant reagent | FLAG peptide | Sigma-Aldrich | Cat#: F3290 | |
| Peptide, recombinant reagent | Ni-NTA agarose | Qiagen | Cat#: 30210 | |
| Peptide, recombinant reagent | Protein A/G sepharose beads | Rockland | ||
| Peptide, recombinant reagent | α-bungarotoxin, Alexa Fluor 647 conjugate | Life Technologies | ||
| Peptide, recombinant reagent | Fura-2 AM | Life Technologies | Cat#: F1221 | |
| chemical compound, drug | Phorbol 12,13-dibutyrate | Sigma-Aldrich | Cat#: P1269 | |
| commercial assay or kit | AdEasy Adenoviral Vector Systems | Stratagene | ||
| commercial assay or kit | QuikChange Lightning Site-Directed Mutagenesis Kit | Stratagene | ||
| software, algorithm | FlowJo | RRID: SCR_008520 | ||
| software, algorithm | PulseFit | HEKA | ||
| software, algorithm | EasyRatioPro | HORIBA | ||
| software, algorithm | GraphPad Prism | RRID: SCR_002798 |
Additional files
-
Transparent reporting form
- https://doi.org/10.7554/eLife.49253.014
