Selective posttranslational inhibition of CaVβ1-associated voltage-dependent calcium channels with a functionalized nanobody

Figures

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  Fig. 1. Rationale for developing post-translational CaVβ-dependent inhibitors for CaV1/CaV2 channels. a Cartoon depicting hypothesized association of CaV1.2 with β2b subunits in adult ventricular myocytes (i) and the expected loss of β2b with gene knockout of this subunit (ii). Bottom, knock out of β2 in adult heart has only minimal impact on whole-cell Ca2+ current making it ambiguous whether most CaV1.2 are associated with β2b in adult ventricular cardiomyocytes. Posttranslational inhibition of cardiac CaV1.2 using a CaVβ-targeted nanobody fused to Nedd4L HECT (CaV-aβlator) domain eliminates CaV1.2 complexes from the membrane and abolishes current (iii), proving that CaV1.2 is stably associated with β2 in adult cardiomyocytes. b Schematic of a scenario where distinct CaVβ isoforms preferentially associate with particular α1-subunit types to mediate different functions (i). Knockdown of one β-isoform may lead to β reshuffling that lessens the functional impact of elimination of the particular CaVβ subunit (ii). By contrast, targeted posttranslational inhibition of the channel complex based on the β isoform would yield a qualitatively different result that more accurately reflects the…

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  Fig. 2. Identification of nb.E8 as a CaVβ1-selective nanobody. a Schematic of fluorescence resonance energy transfer (FRET) pair, Venus-nb.F3 + Cerulean-CaVβ. b Flow cytometry FRET (flow-FRET) scatter plots with FRET efficiency (ED) plotted vs free nb.F3 concentration. The blue lines fit a 1:1 binding model to the data. Data show nb.F3 binds all four CaVβ subunit isoforms. Kd,EFF, effective dissociation constant. c Schematic of Venus-nb.E8 + Cerulean-CaVβ. d Flow-FRET scatter plots and binding curves show nb.E8 exclusively binds CaVβ1. e Top, schematic of CaVβ structural organization. Conserved src homology 3 (SH3, orange) and nucleotide kinase (NK, green) domains are bounded by variable N- and C-termini, and separated by a HOOK domain. Bottom, cryoEM structure of CaVβ1a (PDB: 5GJV). f Flow-FRET scatter plots and binding curves for Cerulean-nb.E8 co-expressed with Venus-tagged β1b[NT-SH3-HOOK] (left), β1b[NK-CT] (middle), and β1b[SH3-HOOK] (right). g Alignment of region within SH3 domains of human CaVβ subunits. Highlighted region is a 21-residue sequence (η1) that shows some variability amongst CaVβ isoforms. h Flow-FRET scatter plots and binding curves for Cerulean-nb.E8 co-expressed…

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  Fig. 3. Crystal structures of nb.F3 and nb.E8 in complex with CaVβ1. a Cartoon diagram of the nb.F3:CaVβ2a complex. CaVβ2a SH3 (helices (dark green) and β-strands (light green)) and NK (helices (dark blue) and β-strands (cyan)) domains are indicated. Nb.F3 (yellow) is shown with a semi-transparent surface. Nb.F3 Site 1 and Site 2 binding regions are indicated by the red and orange ovals, respectively. b Cartoon diagram of nb.F3:nb.E8:CaVβ1b complex. SH3 (helices (green) and β-strands (lime)) and NK (helices (purple) and β-strands (violet)) domains are indicated. Nb.F3 (yellow) and nb.E8 (yellow orange) are shown with a semi-transparent surface. Site 1 and Site 2 binding regions are indicated by the red and orange ovals, respectively. Light green oval denotes nb.E8 binding site. Lower panel shows view from the SH3 domain N-terminus as indicated by the arrows. ABP indicates AID binding pocket location in (a) and (b), (c) View of nb.F3:CaVβ2a Site 1 interactions. d View of nb.F3:CaVβ2a Site 2 interactions. e View of nb.E8:CaVβ1b interactions. Red ovals indicate CaVβ sites that differ among isoforms. Complementarity determining regions (CDRs) are colored similarly in all panels: CDR1…

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  Fig. 4. Nb.E8 inhibits current amplitude and speeds inactivation in CaVβ1-associated CaV2.2 channels. a Exemplar family of whole cell currents from HEK293 cells expressing reconstituted CaV2.2 channels (α1B+β1b+α2δ-1) in the absence (left) or presence (right) of nb.E8. b Population current density vs voltage (J-V) relationship for cells expressing α1B+β1b+α2δ-1 in the absence (black circles; n = 20 cells examined over 4 independent experiments) or presence (red circles; n = 16 cells examined over 4 independent experiments) of nb.E8. *P = 6.5 × 10−5, two-tailed unpaired t-test. c Fractional current remaining after 20 ms (r20) at various test pulse voltages for cells expressing α1B+β1b+α2δ−1 in the absence (black circles; n = 20 cells examined over 4 independent experiments) or presence (red circles; n = 13 cells examined over 4 independent experiments) of nb.E8. *P = 7.4 × 10−5, two-tailed unpaired t test. d–f Data for α1B+β2b+α2δ−1 ± nb.E8, same format as (a–c). e n = 8 cells examined over 3 independent experiments (black symbols) and n = 6 cells examined over 3 independent experiments (red symbols). f n = 7 cells examined over 3 independent experiments (black symbols) and n = 5 cells…

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  Fig. 5. Nb.E8 inhibits CaV2.2 surface density and single channel Po. a Schematic of BBS-α1B + β1b-YFP ± nb.E8. b Flow cytometry contour plots showing CaV2.2 surface density (Alexa647) vs β1b expression (YFP) in HEK293 cells expressing α1B+β1b+α2δ−1 with either CFP (left) or nb.E8 (right). c Bar charts showing impact of nb.E8 on CaV2.2 surface density (n = 6 over 3 independent experiments for both CFP and nb.E8 groups) and β1b-YFP expression (n = 6 over 3 independent experiments for both CFP and nb.E8 groups). *P = 0.0052 compared to CFP control, two-tailed unpaired t test. d Schematic of cell-attached single channel recording. e Exemplar single channel recordings evoked by slow ramp protocols in HEK293 cells expressing α1B+β1b+α2δ−1 with either CFP (left) or nb.E8 (right). f Ensemble average open probability vs voltage (Po-V) relationships in cells expressing α1B+β1b+α2δ−1 with either CFP (gray) or nb.E8 (red). g Bar charts showing impact of nb.E8 on maximal single-channel open probability, Po,max (n = 5 over 3 independent experiments for CFP, and n = 8 over 3 independent experiments for nb.E8). *P = 0.0017 compared to CFP control, two-tailed unpaired t test. Data are means ± SEM. Source…

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  Fig. 6. Chisel-1 selectively eliminates recombinant CaV2.2 channels reconstituted with CaVβ1. a Schematic showing modular structures of Nedd4L and Chisel-1. b Schematic of α1C + β1b ± Chisel-1. c Left, β1b immunoblot in untransfected cells (UT), or cells transiently transfected with α1C + β1b and CFP, nb.E8 or Chisel-1. Right, Bar charts showing impact of Chisel-1 on normalized β1b expression (n = 5 independent experiments each for CFP, nb.E8, and Chisel-1 groups). d Left, β1b pulldown followed by β1b immunoblot. Middle, β1b pulldown followed by ubiquitin immunoblot. Right, Bar chart showing Chisel-1 enhances ubiquitination of β1b (n = 5 independent experiments each for CFP, nb.E8, and Chisel-1 groups). e Left, α1C immunoblot. Right, impact of Chisel-1 on α1C normalized expression (n = 6 independent experiments each for CFP, nb.E8, and Chisel-1 groups). f Left, α1C pulldown followed by α1C immunoblot. Middle, α1C pulldown followed by ubiquitin immunoblot. Right, Bar chart showing Chisel-1 enhances ubiquitination of α1C (n = 6 independent experiments each for CFP, nb.E8, and Chisel-1 groups). g Exemplar family of whole-cell currents in HEK293 cells expressing α1B+β1b+α2δ−1 in the absence…

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  Fig. 7. Generality of nb.E8 and Chisel-1 selective inhibition of recombinant CaV1/CaV2 channels reconstituted with CaVβ1. a Population current density vs voltage (J-V) relationship for cells expressing α1A+β+α2δ−1 and co-expressed with CFP (black symbols), nb.E8 (cyan symbols) or Chisel-1 (red symbols): α1A+β1b+α2δ−1 (CFP, n = 6; nb.E8, n = 6, Chisel-1, n = 3); α1A+β2b+α2δ−1 (CFP, n = 7; nb.E8, n = 6, Chisel-1, n = 5); α1A+β3+α2δ−1 (CFP, n = 11; nb.E8, n = 6, Chisel-1, n = 6); α1A+β4+α2δ−1 (CFP, n = 10; nb.E8, n = 5, Chisel-1, n = 8). b J-V relationship for cells expressing α1D+β+α2δ−1 with CFP (black symbols), nb.E8 (cyan symbols) or Chisel-1 (red symbols): α1D+β1b+α2δ−1 (CFP, n = 8; nb.E8, n = 9, Chisel-1, n = 5); α1D+β2b+α2δ−1 (CFP, n = 8; nb.E8, n = 8, Chisel-1, n = 7); α1D+β3+α2δ−1 (CFP, n = 3; nb.E8, n = 5, Chisel-1, n = 5); α1D+β4+α2δ−1 (CFP, n = 7; nb.E8, n = 10, Chisel-1, n = 4). c J-V relationship for cells expressing α1E+β+α2δ−1 with CFP (black symbols), nb.E8 (cyan symbols) or Chisel-1 (red symbols): α1E+β1b+α2δ−1 (CFP, n = 6; nb.E8, n = 8, Chisel-1, n = 6); α1E+β2b+α2δ−1 (CFP, n = 7; nb.E8, n = 5, Chisel-1, n = 5); α1E+β3+α2δ−1 (CFP, n = 8; nb.E8, n = 7, Chisel-1, n = 4);…

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  Fig. 8. Chisel-1 selectively eliminates current through endogenous CaVβ1-associated HVACCs. a Schematic of skeletal muscle Cav1.1 complex. b Images of isolated flexor digitorum brevis (FDB) fibers either untransfected (top) or transfected with Chisel-1-P2A-CFP (bottom). c Top, exemplar whole-cell currents from isolated FDB fibers expressing CFP (left) or Chisel-1 (right). Bottom, Population J-V curves from isolated FDB fibers expressing CFP (black squares; n = 13 over 3 independent experiments) or Chisel-1 (red squares; n = 13 over 3 independent experiments). d Top, exemplar gating currents from isolated FDB fibers expressing CFP (left) or Chisel-1 (right). Bottom, Population Q-V curves from isolated FDB fibers expressing CFP (black circles; n = 7 over 2 independent experiments) or Chisel-1 (red circles; n = 8 over 2 independent experiments). *P = 9.32 × 10−5 compared to CFP control, two-tailed unpaired t test. e Schematic of ventricular cardiomyocyte CaV1.2 complex. f Confocal images of cardiomyocytes expressing mCherry (top) or Chisel-1-P2A-mCherry (bottom). g Population J-V curves from isolated ventricular myocytes expressing mCherry (black triangles; n = 13 over 3 independent…

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  Fig. 9. Chisel-1 inhibits Ca2+ influx and excitation-transcription coupling in hippocampal neurons. a Representative hippocampal neuron cultures expressing mCherry (top row) or Chisel-1-P2A-mCherry (bottom row) with immunofluorescence detection of β1b (green) and MAP2 (purple). Images are representative of three independent experiments. b Representative CaVβ isoform immunoblots from hippocampal neurons expressing either mCherry or Chisel-1. Immunoblots are representative of three independent experiments. c Time course of mean 90 KCl-evoked Ca2+ transients in hippocampal neurons expressing either mCherry (black line) or Chisel-1 (red trace). d Bar charts showing impact of Chisel-1 on 90 KCl-evoked Ca2+ transients before and after the addition of CaV1/CaV2 channel blockers (mCherry, n = 20 over 3 independent experiments; Chisel-1, n = 30 over 3 independent experiments). e Top, schematic of experimental protocol for assessing excitation-transcription coupling in cultured hippocampal neurons. Bottom, exemplar images showing phosphoCREB (pCREB), microtubule associated protein 2 (MAP2), and DAPI staining of fixed mCherry-expressing hippocampal neurons exposed to either 5 or 40 mM KCl. NBQX,…