Mapping the interaction surface between CaVβ and actin and its role in calcium channel clearance

Figures

• 

  Fig. 1. Workflow diagram illustrating the strategy used to identify the interaction surface between CaVβ and actin by combining cross-linking mass spectrometry (XL-MS) with protein-protein docking and experimental validation. The overall strategy can be divided in four phases; a pre-phase, named protein preparation, aimed to generate the proteins under study, and XL-MS, modeling and experimental and functional validation phases. The optimization of the XL-reaction conditions was done by testing different amounts of the XL (DSBU and DSSO) at different reaction times, as shown in Supplementary Fig. 1, and visualized by SDS-PAGE (1). Replicas of the optimal XL-reaction conditions were carried out for further analysis (2). The band containing the specific cross-linked product between CaVβ and actin was excised and in gel trypsinized (3). The mix of peptides were subjected to C18 stage tip cleaning (4) and LC-MS/MS (5). XL-MS data analysis was performed using three different search engines (6). Unique intermolecular cross-links that were identified by at least two search engines (7) were considered for filtering using DisVis (8) to obtain a set of self-consistent XL-MS derived distance…

• 

  Fig. 2. Chemical XL-MS and computational analysis define a set of distance constraints for modeling the CaVβ–actin complex. A Domain architecture (top) and crystal structure (bottom) of rat CaVβ2 (UniProtKB Q8VGC3-2; PDB 5V2P). Src homology 3 (SH3) and guanylate kinase (GK) domains forming the protein core are labeled. Numbers represent the first and last residues. B Size exclusion chromatography profile of the CaVβ2-core; inset, purified protein resolved by SDS-PAGE. Numbers denote the size of molecular weight (MW) standards. The experiments were repeated at least 3 times. C Representative SDS-PAGE images of optimal cross-linking reactions for the CaVβ2-core and actin using DSSO, DSBU, and control reaction with only vehicle, no cross-linker (Non-XL) from 5, 4 and 3 independent replicas, respectively. DSSO: lane 1, MW standards; lane 2, CaVβ2-core–actin cross-linking; lane 3, actin alone incubated with DSSO; and lane 4, CaVβ2-core alone incubated with DSSO. DSBU: lane 1, CaVβ2-core–actin cross-linking; lane 2, actin alone incubated with DSBU; and lane 3, CaVβ2-core alone incubated with DSBU. MW standards lane was omitted and the full image is shown in Supplementary Fig. 1. The red arrow…

• 

  Fig. 3. Model of CaVβ2 bound to actin shows an extended contact surface and electrostatic complementarity. A Space-filling and ribbon diagram showing two views related by a 90° rotation of the best-scoring docking model of CaVβ2 complexed with actin. One molecule of CaVβ2 (blue) interacts with two adjacent monomers in one actin protofilament, named n (yellow) and n + 2 (pink). Actin subunits are labeled based on their position along the filament from the pointed end (−) to the barbed end (+). For clarity, CaVβ2 is shown in ribbon mode only in the right panel. The four actin subdomains, SD1–SD4, are labeled in the n monomer. Subdomains in monomer n + 2 are indicated with a prime (ʹ); SD2ʹ and SD3ʹ were omitted. B Surface models showing the contact frequency of CaVβ2 and actin residues. C Same as in B but showing the electrostatic potential map of both proteins. D SDS-PAGE analysis of a representative F-actin cosedimentation assay using the indicated NaCl concentrations. Briefly, the CaVβ2-core was incubated with phalloidin-stabilized actin filaments, the mixture was centrifuged, and the supernatant (S) and pellet (P) fractions separated. The pellet was resuspended in the same volume of…

• 

  Fig. 4. Alanine substitution of predicted PPI hotspots decreases the in vitro affinity of CaVβ2 for actin but not for the AID peptide. A Structure of CaVβ2 (blue ribbon), indicating the eight predicted hotspot residues (red spheres) mutated to alanine in the CaVβ2-core. Two actin subunits interacting with CaVβ2 are shown in space-filling mode, oriented from the barbed end (n + 2, pink) to the pointed end (n, yellow). B Linear diagram of the CaVβ2-core domain, indicating the positions of hotspot mutations and size exclusion chromatography profiles of the hotspot mutants, CaVβ2-core 6Ala (orange) and CaVβ2-core 8Ala (green). The arrow denotes the elution volume for CaVβ2-core WT. C SDS-PAGE of representative F-actin cosedimentation assays for the CaVβ2-core WT (lanes 2–5), CaVβ2-core 6Ala (lanes 6–9), and CaVβ2-core 8Ala (lanes 10–13). MW standard (lane 1). CaVβ2-core-derived proteins were centrifuged either alone (control) or after incubation with polymerized actin (actin). S, supernatant; P, pellet. Asterisks denote bands of CaVβ2-core-derived proteins that pelleted with actin. Each experiment was repeated three times; full images are shown in Supplementary Fig. 11A. D Fraction of the…

• 

  Fig. 5. CaVβ4 R482X hotspot mutations blunt association with actin. A Superimposition of the actin-bound structures of CaVβ2 (cyan ribbon) and CaVβ4 (purple ribbon). Each CaVβ structure represents the average of the four best-scoring models of the selected docking cluster. For clarity, two actin subunits used as a reference for the alignment are shown in space-filling mode (yellow and pink). B Linear diagram and comparison of the in vitro actin-binding affinities by F-actin cosedimentation assay for Cavβ4-core (lanes 2–5) and Cavβ4 R482X (lanes 6–9). Numbers denote the positions of predicted hotspots substituted by alanine in the CaVβ4 R482X background according to human CaVβ4 (UniProtKB O00305-1). S and P, supernatant and pellet fractions, respectively. Single and double black arrows indicate the amount of corresponding protein that cosedimented with actin. The assays were repeated at least 3 times (Supplementary Fig. 15A). C Size exclusion chromatography profiles for CaVβ4 R482X (carrying no Ala substitutions) (black), CaVβ4 R482X 6Ala (orange), and CaVβ4 R482X 8Ala (green). D SDS-PAGE of F-actin cosedimentation assays for CaVβ4 R482X (lanes 2–5), CaVβ4 R482X 6Ala (lanes 6–9), and…

• 

  Fig. 6. An actin-association-deficient CaVβ2 mutant decreases CaV1.2-mediated ionic current but not gating currents. A Schematic of the membrane-associated CaVβ2 fused to mRFP (CaVβ2-mRFP) and laser scanning confocal images of HEK293 cells expressing either the wild-type full-length CaVβ2 (WT) or the eight hotspot mutant (8Ala). Fluorescence images for the indicated CaVβ2 (a;d, magenta) and the plasma membrane marker CellMaskTM (b;e, green); overlapping pixels appear as white (c;f). Scale bar, 15 µm (for all images). B Colocalization analysis between the indicated CaVβ2 protein and CellMaskTM. Box plot shows the mean (dashed line), median (continuous line), interquartile range (25th–75th percentiles, box edges) and whiskers (1.5 × interquartilee range). Each dot represents the Mander’s overlap coefficient (MOC) for a field of view containing between 150 and 200 cells; p-value, two-tailed t-test. C Western blot (WB) of crude lysates from cells expressing either CaVβ2 WT or CaVβ2 8Ala. Lysates from three separate experiments were probed with anti-CaVβ2 and anti-GAPDH antibodies. D Representative ionic current traces and plots of ionic current versus voltage (I/V) and fraction of activated…

• 

  Fig. 7. The CaVβ2 actin association-deficient mutant reduces the number of functional CaV1.2 channels and does not alter the channel conductance or maximal open probability. A Representative ionic current traces, with red lines delineating the time window used for noise analysis and plotting the relationship between variance and mean macroscopic current \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left(\frac{{\sigma }^{2}}{\left\langle I\right\rangle \left({Vm}-{Vrev}\right)}\right)$$\end{document}σ2IVm−Vrev versus \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left(\frac{\left\langle I\right\rangle }{{Vm}-{Vrev}}\right)$$\end{document}IVm−Vrev obtained at different voltages for two representative cells expressing CaV1.2 complexed with either CaVβ2 WT or CaVβ2 8Ala. The number of functional channels n and the unitary…

• 

  Fig. 8. CaVβ4 R482X hotspots mutant keeps CaV1.2-mediated ionic current intact. A Representative laser scanning confocal images of HEK293 cells expressing either CaVβ4 R482X (a;c) or CaVβ4 R482X 8Ala (d;f) fused to mCherry. Plasma membrane was stained with CellMaskTM (b;e, green). Merged images (c;f). Scale bar, 15 µm (for all images). B Western blot of three separate crude lysates from cells expressing either CaVβ4 R482X (--) or the 8Ala mutant (8Ala) probed with anti-CaVβ4 and anti-GAPDH antibodies. Uncropped images are shown in Supplementary Fig. 18A. C Representative ionic current traces (induced by −35, +20, and +45 mV pulses) from cells co-expressing CaV1.2 with either CaVβ4 R482X or CaVβ4 R482X 8Ala. D Plots of ionic current and fraction of activated channels versus voltage, and total charge movement (Qon) from the indicated channel subunit combinations. Ionic currents were elicited by voltages of −50 to +50 mV in 5 mV increments from a holding potential of −90 mV; data are presented as mean ± SEM. In the box plot, the edges represent interquartile ranges (25th–75th percentiles), the continuous and dashed lines, the median and mean values and whiskers denote 1.5× the interquartile…

• 

  Fig. 9. Model for the clearance of corrupted CaV1.2 channels from the plasma membrane. Conformationally impaired CaV1.2 proteins at the plasma membrane can be generated by diverse cellular stimuli, which change the channel environment leading to local unfolding, or by exocytic insertion of misfolded channels that evade cytosolic quality control (biosynthetic misfolding). CaVβ–actin interaction is mandatory for the endocytic removal of corrupted CaV1.2 channels, which prevents their build-up and maintains stable CaV1.2 currents, but not for the insertion of functionally folded channels. PM, plasma membrane.