EMC chaperone-CaV structure reveals an ion channel assembly intermediate

Abstract

Voltage-gated ion channels (VGICs) comprise multiple structural units, the assembly of which is required for function^1,2. Structural understanding of how VGIC subunits assemble and whether chaperone proteins are required is lacking. High-voltage-activated calcium channels (Ca_Vs)^3,4 are paradigmatic multisubunit VGICs whose function and trafficking are powerfully shaped by interactions between pore-forming Ca_V1 or Ca_V2 Ca_Vα₁ (ref. ³), and the auxiliary Ca_Vβ⁵ and Ca_Vα₂δ subunits^6,7. Here we present cryo-electron microscopy structures of human brain and cardiac Ca_V1.2 bound with Ca_Vβ₃ to a chaperone-the endoplasmic reticulum membrane protein complex (EMC)^8,9-and of the assembled Ca_V1.2-Ca_Vβ₃-Ca_Vα₂δ-1 channel. These structures provide a view of an EMC-client complex and define EMC sites-the transmembrane (TM) and cytoplasmic (Cyto) docks; interaction between these sites and the client channel causes partial extraction of a pore subunit and splays open the Ca_Vα₂δ-interaction site. The structures identify the Ca_Vα₂δ-binding site for gabapentinoid anti-pain and anti-anxiety drugs⁶, show that EMC and Ca_Vα₂δ interactions with the channel are mutually exclusive, and indicate that EMC-to-Ca_Vα₂δ hand-off involves a divalent ion-dependent step and Ca_V1.2 element ordering. Disruption of the EMC-Ca_V complex compromises Ca_V function, suggesting that the EMC functions as a channel holdase that facilitates channel assembly. Together, the structures reveal a Ca_V assembly intermediate and EMC client-binding sites that could have wide-ranging implications for the biogenesis of VGICs and other membrane proteins.

Extended Data Fig. 1 |. EMC:Ca_V1.2(ΔC)/Ca_Vβ₃ and Ca_V1.2(ΔC)/Ca_Vβ₃/Ca_Vα₂δ-1 Cryo-EM analysis.

a-d, Exemplars of purified Ca_V1.2(ΔC)/Ca_Vβ₃: a, SEC (Superose 6 Increase 10/300 GL). b, peak fraction SDS-PAGE. c, electron micrograph (~105,000x magnification), and d, 2D class averages. e–h Exemplars of purified Ca_V1.2(ΔC)/Ca_Vβ₃/Ca_Vα₂δ-1: e, SEC (Superose 6 Increase 10/300 GL). f, peak fraction SDS-PAGE. Magenta bars in ‘a’ and ‘e’ mark peak fraction. g, electron micrographs (~105,000x magnification), and h, 2D class averages. i, Workflow for electron microscopy data processing for the Ca_V1.2(ΔC)/Ca_Vβ₃ and Ca_V1.2(ΔC)/Ca_Vβ₃/Ca_Vα₂δ-1 samples. Initial cryoSPARC-3.2 Ab initio reconstruction identified a population of particles containing the EMC:Ca_V1.2(ΔC)/Ca_Vβ₃ complex in the Ca_V1.2(ΔC)/Ca_Vβ₃ sample and populations of particles containing either the EMC:Ca_V1.2(ΔC)/Ca_Vβ₃ complex or Ca_V1.2(ΔC)/Ca_Vβ₃/Ca_Vα₂δ-1 complex in the Ca_V1.2(ΔC)/Ca_Vβ₃/Ca_Vα₂δ-1 sample. Red arrows indicate the three classes that were re-extracted, subjected to multiple rounds of 3D heterogeneous classification, and exported from cryoSPARC-3.2 for further 3D refinement in RELION-3.1. This resulted in two maps for the EMC:Ca_V1.2(ΔC)/Ca_Vβ₃ complex (ECAB Maps 1 and 2) and one for the Ca_V1.2(ΔC)/Ca_Vβ₃/Ca_Vα₂δ-1 complex (CABAD Map 1). Multibody refinement was performed in RELION-3.1 to improve the features of flexible regions of the three maps. This resulted in the final map for the Ca_V1.2(ΔC)/Ca_Vβ₃/Ca_Vα₂δ-1 complex (CABAD Map 2). ECAB Maps 1–2 with improved flexible features were merged (cross correlation = 0.9836) to obtain the final map for the EMC:Ca_V1.2(ΔC)/Ca_Vβ₃ complex (ECAB Map 3). Red boxes indicate the final maps used for model building. For ‘b-c’, N = 3. For ‘f-g’, N = 2.

Extended Data Fig. 2 |. Cryo-EM maps of EMC:Ca_V1.2(ΔC)/Ca_Vβ₃ and Ca_V1.2(ΔC)/Ca_Vβ₃/Ca_Vα₂δ-1 complexes.

EMC:Ca_V1.2(ΔC)/Ca_Vβ₃ complex a, side views and b, lumenal (left) and cytoplasmic (right) views. Subunits are coloured as: EMC1 (light blue), EMC2 (aquamarine), EMC3 (light magenta), EMC4 (Forest), EMC5 (light pink), EMC6 (white), EMC7 (marine), EMC8 (orange), EMC10 (smudge), Ca_V1.2 (bright orange), and Ca_Vβ₃ (lavender). Ca_V1.2(ΔC)/Ca_Vβ₃/Ca_Vα₂δ-1 c, side views and d, extracellular (left) and cytoplasmic (right) views. Subunits are coloured as: Ca_V1.2 (slate), Ca_Vβ₃ (violet), and Ca_Vα₂δ-1 (greencyan). Detergent micelle is clear.

Extended Data Fig. 3 |. EMC:Ca_V1.2(ΔC)/Ca_Vβ₃ binding sites.

a, View of the TM dock interaction. EMC1 TM1 (slate) and Ca_V1.2 VSD I (yellow orange) interface. Interface buries 1051 Ų. Select elements and residues are indicated. EMC1 residues are in italics. b, LigPLOT diagram of EMC1 TM:Ca_V1.2 VSD I interactions showing ionic interactions (dashed lines) and van der Waals contacts ≤ 5Å. c, Sequence comparison of the indicated VSDI sequences for human Ca_V1.2 (HsCa_V1.2 (109–182)) (Uniprot Q13936–20) with rabbit Ca_V1.1 (OcCa_V1.1 (36–109)) (NCBI: NP_001095190.1), and human L-type (HsCa_V1.1 (36–109), HsCa_V1.3 (111–184), and HsCa_V1.4 (77–150)) (NCBI: NP_000060.2, NP_000711.1, and NP_005174.2) and non-L-Type (HsCa_V2.1 (83–156), HsCa_V2.2 (80–153), and HsCa_V2.3 (74–147)) (NCBI: NP_000059.3, NP_000709.1, and NP_001192222.1) channels. Red asterisks indicate residues involved in the cation-π pocket (120 and 123) and salt bridge (161). Red band highlights the residue that coordinates the Ca²⁺ ion in the Ca_Vα₂δ VWA domain. d, Ca_Vβ₃:EMC8 interaction. Callouts show the details of the indicated parts of the Ca_Vβ₃ NK loop interaction with EMC8. e, LigPLOT diagram of Ca_Vβ₃:EMC8 interactions showing ionic interactions (dashed lines) and van der Waals contacts ≤ 5Å. Ca_Vβ:EMC8 hydrogen bond and salt bridge pairs are: Asp220:His208, Ser222:His208, Arg226:Glu166, Lys234:Thr8, Arg240:Asp56/Tyr87, Ser242:Lys204, and Gln279: His208. f, Sequence conservation for the indicated Ca_Vβ elements from the EMC8 (top) and EMC2 (bottom) interaction sites. OcCa_Vβ₃ (Uniprot P54286; 218–243, 277–282; 300–322); HsCa_Vβ₁ (Uniprot Q02641.3; 270–295, 329–334; 352–374); HsCa_Vβ₂ (Uniprot Q08289; 322–347, 381–386; 404–426); RnCa_Vβ₂ (Uniprot Q8VGC3; 318–343, 377–382; 400–422); HsCa_Vβ₃ (Uniprot P54284; 218–243, 277–282; 300–322); HsCa_Vβ₄ (Uniprot O00305; 260–285, 319–324; 342–364). g, Superposition of rat Ca_Vβ₃ alone (violet, PDB:1VYU, chain B) and Ca_Vβ₃ from the EMC complex. Boundaries of the disordered part of the T218-A243 loop in Ca_Vβ₃, and Q301, and ABP, are indicated, (RMSD_Cα = 1.39Å). h, LigPLOT diagram of Ca_Vβ₃:EMC2 interactions showing van der Waals contacts ≤ 5Å.

Extended Data Fig. 4 |. Ca_V1.2 structural details.

a–d, Structures of the indicated VSDs from the Ca_V1.2(ΔC)/Ca_Vβ₃/Ca_Vα₂δ-1 complex. Gating charge residues, anionic counter charges (An1 and An2) and aromatic site of the charge transfer centre^,, are shown. e, LigPLOT diagram of the Ca_Vα₂δ-1 leucine binding site showing hydrogen bonds and ionic interactions (dashed lines) and van der Waals contacts ≤ 5Å. f, LigPLOT diagram of blocking lipid: Ca_V1.2 showing van der Waals contacts ≤ 5Å. Domain I (yellow orange), Domain II (dark red), and Domain III (green) residues are indicated.

Extended Data Fig. 5 |. Conformational changes between EMC-bound and Ca_Vα₂δ-bound Ca_V1.2(ΔC)/Ca_Vβ₃.

Superposition of Ca_V1.2 from the EMC:Ca_V1.2(ΔC)/Ca_Vβ₃ and Ca_V1.2(ΔC)/Ca_Vβ₃/Ca_Vα₂δ-1 complexes showing a, VSD conformational changes. b, PD conformational changes showing the superposition from ‘a’. Insets show each PD. Elements Ca_V1.2 from the EMC complex are: VSD I/PD I (yellow orange), VSD II/PD II (firebrick), VSD III/PD III (lime), VSD IV/PD IV (marine). Ca_V1.2 (slate) and Ca_Vβ₃ (violet) from Ca_V1.2(ΔC)/Ca_Vβ₃/Ca_Vα₂δ-1 and Ca_Vβ₃ from the EMC (light teal) are semi-transparent. c, Superposition of Ca_Vβ₃ and the Ca_V1.2 AID helix from the EMC:Ca_V1.2(ΔC)/Ca_Vβ₃ and Ca_V1.2(ΔC)/Ca_Vβ₃/Ca_Vα₂δ-1 complexes. Location of EMC8 is indicated by the orange oval. Red arrows in ‘a-c’ indicate conformational changes between Ca_V1.2(ΔC)/Ca_Vβ₃/Ca_Vα₂δ-1 and EMC:Ca_V1.2(ΔC)/Ca_Vβ₃. d, Comparison of IIS0 and surrounding regions in the EMC complex (VSDII, firebrick; AID (yellow orange), and Ca_Vβ₃ (light teal)) their corresponding elements in Ca_V1.2(ΔC)/Ca_Vβ₃/Ca_Vα₂δ-1 (slate). e, Interactions between VSD II:PD III in the Ca_Vβ:AID:VSD II: PD III subcomplex from the EMC:Ca_V1.2(ΔC)/Ca_Vβ₃ structure.

Extended Data Fig. 6 |. Ca_V1.2 pore domain superposition for EMC-bound and Ca_Vα₂δ-bound Ca_V1.2(ΔC)/Ca_Vβ₃.

Pore domains from the EMC-bound complex are: PD I (yellow orange), PD II (firebrick), PD III (lime), and PD IV (marine). Pore domains from Ca_V1.2(ΔC)/Ca_Vβ₃/Ca_Vα₂δ-1 are slate. Calcium ions are from Ca_V1.2(ΔC)/Ca_Vβ₃/Ca_Vα₂δ-1. Selectivity filter (SF), hydrophobic cavity, and inner gate regions and select residues are indicated.

Extended Data Fig. 7 |. Mutually exclusive interactions of the EMC holdase and Ca_Vα₂δ with the core Ca_V1.2/Ca_Vβ₃ complex and ordering of the Ca_V1.2 pore and VSDs by Ca_Vα₂δ.

a, Superposition of Ca_Vα₂δ-1 (semi-transparent, aquamarine) from the Ca_V1.2(ΔC)/Ca_Vβ₃/Ca_Vα₂δ-1 structure with the EMC:Ca_V1.2/Ca_Vβ₃ complex. EMC1 surface is shown. Red oval highlights clash regions. Colours of the EMC:Ca_V1.2/Ca_Vβ₃ complex are as in Fig. 1a. Grey bars denote the membrane. b, Close up view of clash between EMC1 (light blue) and Ca_Vα₂δ-1 (aquamarine). EMC1, EMC3 (magenta), EMC5 (pink), EMC6 (white), VSD I (yellow orange), PD II (firebrick), and PD III (lime) from the EMC:Ca_V1.2/Ca_Vβ₃ complex are shown. Red oval highlights clash regions. Ca_Vα₂δ-1 domains are indicated. Divalent staple is indicated. SF calcium ions are from Ca_V1.2(ΔC)/Ca_Vβ₃/Ca_Vα₂δ-1 and mark the location of the pore in the Ca_Vα₂δ-assembled channel. c, Superposition of VSD I (yellow orange), PD II (firebrick), and PD III (lime) from the EMC complex (semi-transparent) and their corresponding parts from Ca_V1.2(ΔC)/Ca_Vβ₃/Ca_Vα₂δ-1 (slate). Ca_Vα₂δ-1 (aquamarine) is shown as a semi-transparent surface. Calcium ions are from Ca_V1.2(ΔC)/Ca_Vβ₃/Ca_Vα₂δ-1. Left inset shows the coordination of the divalent staple by the VWA domain MIDAS and D151 in Ca_V1.2(ΔC)/Ca_Vβ₃/Ca_Vα₂δ-1. Red distance shows the position of Ca_V1.2 D151 in the EMC complex relative to the calcium ion in the Ca_Vα₂δ complex. Asterisks mark positions where coordinated alanine mutation impair the ability of Ca_Vα₂δ to enhance Ca_V currents and surface expression. Right inset shows the extensive contacts between PD II and PD III loops with Ca_Vα₂δ in the Ca_V1.2(ΔC)/Ca_Vβ₃/Ca_Vα₂δ-1 complex. The PD III loops are disordered in the EMC:Ca_V1.2/Ca_Vβ₃ complex. Ca_Vα₂δ-1 residues are in italics. d, Schematic showing of the conformational changes and interaction sites in the exchange between the EMC:Ca_V/Ca_Vβ holdase complex and assembled Ca_V/Ca_Vβ/Ca_Vα₂δ channel. Black ovals indicate key interaction sites in each complex.

Extended Data Fig. 8 |. Exemplar purification of Ca_V subunit combinations.

a-c Superose 6 Increase 10/300 GL chromatogram and peak fraction SDS-PAGE for: a, Ca_V1.2(ΔC)/Ca_Vβ₃ and b, Ca_V1.2(ΔC). c, Relative detection by mass spectrometry from ‘a’ and ‘b’ of EMC proteins with respect to Ca_V1.2(ΔC) across 3 replicates. d, Superose 6 Increase 10/300 GL chromatogram and peak fraction SDS-PAGE for Ca_Vβ₃. Ca_Vβ₃-NK and Ca_Vβ₃-SH3 are Ca_Vβ₃ proteolytic fragments. e, Absolute detection of EMC proteins and Ca_Vβ₃ by mass spectrometry following expression and purification of Ca_Vβ₃. f, Superose 6 Increase 10/300 GL chromatogram and peak fraction SDS-PAGE for Ca_V1.2/Ca_Vβ₃. Magenta bars in ‘a’ ‘b’, ‘d’, and ‘f’ mark peak fraction. g, Absolute detection of Ca_V1.2, Ca_V1.2(ΔC), Ca_Vβ₃, and EMC proteins from ‘a’ and ‘f’. Error bars are calculated as SEM. ND denotes not detected.

Extended Data Fig. 9 |. Functional and biochemical characterization of Ca_V mutants.

a, Voltage dependent activation (left) and I-V relationships (right) for the indicated channels. Parentheses indicate ‘n’ independent cells examined over >2 independent experiments. b, Superose 6 Increase 10/300 GL chromatogram and peak fraction SDS-PAGE for Ca_V1.2(ΔC)/Ca_Vβ₃(11A). Magenta bar marks peak fraction. c, Relative detection by mass spectrometry from ‘b’ of EMC proteins with respect to Ca_V1.2(ΔC) across 3 replicates. Data for Ca_V1.2(ΔC)/Ca_Vβ₃ are from Extended Data Fig. 8c. ‘ND’ indicates ‘not detected. Data in ‘a’ and ‘c’ are presented as mean ± SEM.

Fig. 1 |. The structure of the EMC–Ca_V1.2(ΔC)–Ca_Vβ₃ complex.

a, Cartoon structure side views of the EMC–Ca_V1.2(ΔC)–Ca_Vβ₃ complex. EMC1, EMC2, EMC3, EMC4, EMC5, EMC6, EMC7, EMC8, EMC10, CaV1.2 and Ca_Vβ₃ are shown. EMC subunits are shown with semi-transparent surfaces. The arrows indicate dimensions. b, The location of the TM dock and Cyto dock EMC client-binding sites. Ca_V1.2 components interacting with the EMC are shown as cylinders. The AID helix is also shown. The grey bars in a and b denote the membrane. c, Lumenal view of the EMC–Ca_V1.2(ΔC)–Ca_Vβ₃ complex transmembrane elements. EMC components are shown as cylinders. Ca_V1.2(ΔC) domains are coloured as domain I (yellow orange), domain II (dark red), domain III (lime) and domain IV (deep blue). VSD and PD elements are labelled. The red oval in b and c shows the TM dock site. The pale purple circle shows the site of interaction of the EMC1 brace/crossbar helix with Ca_V1.2 VSD I and PD II (outlined). d, Side view of the TM dock–VSD I interaction. Insets: details of interactions in the of the EMC1 brace/crossbar helix with Ca_V1.2 VSD I and PD II. f, Details of the Cyto dock. Ca_Vβ and AID helix are shown as cylinders. EMC2, EMC3, EMC4, EMC5, EMC6 and EMC8 are shown as surfaces. Insets: details of the EMC2 and EMC8 sites.

Fig. 2 |. The structure of the Ca_V1.2(ΔC)–Ca_Vβ₃–Ca_Vα₂δ-1 channel complex.

a, Side views of Ca_V1.2(ΔC)–Ca_Vβ₃–Ca_Vα₂δ-1. Leucine and blocking lipid are shown as space filling. The grey bars denote the membrane. b, Details of the Ca_Vα₂δ-1 Cache1 ligand-binding site. Leucine (purple) and contacting side chains from Ca_Vα₂δ1 (green cyan) are shown as sticks. c, Details of the blocking-lipid-binding site. Ca_V1.2 PDs are shown as cylinders and coloured as follows: PD I (yellow orange), PD II (dark red), PD III (lime) and PD IV (deep blue). Lipid-contact residues are shown as sticks. Blocking lipid is shown as space filling (left) and sticks (right). d, Interaction between the blocking lipid and SF ions. Hallmark SF calcium-binding residues are shown as sticks (purple). SF calcium ions are indicated.

Fig. 3 |. EMC interactions remodel Ca_V structure and extract PD III.

a, Superposition of Ca_V1.2–Ca_Vβ₃ from the EMC and Ca_V1.2(ΔC)–Ca_Vβ₃–Ca_Vα₂δ-1 complexes. Ca_V1.2 elements from the EMC complex are coloured as follows: VSD I/PD I (yellow orange), VSD II/PD II (dark red), VSD III/PD III (lime) and VSD IV/PD IV (deep blue). Ca_V1.2 (purple) and Ca_Vβ₃ (magenta) from Ca_V1.2(ΔC)–Ca_Vβ₃–Ca_Vα₂δ and Ca_Vβ₃ from the EMC (light teal) are semi-transparent. The red arrows indicate conformational changes between Ca_V1.2(ΔC)–Ca_Vβ₃–Ca_Vα₂δ-1 and EMC–Ca_V1.2(ΔC)–Ca_Vβ. Coloured ovals highlight key domains reshaped by the EMC. b, Superposition of the S4 and S4–S5 linker–PD III elements from the EMC–Ca_V1.2(ΔC)–Ca_Vβ₃ complex (lime) and Ca_V1.2(ΔC)–Ca_Vβ3–Ca_Vα₂δ-1 (purple). The Cα–Cα distance for Val1019 is indicated. c, Interactions within the Ca_Vβ–AID–VSD II–PD III subcomplex in EMC–Ca_V1.2(ΔC)–Ca_Vβ₃. The grey bars in a and c denote the membrane.

Fig. 4 |. EMC association causes Ca_V1.2 pore structural changes.

a, Ca_V1.2 pore profile comparison for EMC–Ca_V1.2(ΔC)–Ca_Vβ₃ (orange) and Ca_V1.2(ΔC)–Ca_Vβ₃–Ca_Vα₂δ-1 (blue), calculated using HOLE. EMC: SF = 1.78 Å, inner gate = 1.98 Å; Ca_V1.2: SF = 0.65 Å, inner gate = 0.98 Å. b, Side views of EMC–Ca_V1.2(ΔC)–Ca_Vβ₃ (left) and Ca_V1.2(ΔC)–Ca_Vβ₃–Ca_Vα₂δ (right) pore profiles calculated using MOLE. The SF, central cavity and hydrophobic gate regions are indicated. SF and intracellular gate residues are shown. c, Comparison of the Ca_V1.2 SF filter regions in the EMC–Ca_V1.2(ΔC)–Ca_Vβ₃ pore (PD I, PD II, PD III and PD IV) and Ca_V1.2(ΔC)–Ca_Vβ₃–Ca_Vα₂ δ-1 (semi-transparent, purple). Ca²⁺−1 from Ca_V1.2(ΔC)–Ca_Vβ₃–Ca_Vα₂δ-1 is shown as a white sphere. d,e, View of the Ca_V1.2 intracellular gate from EMC–Ca_V1.2(ΔC)–Ca_Vβ₃ (d) and Ca_V1.2(ΔC)–Ca_Vβ₃–Ca_Vα₂δ-1 (e). GDN is shown as sticks. PDs are coloured as follows: PD I (yellow orange), PD II (dark red), PD III (lime) and PD IV (deep blue). f,g, Side views of the EMC–Ca_V1.2(ΔC)–Ca_Vβ₃ pore showing global (f) and detailed (g) views. SF glutamates in e and GDN-contacting residues in f are shown as sticks. PD elements are labelled.

Fig. 5 |. Disruption of EMC–channel interactions affects Ca_V function.

a,b, Cartoon diagrams showing the location of TM dock 3A mutants (a), and Cyto dock 5A (magenta), Δ220–240 (red) and 11A (sticks) mutants (b). c, Current densities at +20 mV for the indicated channels and HEK293FT cell lines. Data are mean ± s.e.m. The values in parentheses indicate n independent cells examined over ≥2 independent experiments. Statistical significance was assessed using two-sided unpaired t-tests between Ca_v1.2(ΔC)–Ca_vβ₃ and mutants. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P = 0.01–0.05; NS, P > 0.05. d, Exemplar Ca²⁺ currents for HEK293 cells expressing the indicated channels in the presence of Ca_Vα₂δ-1. Insets: the stimulation protocol and currents at +20 mV. e, Cartoon of the EMC–Ca_V1.2(ΔC)–Ca_Vβ₃ complex showing the following selected elements: EMC1, EMC2, EMC3, EMC5, EMC6, EMC8, Ca_V1.2 VSD I and PD II, and Ca_Vβ₃. The arrows indicate the TM dock and Cyto dock. Surfaces are shown for EMC5 and EMC6. f, Exemplar Ca_V1.2(ΔC)–Ca_Vβ₃–Ca_Vα₂δ-1 Ca²⁺ currents for HEK293FT and HEK293FT EMC5-knockout and EMC6-knockout cells. Insets: the protocol and currents at +20 mV.