Structure, gating, and pharmacology of human CaV3.3 channel

Fig. 1. Architecture of the CaV3.3 channel. a The activation (right) and inactivation (left) properties of the truncated CaV3.3EM and full-length wild type (WT) CaV3.3WT. Normalized conductance (G/Gmax) for the activation properties is applied by a held at −100 mV and then a series of 200 ms voltage steps from −100 mV to +20 mV in 10 mV increments. The ion current (I/Imax) for inactivation (left) properties are assessed with a 3.6 s holding voltages ranging from −110 mV to 0 mV (10 mV increments) followed by a 150 ms test pulse at −20 mV. N cells were analyzed (WT, n = 7; EM, n = 8). Data are presented as mean values +/– SEM. Source data are provided as a Source Data file. b The overall structure of the CaV3.3apo. The domains of CaV3.3 are colored as DI in purple, DII in green, DIII in blue, and DIV in salmon. A cation ion in the selectivity filter vestibule was presented as a gray sphere. c The ion permeation path in the pore domain. The selectivity filter and S5-S6 helices are shown in cartoon and viewed in parallel to the membrane plane. The ion-conducting pathway was calculated by the program HOLE and illustrated by gray dots in the left panel. The selectivity filter ring of four…

Fig. 2. Positive charged S6Cyto region is critical for the channel gating. a The pore domain segments in the DI/DIII (left panel) and DII/DIV (right panel) of the CaV3.3 (colored), superimposed with the corresponding region of CaV3.1 structure (gray). b Zoom-in view highlights the structural comparison S6II helix between CaV3.3apo and CaV3.1 (PDB ID: 6KZO) (gray). The shifts of the side chain of residue on S6II helix between CaV3.3apo and CaV3.1 undergoing axial rotation are indicated by red arrows. c Sequence alignment of S6III helix of all human CaV members, numbered according to full-length subunits. The secondary structure of CaV2.2 and CaV3.3 marked above and below the sequence alignment, respectively. The dashes represent gaps. The positively charged residues on S6III are shaded in blue. Residues involved in CaV3.33G are marked by red triangle. d The pore domain segments in the DI/DIII of the CaV3.3apo (colorful) and CaV2.2 (PDB ID: 7VFS) (yellow) are superimposed. The S6Cyto and S6TM regions are indicated. The positively charged residues on the S6Cyto region are shown as blue spheres. e. Normalized conductance-voltage (G/V) and current-voltage(I/V) relationship for the CaV3.3WT…

Fig. 3. Structure basis for blockade of CaV3.3 by mibefradil. a Chemical structures of MIB. b The cryo-EM density shown in blue mesh for MIB in sticks. c The overall structure of the CaV3.3MIB complex. The domains of CaV3.3MIB are colored as DI in purple, DII in green, DIII in blue, and DIV in salmon. The MIB in the pore domain is presented as pink spheres. d, e Detailed binding sites for MIB showing interactions between MIB and CaV3.3. The side chains of key residues are displayed on sticks and the hydrophobic side chains are overlaid with transparent surfaces. Black dashed lines indicated potential hydrogen bonds. Phospholipids entering through fenestrations are shown in gray. f Comparison of the MIB binding sites of CaV3.3 (pink) with CaV1.1 (PDB ID: 5GJV) (neon green).

Fig. 4. Structure basis for blockade of CaV3.3 by otilonium bromids. a Chemical structures of OB. b The cryo-EM density shown in blue mesh for OB in sticks. c Hydrophobic residues which surrounding the DII-DIII fenestration site and penetrated by OB are shown in sticks and overlaid with transparent surfaces viewed in facing the DII-DIII fenestration site. OB is shown as brown sticks. d Detailed binding sites of OB in the pore domain. The side chains of key residues are displayed in sticks and overlaid with transparent surfaces. Black dashed lines indicated potential hydrogen bonds. Phospholipids entering through other fenestrations are shown as gray sticks. e Binding site of OB in CaV3.3OB with CaV1.1 (PDB ID: 5GJV) (neon green) structures. The domains of CaV3.3OB are colored as DI in purple, DII in green, DIII in blue, and DIV in salmon. Extracellular view sectioned below the selectivity filter indicates OB penetrates through the DII-DIII fenestration site of CaV3.3OB (left panel) and CaV1.1(middle panel). Comparison of key residues in the pore domain between CaV3.3OB and CaV1.1 is shown in the right panel.

Fig. 5. Structure basis for blockade of CaV3.3 by pimozide. a Chemical structures of pimozide. b The cryo-EM density shown in blue mesh for PMZ in sticks. c Cross-sectional view, showing the open selectivity filter, fenestrations, pore domain where PMZ (blue spheres) located and intracellular gate. Phospholipids entering through other fenestrations are displayed as white sticks. d Detailed binding sites for pimozide. The side chains of key residues are displayed in sticks and the hydrophobic side chains are overlaid with transparent surfaces. Black dashed lines indicated potential hydrogen bonds. e Comparison of CaV3.3PMZ with CaV3.3apo (wheat). The shifts of the backbone of S6III helix and side chain of L1423 and F1426 and phosphate group of the phospholipid are indicated by red arrows.