Structural basis for activation of voltage sensor domains in an ion channel TPC1

Abstract

Voltage-sensing domains (VSDs) couple changes in transmembrane electrical potential to conformational changes that regulate ion conductance through a central channel. Positively charged amino acids inside each sensor cooperatively respond to changes in voltage. Our previous structure of a TPC1 channel captured an example of a resting-state VSD in an intact ion channel. To generate an activated-state VSD in the same channel we removed the luminal inhibitory Ca²⁺-binding site (Ca_i²⁺), which shifts voltage-dependent opening to more negative voltage and activation at 0 mV. Cryo-EM reveals two coexisting structures of the VSD, an intermediate state 1 that partially closes access to the cytoplasmic side but remains occluded on the luminal side and an intermediate activated state 2 in which the cytoplasmic solvent access to the gating charges closes, while luminal access partially opens. Activation can be thought of as moving a hydrophobic insulating region of the VSD from the external side to an alternate grouping on the internal side. This effectively moves the gating charges from the inside potential to that of the outside. Activation also requires binding of Ca²⁺ to a cytoplasmic site (Ca_a²⁺). An X-ray structure with Ca_a²⁺ removed and a near-atomic resolution cryo-EM structure with Ca_i²⁺ removed define how dramatic conformational changes in the cytoplasmic domains may communicate with the VSD during activation. Together four structures provide a basis for understanding the voltage-dependent transition from resting to activated state, the tuning of VSD by thermodynamic stability, and this channel's requirement of cytoplasmic Ca²⁺ ions for activation.

Keywords: X-ray crystallography; cryo-EM; ion channel; two pore channnel; voltage sensor.

Fig. 1.

Cryo-EM Structure of the AtTPC1_DDE–saposin A–Fab complex. (A) Side (Left) and top-down (Right) views of cryo-EM density. The composite map is colored to highlight high-resolution features of TPC1 (blue, EMDB entry no. 8957), VSD2 in state 1 (cyan, EMDB entry no. 8958) and state 2 (pink, EMDB entry no. 8960), and the Fab variable domains (orange, EMDB entry no. 8956). Unsharpened density is shown at low contour (transparent gray). Membrane boundaries (black bars) defined by the nanodisc are marked. (B) Overlay of AtTPC1_WT (red, PDB ID code 5DQQ), state 1 (blue), and state 2 (pink). Ca_i²⁺ and Ca_a²⁺ denote sites of luminal inhibition and cytoplasmic activation by Ca²⁺ ions. Dash lines around Ca_a²⁺ indicate hypothetical position in cryo-EM structure. Ca²⁺ ions shown as colored balls.

Fig. 2.

Three states of VSD2. View of VSD2 in the membrane plane from the center of the channel looking outward that illustrates the rotation and twisting of VSD2 helices S7, S8, S9, and S10. Connections to the pore domains are omitted for clarity. S10 is highlighted in each state with a different color than the other helices. Gating charges R1–R3 (R537, R540, and R543) and CT residue Y475 are shown. (Left) Resting-state AtTPC1_WT (red, PDB ID code 5DQQ), (Center) AtTPC1_DDE state 1 (blue), and (Right) AtTPC1_DDE state 2 (pink).

Fig. 3.

Activation of the voltage sensor. Side views of a common slice perpendicular to the membrane surface of VSD2 in AtTPC1_WT resting state (red, PDB ID code 5DQQ) and AtTPC1_DDE state 1 (blue) and state 2 (pink). S10 shown with gating charges R1–R3. The CT Cα position (black ball) is marked. Based on a structural alignment with respect to the pore helices S6–S7 of each structure.

Fig. 4.

Ion permeation pathway. (A and B) Orthogonal side views through pore helices (A) S5–S6 (pore 1) and (B) S11–S12 (pore 2) of the channel homodimer overlaid with high-resolution cryo-EM density (gray mesh). (C) HOLE plot of pore radii along central channel coordinate of AtTPC1_DDE (red) and AtTPC1_WT (blue). (D) Top-down view through central pore. Gate residues Y305, L673, and F676 are shown. (E–G) Side views through the selectivity filter in (E) pore 1, (F) pore 2, and (G) an overlay of pore 2 of AtTPC1_DDE (blue) and AtTPC1_WT (pink). (Upper) E605 and D606 and (Lower) S265, T263, T264, V628, M629, and N631 selectivity filter residues are shown. Density for lipids (SI Appendix, Fig. S7) and ions is omitted for clarity.

Fig. 5.

Dynamics of cytoplasmic domains. (A) Overlay of (Left) AtTPC1_WT and (Right) AtTPC1_DA crystal structures colored by B-factor value (100–450 Ų) (see Methods and SI Appendix, Supplementary Discussion). (B) Side views of AtTPC1_WT (red) and AtTPC1_DDE (blue). (C) Views from (Top Left) side and (Top Right) bottom of EF3 for overlaid AtTPC1_DDE (blue) and AtTPC1_WT crystal structures (red, PDB ID code 5DQQ). CTD interactions are omitted for clarity. Ca²⁺-ion binding sites in (Bottom Right) EF1–EF2 and (Bottom Left) EF3–EF4. (D) View of EF3-CTD and EF3-EF4 interactions and connection to VSD2. (E) Structure of the CTD in AtTPC1_DDE and a possible coupling pathway to the pore gate and Ca_a²⁺. Dashed lines indicate the hypothetical Ca_a²⁺ ion position.

Fig. 6.

Mechanism of TPC1 channel activation. Schematic summarizing the conformations of VSD2, EF3, CTD, and selectivity filter (SF) observed in crystal and cryo-EM structures under high and low effective luminal Ca²⁺-ion concentrations. A potential model for coupling between luminal inhibition Ca_i²⁺, cytoplasmic activation Ca_a²⁺, voltage sensing, and channel gating is shown. Helices and ions that move between the AtTPC1_WT resting state (red), AtTPC1_DDE state 1 (cyan), AtTPC1_DDE state 2 (pink), and hypothetical activated-open state (purple) are colored. Ca²⁺ ions are shown as green balls. Gating charges that change position with respect to the CT (dashed line) between states are colored red.