On the structure and mechanism of two-pore channels

Abstract

In eukaryotes, two-pore channels (TPC1-3) comprise a family of ion channels that regulate the conductance of Na⁺ and Ca²⁺ ions across cellular membranes. TPC1-3 form endolysosomal channels, but TPC3 can also function in the plasma membrane. TPC1/3 are voltage-gated channels, but TPC2 opens in response to binding endolysosome-specific lipid phosphatidylinositol-3,5-diphosphate (PI(3,5)P₂ ). Filoviruses, such as Ebola, exploit TPC-mediated ion release as a means of escape from the endolysosome during infection. Antagonists that block TPC1/2 channel conductance abrogate filoviral infections. TPC1/2 form complexes with the mechanistic target of rapamycin complex 1 (mTORC1) at the endolysosomal surface that couple cellular metabolic state and cytosolic nutrient concentrations to the control of membrane potential and pH. We determined the X-ray structure of TPC1 from Arabidopsis thaliana (AtTPC1) to 2.87Å resolution-one of the two first reports of a TPC channel structure. Here, we summarize these findings and the implications that the structure may have for understanding endolysosomal control mechanisms and their role in human health.

Keywords: ion channels; lysosome; membrane protein; structure; transport.

Figure 1. Overview of the AtTPC1 Structure

Views down (top) long and short channel axes, and (bottom) top-down through the central channel of AtTPC1 (PDB 5DQQ; ref. [20]). Boundaries for endolysosome/vacuole (E), membrane (M), and cytoplasm (C) are shown. Ca²⁺-ions, including the sites for luminal inhibition (Ca_i²⁺) and cytoplasmic activation (Ca_a²⁺), are shown as green spheres.

Figure 2. TPC Activation Mechanisms

A) Summary of luminal/extracellular and cytoplasmic agents and processes that modulate TPC channel opening. B) Surface renderings of a (left) symmetrical tetrameric channel (Na_vAb; PDB 3RVY; ref. [56]) versus (right) the asymmetric tandem TPC1 dimer (AtTPC1; PDB 5DQQ; ref. [20]). Outward arrows highlight potential differential contributions from the sensory and pore domains to channel activation.

Figure 3. Mechanism of Voltage-sensing

A) Comparison of (left) resting-state AtTPC1 (PDB 5DQQ; ref. [20]) and (right) active-state Na_vAb (PDB 3RVY; ref. [56]) structures. View through the (Top) membrane, (Middle) luminal/extracellular, and (Bottom) cytoplasmic faces of the VSD. The charge transfer center (CT) location (dash line) in S2 and gating charges (R1-R4) in S4 are shown. B) A hypothetical pathway for gating charge movement in the VSD from the cytoplasmic leaflet in the resting-state through the membrane to the luminal leaflet in the active-state.

Figure 4. TPC Pharmacology

A) Predicted binding site for DHP (red) Ca_v blockers based on sequence homology with Ca_vs overlaid onto the AtTPC1 structure bound to non-DHP molecule trans-NED-19 (PDB 5DQQ; ref. [20]). B) Molecular structure of representative pharmacophores that block TPC channels.

Figure 5. Model for TPC1 Activation and Regulation

Hypothetical model for TPC1 activation by membrane potential (ΔΨ) and cytosolic Ca²⁺ ions (Ca_a²⁺), and inhibition by luminal Ca²⁺ ions (Ca_i²⁺) and phosphorylation (*) overlaid onto the AtTPC1 structure (PDB 5DQQ; ref. [20]). A possible mechanism for conformational coupling between voltage-sensor VSD2 and EF-hand domain EF2 during channel activation is shown. Hypothetical permeating ions are shown as spheres (magenta). The transparency of VSD1 is increased for clarity.