Structure, inhibition and regulation of two-pore channel TPC1 from Arabidopsis thaliana

Two-pore channels (TPCs) comprise a subfamily (TPC1-3) of eukaryotic voltage-and ligand-gated cation channels(1,2) with two non-equivalent tandem pore-forming subunits that dimerize to form quasi-tetramers. Found in vacuolar(3) or endolysosomal(4) membranes, they regulate the conductance of sodium(5) and calcium(3,6) ions, intravesicular pH(5), trafficking(7) and excitability(8,9). TPCs are activated by a decrease in transmembrane potential(1,3,9,10) and an increase in cytosolic calcium concentrations(1,10), are inhibited by low luminal pH and calcium(11), and are regulated by phosphorylation(12,13). Here we report the crystal structure of TPC1 from Arabidopsis thaliana at 2.87 angstrom resolution as a basis for understanding ion permeation(3,4,10), channel activation(1,5,10), the location of voltage-sensing domains(1,9,10) and regulatory ion-binding sites(11,14). We determined sites of phosphorylation3,4 in the amino-terminal and carboxy-terminal domains that are positioned to allosterically modulate cytoplasmic Ca2+ activation. One of the two voltage-sensing domains (VSD2) encodes voltage sensitivity and inhibition by luminal Ca2+ and adopts a conformation distinct from the activated state observed in structures of other voltage-gated ion channels(15,16). The structure shows that potent pharmacophore trans-Ned-19 (ref. 17) acts allosterically by clamping the pore domains to VSD2. In animals, Ned-19 prevents infection by Ebola virus and other filoviruses, presumably by altering their fusion with the endolysosome and delivery of their contents into the cytoplasm(7).