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

The X-ray crystal structure of a two-pore channel from Arabidopsis thaliana is reported, revealing the mechanisms of ion permeation, inhibition channel activation, and location of regulatory sites and voltage-sensing domains. Characterization of the two-pore channel AtTPC1 The X-ray crystal structure of the two-pore channel AtTPC1 from Arabidopsis thaliana reveals the structure and mechanism of voltage gating of a type of cation-selective ion channel ubiquitously expressed in the organelles of animal and plant cells. AtTPC1 is activated by both voltage and cytosolic Ca 2+ , and voltage activation can be inhibited by luminal Ca 2+ . Youxing Jiang and colleagues determined the crystal structure of AtTPC1 to 3.3 Å resolution and find that, as predicted, two AtTPC1 subunits make up the functional channel. Alexander Kintzer and Robert Stroud report the AtTPC1 crystal structure at 2.87 Å resolution, revealing the mechanisms of ion permeation, channel activation, and location of regulatory sites and voltage-sensing domains. 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 Å 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 phosphorylation 3 , 4 in the amino-terminal and carboxy-terminal domains that are positioned to allosterically modulate cytoplasmic Ca 2+ activation. One of the two voltage-sensing domains (VSD2) encodes voltage sensitivity and inhibition by luminal Ca 2+ 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