Voltage-gating and cytosolic Ca²⁺ activation mechanisms of arabidopsis two-pore channel AtTPC1

Arabidopsis thaliana two-pore channel AtTPC1 is a voltage-gated, Ca²⁺-modulated, nonselective cation channel that is localized in the vacuolar membrane and responsible for generating slow vacuolar (SV) current. Under depolarizing membrane potential, cytosolic Ca²⁺ activates AtTPC1 by binding at the EF-hand domain, whereas luminal Ca²⁺ inhibits the channel by stabilizing the voltage-sensing domain II (VSDII) in the resting state. Here, we present 2.8 to 3.3 Å cryoelectron microscopy (cryo-EM) structures of AtTPC1 in two conformations, one in closed conformation with unbound EF-hand domain and resting VSDII and the other in a partially open conformation with Ca²⁺-bound EF-hand domain and activated VSDII. Structural comparison between the two different conformations allows us to elucidate the structural mechanisms of voltage gating, cytosolic Ca²⁺ activation, and their coupling in AtTPC1. This study also provides structural insight into the general voltage-gating mechanism among voltage-gated ion channels.