Abstract
Mammalian two-pore channels (TPCs) regulate the physiological functions of the endolysosome. Here we present cryo-EM structures of human TPC2 (HsTPC2), a phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2)-activated, Na+ selective channel, in the ligand-bound and apo states. The apo structure captures the closed conformation, while the ligand-bound form features the channel in both open and closed conformations. Combined with functional analysis, these structures provide insights into the mechanism of PI(3,5)P2-regulated gating of TPC2, which is distinct from that of TPC1. Specifically, the endolysosome-specific PI(3,5)P2 binds at the first 6-TM and activates the channel - independently of the membrane potential - by inducing a structural change at the pore-lining inner helix (IS6), which forms a continuous helix in the open state but breaks into two segments at Gly317 in the closed state. Additionally, structural comparison to the voltage-dependent TPC1 structure allowed us to identify Ile551 as being responsible for the loss of voltage dependence in TPC2.
Highlights
Two-pore channels (TPCs) belong to the voltage-gated ion channel superfamily and function as a homodimer, with each subunit containing two homologous Shaker-like 6-TM repeats
The single particles of HsTPC2 in the presence of PI(3,5)P2 can be partitioned into two classes of particles, and 3D refinement yielded HsTPC2 structures in the open and closed conformations at 3.7 Aand 3.4 Aresolution, respectively
We reveal the structural mechanism of how PI(3,5)P2 activates human TPC2
Summary
Two-pore channels (TPCs) belong to the voltage-gated ion channel superfamily and function as a homodimer, with each subunit containing two homologous Shaker-like 6-TM repeats. TPC functions have been shown to be associated with a plethora of physiological processes, including mTOR-dependent nutrient sensing (Cang et al, 2013), Ebola infection (Sakurai et al, 2015) and autophagy (Fernandez et al, 2016; Pereira et al, 2011). Despite their physiological importance, the biophysical properties of mammalian TPC channels are still under debate. The plasma membrane-localized PI(4,5)P2 cannot activate the TPC channels; the lipid isoform-dependent gating property defines the compartment
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