Structure Inhibition and Regulation of a Two-Pore Channel TPC1

Abstract

Membrane transport serves vital functions in the cell, providing nutrients for growth, relaying electrical signals, evading pathogens, and maintaining homeostasis. Voltage- and ligand-gated ion channels propagate cellular electrical signals by coupling changes in membrane potential and the binding of molecules to channel opening and selective passage of ions through the membrane barrier. Two-pore channels (TPCs) are intracellular ion channels that integrate changes in membrane potential, second messengers, and phosphorylation to control endolysosomal trafficking, autophagy, cellular ion and amino acid homeostasis, and ultra-long action potential-like signals. They broadly impact human diseases related to trafficking including filoviral infections, Parkinson's disease, obesity, fatty liver disease, and Alzheimer's disease. The response of TPCs to multiple cellular inputs suggests a multi-state gating mechanism. Nevertheless, the mechanisms that govern cycles of activation and deactivation or ‘gating’ in the channel remain poorly understood. To understand the bases for ion permeation, channel activation, the location of voltage-sensing domains and regulatory ion-binding sites, and phosphoregulation, we determined the crystal structure of TPC1 from Arabidopsis thaliana to 2.87A resolution. This reveals for the first time how TPC channels assemble as ‘quasi-tetramers’ from two non-equivalent tandem pore-forming subunits. We determined sites of phosphorylation in the N-terminal and C-terminal domains that are positioned to allosterically modulate channel activation by cytoplasmic calcium. One of the two voltage sensing domains (VSD2) encodes voltage sensitivity and inhibition by luminal calcium locks VSD2 in a ‘resting’ conformation, distinct from the activated VSDs observed in structures of other voltage-gated ion channels. The structure shows how potent pharmacophore trans-Ned-19 allosterically acts to inhibit channel opening. In animals, trans-Ned-19 prevents infection by Ebola virus and Filoviruses by blocking fusion of the viral and endolysosomal membranes, thereby preventing delivery of their RNA into the host cytoplasm. The structure of TPC1 paves the way for understanding the complex function of these channels and may aid the development of antiviral compounds.