BPS2026 - Structural and functional characterization of TPC2 modulation and a novel gain-of-function mutation in TPC1

Two pore channels (TPCs) are evolutionarily conserved, non-selective cation channels expressed throughout the endo-lysosomal network. They regulate key cellular processes, such as, autophagy, lysosomal exocytosis, phagocytosis, metabolism, and signal transduction. In humans, two isoforms have been identified, TPC1 and TPC2, both of which are activated by the endo-lysosomal phosphoinositide PI(3,5)P 2 and by NAADP (nicotinic acid adenine dinucleotide phosphate). TPC1 is additionally activated by the phosphoinositide PI(3)P. Pharmacological modulation of TPC2 activity holds the potential to treat on the one hand, neurodegenerative (lysosomal storage) diseases (TPC2 activation), while on the other hand, by antagonizing TPC2 activity to provide new approaches to anticancer and antiviral treatments. While the impact of TPC2 in pathophysiology has been studied by now to a fairly detailed level, roles of TPC1 in disease remain less well studied. We identified the TPC2 binding sites of both agonists (TPC2-A1-N and its analog SGA-111, mimicking endogenous NAADP activation) and the antagonist SG-094, a tetrandrine derivative, discovered by cryo-EM and validated via patch-clamp electrophysiology and Ca 2+ -imaging. We also discovered a novel, disease causing TPCN1 mutation, causing an isoleucine-to-threonine substitution (I→T). Overexpressed hTPC1 I/T in HEK cells showed increased Na + currents upon PI(3)P activation, representing the first gain-of-function mutation of human TPC1. Together, these findings advance the structural and functional understanding of TPCs, establish a disease link for TPC1, and provide a framework for structure-guided development of selective TPC modulators with potential therapeutic applications.