Endosomal TPC1-mediated Ca²⁺ nanodomains regulate transferrin receptor trafficking and iron homeostasis

Two-pore channel 1 (TPC1) is an endosomal Na⁺/Ca²⁺-selective channel implicated in membrane trafficking, endosome tubulation, and excitability, but how TPC1 regulates membrane trafficking is unknown. Using TPC1-null human cells, we demonstrate that TPC1 drives transferrin receptor (TfR) trafficking and recycling via Ca²⁺, and not Na⁺ fluxes or endosomal pH changes, since channel-targeted Ca²⁺-buffers inhibited trafficking, whereas a Na⁺-deficient Ca²⁺-permeable TPC1 mutant fully supported trafficking. TPC1 was unique since other Ca²⁺ sources did not support TfR trafficking. TPC1 activity depended on the lipid PI(3,5)P₂, since trafficking was impaired by a lipid-insensitive TPC1 or inhibitors of lipid synthesis. Finally, a corollary of this reduced TfR trafficking is an iron-deficiency and storage phenotype in TPC1-deficient HeLa cells and mice. Our findings highlight endosomes as unique Ca²⁺ stores mobilized by a phosphoinositide-induced TPC1 channel that generates local Ca²⁺ nanodomains crucial for maintaining TfR trafficking and consequent iron homeostasis.