Investigating The Localisation and Function of TRPM2 in Endomembranes

Abstract

TRPM2 is a non-specific cation permeable channel that regulates Ca2+ linked to stress regulated pathogenesis of neurodegenerative disease. The endomembrane localisation and function of TRPM2 are unclear and controversial. Knockdown-validated immunofluorescence in HeLa cells, showed TRPM2 homogenously distributed in cytoplasmic punctae. These punctae co-localised to retromer coats, involved in retrieving receptors such as CI-MPR from endolysosomes to Golgi. Retromer knockdown disturbed this colocalisation. Importantly immunofluorescence, biotinylation experiments and a clathrin inhibitor (Pitstop-2) studies failed to reveal plasma membrane pools. TRPM2-siRNA induced accumulation of CIMPR in swollen late-endosomes. TRPM2 immunosedimented 75% of endogenous core retromer component, VPS35 and direct interaction was confirmed using bacterially expressed TRPM2 C- and N-termini. Consistent with molecular docking studies, TRPM2 did not bind cargo-binding VPS26, which agrees with the absence of retromer binding motifs. Thus, TPRM2 likely forms a structural element of retromer functioning to regulate retrograde trafficking to the Golgi. Additional immunofluorescence localisations and live imaging showed retromer-associated punctae moving along the ER-reticulum. Subcellular fractionation confirmed ER localisation and calcium responsiveness to TRPM2 agonist ADPR. The ER pool was resistant to prolonged cycloheximide while combined mass spectroscopy, motif searches and bacterial proteins interaction studies confirmed TRPM2 binding to the COPI complex, involved in Golgi to ER retrieval. Upon TRPM2 knockdown the membrane protein VSVG remained trapped in the ER bound to chaperones, an effect that was countered by the expression of siRNA-resistant WT but not a calcium TRPM2 mutant (linked to Parkinson's disease). Our studies suggest that TRPM2 is a hub for calcium-mediated stress regulation of membrane traffic where subdomains of the ER containing TRPM2 associate directly with retromer and regulate endosomal tubule formation. In concert, ER-located TRPM2 regulates ER-chaperone function and protein export via as yet poorly defined calcium-based mechanisms. They open v up new understanding of neurodegeneration mechanisms where TRPM2 is of known importance.