Functional characterization of the transient receptor potential melastatin 2 (TRPM2) cation channel from Nematostella vectensis reconstituted into lipid bilayer

Transient receptor potential melastatin 2 (TRPM2) cation channel activity is required for insulin secretion, immune cell activation and body heat control. Channel activation upon oxidative stress is involved in the pathology of stroke and neurodegenerative disorders. Cytosolic Ca 2+ , ADP-ribose (ADPR) and phosphatidylinositol-4,5-bisphosphate (PIP 2 ) are the obligate activators of the channel. Several TRPM2 cryo-EM structures have been resolved to date, yet functionality of the purified protein has not been tested. Here we reconstituted overexpressed and purified TRPM2 from Nematostella vectensis (nvTRPM2) into lipid bilayers and found that the protein is fully functional. Consistent with the observations in native membranes, nvTRPM2 in lipid bilayers is co-activated by cytosolic Ca 2+ and either ADPR or ADPR-2′-phosphate (ADPRP). The physiological metabolite ADPRP has a higher apparent affinity than ADPR. In lipid bilayers nvTRPM2 displays a large linear unitary conductance, its open probability (P o ) shows little voltage dependence and is stable over several minutes. P o is high without addition of exogenous PIP 2 , but is largely blunted by treatment with poly- l -Lysine, a polycation that masks PIP 2 headgroups. These results indicate that PIP 2 or some other activating phosphoinositol lipid co-purifies with nvTRPM2, suggesting a high PIP 2 binding affinity of nvTRPM2 under physiological conditions.