Species-Specific Regulation of TRPM2 by PI(4,5)P(2) via the Membrane Interfacial Cavity

The human apoptosis channel TRPM2 is stimulated by intracellular ADR-ribose and calcium. Recent studies show pronounced species-specific activation mechanisms. Our aim was to analyse the functional effect of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)), commonly referred to as PIP(2), on different TRPM2 orthologues. Moreover, we wished to identify the interaction site between TRPM2 and PIP(2). We demonstrate a crucial role of PIP(2), in the activation of TRPM2 orthologues of man, zebrafish, and sea anemone. Utilizing inside-out patch clamp recordings of HEK-293 cells transfected with TRPM2, differential effects of PIP(2) that were dependent on the species variant became apparent. While depletion of PIP(2) via polylysine uniformly caused complete inactivation of TRPM2, restoration of channel activity by artificial PIP(2) differed widely. Human TRPM2 was the least sensitive species variant, making it the most susceptible one for regulation by changes in intramembranous PIP(2) content. Furthermore, mutations of highly conserved positively charged amino acid residues in the membrane interfacial cavity reduced the PIP(2) sensitivity in all three TRPM2 orthologues to varying degrees. We conclude that the membrane interfacial cavity acts as a uniform PIP(2) binding site of TRPM2, facilitating channel activation in the presence of ADPR and Ca(2+) in a species-specific manner.