Two-pore channel blockade by phosphoinositide kinase inhibitors YM201636 and PI-103 determined by a histidine residue near pore-entrance

Human two-pore channels (TPCs) are endolysosomal cation channels and play an important role in NAADP-evoked Ca 2+ release and endomembrane dynamics. TPCs are activated by PI(3,5)P 2 , which is synthesized by PIKfyve. Here, we found that YM201636, a widely used PIKfyve inhibitor, potently inhibits PI(3,5)P 2 -induced human TPC2 channel Na + currents in a dose-dependent manner with an IC 50 of 0.16 μM. YM201636 also effectively inhibits the currents of a constitutively open TPC2 mutant channel, whereas it exerts no effect when applied in the channel’s closed state. A YM201636 analog, PI-103, an inhibitor of PI3K and mTOR, also inhibits human TPC2 with an IC 50 of 0.64 μM. Substitution of the pore-constriction residues Leu690, Leu694 and Tyr312 by Ala reduced the effectiveness of YM201636 and PI-103 in channel inhibition. Interestingly, a His699 residue located near the cytosolic pore entrance is a key determinant for the TPC2 channels’ sensitivity to YM201636 and PI-103, whose substitution with a Phe in the human TPC1 largely accounts for the channel’s loss of inhibition by YM201636. Molecular dynamic simulation and docking analysis of YM201636’s binding to TPC2 indicated that His699 can interact with the inhibitor and facilitate the channel-blockade at the cytosolic end of the pore. We conclude that YM201636 and PI-103 directly block the TPC2’s open-state channel pore at the cytosolic entrance where the His699 is a key determinant for the blockers’ access to the pore. These findings identify two new potent TPC2 channel blockers, reveal a channel pore entrance blockade mechanism, and provide a new protein target in interpreting the pharmacological effects of these two widely used phosphoinositide kinase inhibitors.