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

ABSTRACT: Human two-pore channels (TPCs) are endolysosomal cation channels and play an important role in NAADP-evoked Ca²⁺ release and endomembrane dynamics. We found that YM201636, a PIKfyve inhibitor, potently inhibits PI(3,5)P₂-activated human TPC2 with an IC₅₀ of 0.16 μM. YM201636 also effectively inhibits NAADP-activated TPC2 and a constitutively-open TPC2 L690A/L694A mutant channel; whereas it exerts little effect when applied in the channel's closed state. PI-103, a YM201636 analog and an inhibitor of PI3K and mTOR, also inhibits human TPC2 with an IC₅₀ of 0.64 μM. With mutational, virtual docking, and molecular dynamic simulation analyses, we found that YM201636 and PI-103 directly block the TPC2's open-state channel pore at the bundle-cross pore-gate region where a nearby H699 residue is a key determinant for channel's sensitivity to the inhibitors. H699 likely interacts with the blockers around the pore entrance and facilitates their access to the pore. Substitution of a Phe for H699 largely accounts for the TPC1 channel's insensitivity to YM201636. These findings identify two potent TPC2 channel blockers, reveal a channel pore entrance blockade mechanism, and provide an ion channel target in interpreting the pharmacological effects of two commonly used phosphoinositide kinase inhibitors.

SUBMITTER: Du C

PROVIDER: S-EPMC9308409 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Aquaporin-4 (AQP4), the primary water channel in glial cells of the mammalian brain, plays a critical role in water transport in the central nervous system. Previous experiments have shown that the water permeability of AQP4 depends on the cholesterol content in the lipid bilayer, but it was not clear whether changes in permeability were due to direct cholesterol-AQP4 interactions or to indirect effects caused by cholesterol-induced changes in bilayer elasticity or bilayer thickness. To determine the effects resulting only from bilayer thickness, here we use a combination of experiments and simulations to analyze AQP4 in cholesterol-free phospholipid bilayers with similar elastic properties but different hydrocarbon core thicknesses previously determined by x-ray diffraction. The channel (unit) water permeabilities of AQP4 measured by osmotic-gradient experiments were 3.5 ± 0.2 × 10(-13) cm(3)/s (mean ± SE), 3.0 ± 0.3 × 10(-13) cm(3)/s, 2.5 ± 0.2 × 10(-13) cm(3)/s, and 0.9 ± 0.1 × 10(-13) cm(3)/s in bilayers containing (C22:1)(C22:1)PC, (C20:1)(C20:1)PC, (C16:0)(C18:1)PC, and (C13:0)(C13:0)PC, respectively. Channel permeabilities obtained by molecular dynamics (MD) simulations were 3.3 ± 0.1 × 10(-13) cm(3)/s and 2.5 ± 0.1 × 10(-13) cm(3)/s in (C22:1)(C22:1)PC and (C14:0)(C14:0)PC bilayers, respectively. Both the osmotic-gradient and MD-simulation results indicated that AQP4 channel permeability decreased with decreasing bilayer hydrocarbon thickness. The MD simulations also suggested structural modifications in AQP4 in response to changes in bilayer thickness. Although the simulations showed no appreciable changes to the radius of the pore located in the hydrocarbon region of the bilayers, the simulations indicated that there were changes in both pore length and ?-helix organization near the cytoplasmic vestibule of the channel. These structural changes, caused by mismatch between the hydrophobic length of AQP4 and the bilayer hydrocarbon thickness, could explain the observed differences in water permeability with changes in bilayer thickness.

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

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