Department of Anesthesiology & Perioperative Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
Neuroscience and Biochemistry & Cell Biology Programs, The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, TX, USA.
Commun Biol. 2022 Jul 23;5(1):738. doi: 10.1038/s42003-022-03701-5.
Human two-pore channels (TPCs) are endolysosomal cation channels and play an important role in NAADP-evoked Ca release and endomembrane dynamics. We found that YM201636, a PIKfyve inhibitor, potently inhibits PI(3,5)P-activated human TPC2 with an IC 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 IC 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.
人源双孔通道（TPCs）是内溶酶体阳离子通道，在 NAADP 诱导的 Ca2+释放和内膜动力学中发挥重要作用。我们发现，PIKfyve 抑制剂 YM201636 对人源 TPC2 的抑制作用很强，IC50 为 0.16 μM。YM201636 还能有效抑制 NAADP 激活的 TPC2 和组成型开放的 TPC2 L690A/L694A 突变体通道；而当通道处于关闭状态时，其作用很小。PI-103 是 YM201636 的类似物，也是 PI3K 和 mTOR 的抑制剂，对人源 TPC2 的抑制作用也很强，IC50 为 0.64 μM。通过突变、虚拟对接和分子动力学模拟分析，我们发现 YM201636 和 PI-103 直接阻断 TPC2 的开放状态通道孔，位于束交叉孔门区域，附近的 H699 残基是通道对抑制剂敏感性的关键决定因素。H699 可能与孔入口周围的抑制剂相互作用，促进其进入孔道。H699 突变为苯丙氨酸在很大程度上解释了 TPC1 通道对 YM201636 的不敏感性。这些发现确定了两种有效的 TPC2 通道阻断剂，揭示了通道孔入口阻断机制，并为解释两种常用的磷酸肌醇激酶抑制剂的药理学作用提供了一个离子通道靶点。
