Department of Physiology, University of Texas Southwestern Medical Center, Dallas, United States.
Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States.
Elife. 2019 Mar 12;8:e45222. doi: 10.7554/eLife.45222.
Mammalian two-pore channels (TPCs) regulate the physiological functions of the endolysosome. Here we present cryo-EM structures of human TPC2 (HsTPC2), a phosphatidylinositol 3,5-bisphosphate (PI(3,5)P)-activated, Na selective channel, in the ligand-bound and apo states. The apo structure captures the closed conformation, while the ligand-bound form features the channel in both open and closed conformations. Combined with functional analysis, these structures provide insights into the mechanism of PI(3,5)P-regulated gating of TPC2, which is distinct from that of TPC1. Specifically, the endolysosome-specific PI(3,5)P binds at the first 6-TM and activates the channel - independently of the membrane potential - by inducing a structural change at the pore-lining inner helix (IS6), which forms a continuous helix in the open state but breaks into two segments at Gly317 in the closed state. Additionally, structural comparison to the voltage-dependent TPC1 structure allowed us to identify Ile551 as being responsible for the loss of voltage dependence in TPC2.
哺乳动物双孔通道（TPCs）调节内溶酶体的生理功能。在这里，我们呈现了人源 TPC2（HsTPC2）的冷冻电镜结构，这是一种磷脂酰肌醇 3,5-二磷酸（PI(3,5)P）激活的、钠离子选择性通道，处于配体结合和无配体状态。无配体状态捕获了关闭构象，而配体结合形式则显示了通道的开放和关闭构象。结合功能分析，这些结构提供了对 PI(3,5)P 调节 TPC2 门控机制的深入了解，这与 TPC1 的机制不同。具体来说，内溶酶体特异性的 PI(3,5)P 结合在第一个 6-TM 上，并通过诱导位于孔衬内层螺旋（IS6）的结构变化，在不依赖于膜电位的情况下激活通道，在开放状态下，IS6 形成连续的螺旋，而在关闭状态下，IS6 在 Gly317 处断裂成两个片段。此外，与电压依赖性 TPC1 结构的结构比较使我们能够确定 Ile551 是 TPC2 中电压依赖性丧失的原因。
