Transmembrane protein 16F (TMEM16F) is a ubiquitously expressed Ca²⁺-activated phospholipid scramblase that also functions as a largely non-selective ion channel. Though recent structural studies have revealed the closed and intermediate conformations of mammalian TMEM16F (mTMEM16F), the open and conductive state remains elusive. Instead, it has been proposed that an open hydrophilic pathway may not be required for lipid scrambling. We previously identified an inner activation gate, consisting of F518, Y563, and I612, and showed that charged mutations of the inner gate residues led to constitutively active mTMEM16F scrambling. Herein, atomistic simulations show that lysine substitution of F518 and Y563 can indeed lead to spontaneous opening of the permeation pore in the Ca²⁺-bound state of mTMEM16F. Dilation of the pore exposes hydrophilic patches in the upper pore region, greatly increases the pore hydration level, and enables lipid scrambling. The putative open state of mTMEM16F resembles the active state of fungal scramblases and is a meta-stable state for the wild-type protein in the Ca²⁺-bound state. Therefore, mTMEM16F may be capable of supporting the canonical in-groove scrambling mechanism in addition to the out-of-groove one. Further analysis reveals that the in-groove phospholipid and ion transduction pathways of mTMEM16F overlap from the intracellular side up to the inner gate but diverge from each other with different exits to the extracellular side of membrane.

跨膜蛋白 16F (TMEM16F) 是一种普遍表达的 Ca ²⁺激活的磷脂扰乱酶，也可作为非选择性离子通道。尽管最近的结构研究揭示了哺乳动物 TMEM16F (mTMEM16F) 的闭合和中间构象，但开放和导电状态仍然难以捉摸。相反，有人提出，脂质加扰可能不需要开放的亲水途径。我们之前鉴定了一个由 F518、Y563 和 I612 组成的内部激活门，并表明内门残基的带电突变导致组成型活性 mTMEM16F 扰乱。在此，原子模拟表明F518和Y563的赖氨酸取代确实可以导致mTMEM16F的Ca ²⁺结合状态下渗透孔的自发打开。毛孔的扩张暴露了上部毛孔区域的亲水斑块，大大增加了毛孔水合水平，并使脂质扰乱成为可能。^{mTMEM16F 的推定开放状态类似于真菌扰乱酶的活性状态，并且是处于 Ca 2+}结合状态的野生型蛋白质的亚稳定状态。因此，除了槽外加扰机制之外，mTMEM16F 还可能能够支持规范的槽内加扰机制。进一步分析表明，mTMEM16F 的槽内磷脂和离子转导途径从细胞内侧到内门重叠，但彼此分叉，具有不同的出口到膜的细胞外侧。