Transmembrane protein 16 F (TMEM16F) is a Ca²⁺-activated homodimer which functions as an ion channel and a phospholipid scramblase. Despite the availability of several TMEM16F cryogenic electron microscopy (cryo-EM) structures, the mechanism of activation and substrate translocation remains controversial, possibly due to restrictions in the accessible protein conformational space. In this study, we use atomic force microscopy under physiological conditions to reveal a range of structurally and mechanically diverse TMEM16F assemblies, characterized by variable inter-subunit dimerization interfaces and protomer orientations, which have escaped prior cryo-EM studies. Furthermore, we find that Ca²⁺-induced activation is associated to stepwise changes in the pore region that affect the mechanical properties of transmembrane helices TM3, TM4 and TM6. Our direct observation of membrane remodelling in response to Ca²⁺ binding along with additional electrophysiological analysis, relate this structural multiplicity of TMEM16F to lipid and ion permeation processes. These results thus demonstrate how conformational heterogeneity of TMEM16F directly contributes to its diverse physiological functions.

跨膜蛋白 16 F (TMEM16F) 是一种 Ca ²⁺激活的同二聚体，具有离子通道和磷脂扰乱酶的功能。尽管已有多种 TMEM16F 低温电子显微镜 (cryo-EM) 结构，但激活和底物易位的机制仍然存在争议，可能是由于可及的蛋白质构象空间的限制。在这项研究中，我们在生理条件下使用原子力显微镜揭示了一系列结构和机械上多样化的 TMEM16F 组件，其特征是可变的亚基间二聚化界面和原聚体方向，这些都逃脱了之前的冷冻电镜研究。此外，我们发现Ca ²⁺诱导的活化与孔区域的逐步变化相关，影响跨膜螺旋TM3、TM4 和TM6 的机械性能。我们对 Ca ²⁺结合响应的膜重塑的直接观察以及额外的电生理学分析，将 TMEM16F 的这种结构多样性与脂质和离子渗透过程联系起来。因此，这些结果证明了 TMEM16F 的构象异质性如何直接有助于其多样化的生理功能。