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Cryo-EM Studies of TMEM16F Calcium-Activated Ion Channel Suggest Features Important for Lipid Scrambling

SUMMARY As a Ca2+-activated lipid scramblase and ion channel that mediates Ca2+ influx, TMEM16F relies on both functions to facilitate extracellular vesicle generation, blood coagulation, and bone formation. How a bona fide ion channel scrambles lipids remains elusive. Our structural analyses revealed the coexistence of an intact channel pore and PIP2-dependent protein conformation changes leading to membrane distortion. Correlated to the extent of membrane distortion, many tightly bound lipids are slanted. Structure-based mutagenesis studies further reveal that neutralization of some lipid-binding residues or those near membrane distortion specifically alters the onset of lipid scrambling, but not Ca2+ influx, thus identifying features outside of channel pore that are important for lipid scrambling. Together, our studies demonstrate that membrane distortion does not require open hydrophilic grooves facing the membrane interior and provide further evidence to suggest separate pathways for lipid scrambling and ion permeation.

(A) Representative trace of inside-out patch from HEK293 cells transiently transfected with full-length TMEM16F. The membrane potential was held at 80 mV, and patches were exposed to increasing Ca 2+ concentrations as indicated. (M, N) FSC curves of free (black), work (red) and sum (blue) for processing of Ca 2+ -bound (M) and Ca 2+ -free (N) TMEM16F in digitonin. Curves with resolution corresponding to FSC = 0.5 are shown.
(E-N) Pairwise superimposition of TM6-TM8 with Ca 2+ coordinating residues (E-J) or pairwise superimposition of TM3-TM8 as pore-lining helices (K-N) in Ca 2+ -bound TMEM16F in digitonin (green, from this study, or grey, for 6QP6), Ca 2+ -free TMEM16F in digitonin (yellow), Ca 2+ -bound TMEM16F in nanodiscs supplemented with PIP 2 (orange for class 1, blue for class 2), or Ca 2+ -bound TMEM16F in nanodiscs without PIP 2 (purple, for 6QPC). (A-F) Application of paraformaldehyde (PFA) for chemical induction of GPMV generation in Ca 2+ -free extracellular solution followed with elevation of extracellular Ca 2+ concentration to 2 mM ten minutes later revealed robust Ca 2+ influx in cells expressing TMEM16F-mCherry but not control cells expressing TMEM16A-mCherry or soluble mCherry (mCh-cyto) (A, B). The Ca 2+ rise in the absence of extracellular Ca 2+ does not require TMEM16F (C, D); this Ca 2+ release from internal store is induced by the PFA treatment (E, F).
(I) Surface expression of TMEM16F tagged with mScarlet. (D, E) Local resolution of TMEM16F in PIP 2 supplemented nanodiscs (D for class 1, E for class 2), as estimated by RELION and shown with pseudo-color representation of resolution.
(F, G) FSC curves of two independently refined maps before (black) and after (red) post-processing in RELION for TMEM16F in PIP 2 supplemented nanodiscs (F for class 1, G for class 2). Curves with resolution corresponding to FSC = 0.143 are shown.
(H, I) FSC curves of free (black), work (red) and sum (blue) for processing of Ca 2+ -bound TMEM16F in PIP 2 supplemented nanodics (H for class 1, I for class 2). Curves with resolution corresponding to FSC = 0.5 are shown.
(J, K) dFSC from different Fourier cones for TMEM16F in PIP 2 supplemented nanodiscs (J for class 1, K for class 2). Each curve indicates a different direction.
(L) Representative cryo-EM densities of the ten transmembrane helices TM1-TM10 of TMEM16F (class 1) in PIP 2 supplemented nanodiscs. The electron microscopy densities are shown in blue and the model is shown as sticks colored according to atom type: C, light grey; N, blue; O, red; and S, yellow. (A) Ca 2+ -bound TMEM16F in digitonin. Starting with 3D classification of 1,261,881 particles, we picked class 3, 5, and 6 of the seven classes for 2D classification. After 2D classification was applied to each class, particles from the best 2D classes were pooled together for another round of 3D classification, to yield three classes for 3D refinement. The class with 170,824 particles had a resolution of 3.8 Å. After being masked, they reached a resolution of 3.7 Å.
(B) Ca 2+ -free TMEM16F in digitonin. Starting with 3D classification of 1,182,627 particles, we picked class 1, 2, and 5 of the five classes for 2D classification. After 2D classification was applied to each class, 324,624 particles from the best 2D classes were processed for another round of 3D classification and 3D refinement ending with a resolution of 4.3 Å. After being masked they reached a resolution of 3.98 Å.
(C) Ca 2+ -bound TMEM16F in PIP 2 supplemented nanodiscs. All 2,355,541 picked particles were subdivided into four groups, and 3D classification was applied to each group to generate 3 classes. 2D classification was done for the best of the three classes of each group. Particles from the good 2D classes were pooled together (total 1,056,110 particles) and applied for another round of 3D classification to yield three classes. Class 1 and class 2 were picked for further 2D classification.
Particles from the resulting good 2D classes were pooled together within each class and used for 3D refinement, resulting in a resolution of 3.61 Å for class 1 and 3.66 Å for class 2. After being masked, they reached a resolution of 3.15 Å for class 1 and 3.27 Å for class 2.
(D) Ca 2+ -bound TMEM16F in nanodiscs without PIP 2 supplement. Starting with 3D classification of 1,437,602 particles, we picked class 1, 5 and 6 of the seven classes for 2D classification. After another round of 2D classification of these three classes combined, we picked 268,416 particles for 3D refinement ending with with a resolution of 6.62 Å. (B) Transmembrane helices and loops of TMEM16F in PIP 2 supplemented nanodiscs (class 1) (orange) with lipids (magenta) and one Ca 2+ ion (green sphere) in each monomer, overlaid on the electron density map (sharpened, in light grey).
(K, L) The basic residues in the N-terminal domain of TMEM16F that are crucial for PIP 2 modulation of channel activity are near the TM6-TM7 loop of Ca 2+ -freeTMEM16F in digitonin (K) and Ca 2+ -bound TMEM16F in nanodiscs supplemented with PIP 2 (L). Residues in red boxes are identical in all four sequences, and those in orange boxes are highly conserved. TMEM16F residues in blue are tested in mutagenesis studies.  Position  16F  pre-TM1 elbow  TM3  TM3/TM5-TM6 loop  TM3/TM5-TM6 loop  TM4  TM4  TM4-TM5 loop  TM6  TM6  TM6  TM8  TM9-TM10 loop  TM9-TM10