An inner activation gate controls TMEM16F phospholipid scrambling

Abstract

Transmembrane protein 16F (TMEM16F) is an enigmatic Ca 2+ -activated phospholipid scramblase (CaPLSase) that passively transports phospholipids down their chemical gradients and mediates blood coagulation, bone development and viral infection. Despite recent advances in the structure and function understanding of TMEM16 proteins, how mammalian TMEM16 CaPLSases open and close, or gate their phospholipid permeation pathways remains unclear. Here we identify an inner activation gate, which is established by three hydrophobic residues, F518, Y563 and I612, in the middle of the phospholipid permeation pathway of TMEM16F-CaPLSase. Disrupting the inner gate profoundly alters TMEM16F phospholipid permeation. Lysine substitutions of F518 and Y563 even lead to constitutively active CaPLSases that bypass Ca 2+ -dependent activation. Strikingly, an analogous lysine mutation to TMEM16F-F518 in TMEM16A (L543K) is sufficient to confer CaPLSase activity to the Ca 2+ -activated Cl − channel (CaCC). The identification of an inner activation gate can help elucidate the gating and permeation mechanism of TMEM16 CaPLSases and channels.

Specific PIP2Binding Promotes Calcium Activation of TMEM16A Chloride Channels

“…We note that the observed movement of TMs 3-6 is similar to the previously proposed "clam shell" model of TMEM16F activation. 17 However, the degree of TMs movement is smaller in TMEM16A compared to that of TMEM16F lipid scramblase (e.g., see Fig. 1D).…”

“…3,7 Our previous study of TMEM16F lipid scramblase has suggested a conserved "clam-shell" model of activation for the TMEM16 family proteins. 17 Whether a TMEM16 protein has channel and/or scramblase activities is likely determined by how wide the same pore may be dilated (e.g., see Fig. S1), as governed by the balance of interactions at the pore and various allosteric regulatory sites.…”

“…S1), as governed by the balance of interactions at the pore and various allosteric regulatory sites. For example, while the fully activated state of the wild-type TMEM16A with both Ca 2+ and PIP2 bound can only accommodate ion permeation, a single inner gate L543K mutation could convert TMEM16A CaCC into a lipid scramblase, 17 presumably by forcing additional dilation of the pore. However, our current study suggests that the permeation pathways of ions and lipids through the pore do not fully overlap.…”

“…However, the movement of lipid head groups through the TMEM16F pore follows the so-called 'credit card' model, where lipid head groups exit or enter the pore directly from the membrane from the extracellular side, without involving the extracellular vestibules. 17,44 It is not clear if the ion permeation pathway will coincide with or diverge from the lipid pathway when the pore is dilated enough for scramblase activities. 45 Nonetheless, the existence of alternative potential ion pathways may offer fascinating possibilities of how TMEM16 family ion channels and lipid scramblases may be regulated in biological processes.…”

“…These conserved residues have been shown to be part of an "inner gate" that is likely conserved in all TMEM16 family proteins. 17 Comparison of the Ca 2+ -bound and free structures reveals the Ca 2+ activation mechanism of TMEM16A. 14,15 In the Ca 2+ -free state, the lower half of TM6 bends towards TM4 near G644 and is unfolded beyond G656, which occludes the lower pore region and potentially blocks the entry of permeating ions (Fig.…”