Cryo-EM structures and functional characterization of the lipid scramblase TMEM16F

Abstract

The lipid scramblase TMEM16F initiates blood coagulation by catalyzing the exposure of phosphatidylserine in platelets. The protein is part of a family of membrane proteins, which encompasses calcium-activated channels for ions and lipids. Here, we reveal features of TMEM16F that underlie its function as lipid scramblase and ion channel. The cryo-EM structures of TMEM16F in Ca 2+ -bound and Ca 2+ -free states display a striking similarity to the scramblingincompetent anion channel TMEM16A, yet with distinct differences in the catalytic site and in the conformational changes upon activation. In conjunction with functional data, we demonstrate the relationship between ion conduction and lipid scrambling. Although activated by a common mechanism, which likely resembles an equivalent process defined in the homologue nhTMEM16, both functions appear to be mediated by alternate protein conformations, which are at equilibrium in the ligand-bound state. Keywords:TMEM16 family, lipid scramblase, membrane protein structure, transport mechanism, cryo-EM, single particle of the family, TMEM16F appears, with respect to phylogenetic relationships, as intermediate between the two proteins. Although working as lipid scramblase (Suzuki et al., 2010; Watanabe et al., 2018), it is closer related to the ion channel TMEM16A than to nhTMEM16 ( Figure S1A).Moreover, whereas scrambling-related ion conduction was found to be a feature of several family members (Lee et al., 2016;Malvezzi et al., 2018; Whitlock and Hartzell, 2016b), TMEM16F is the only lipid scramblase for which instantaneous calcium-activated currents were recorded in excised patches (Yang et al., 2012). To better understand how the small sequence differences in TMEM16F give rise to its distinct functional properties, we determined its structure by cryo-EM in Ca 2+ -bound and Ca 2+ -free states, both in a detergent and in a lipid environment. In parallel, we characterized the lipid transport properties of TMEM16F in vitro after reconstitution of the protein into liposomes, as well as ion conduction in transfected cells by electrophysiology. Collectively, our study reveals the architecture of TMEM16F, defines conformational changes upon ligand binding and suggests potential mechanisms for ion and lipid movement. In the most plausible scenario, both transport processes, although activated by the same mechanism, are mediated by distinct protein conformations which are at equilibrium in a calcium-bound state. RESULTS Functional properties of TMEM16FIn our study, we explored the relationship of the TMEM16F structure to its diverse functional properties. For this purpose, we have expressed TMEM16F in HEK293 cells and purified it in the detergent digitonin ( Figures S1B and S1C). To confirm that the purified protein has retained its activity as a lipid scramblase, we have investigated lipid transport with proteoliposomes using an assay that was previously established for fungal TMEM16 scramblases (Malvezzi et al., 2013;Ploier and Menon, 2016). Our data reveals a Ca 2+ -induced ...

Dynamic Change of Electrostatic Field in TMEM16F Permeation Pathway Shifts Its Ion Selectivity

“…The rapid decrease of current rendered it difficult to record the reversal potential of wild-type TMEM16F in 1 mM Ca 2+ , because when we switched the bath (equivalent to intracellular solution) from 150 mM NaCl to 15 mM NaCl, the currents recorded from Tmem16ftransfected cells were indistinguishable from currents endogenous to HEK293 cells ( Figure 1-figure supplement 1A, B, C). Consistent with previous reports (6,9), TMEM16F current induced by 15 µM Ca 2+ was selective for Na + over Cl -( Figure 1E).…”

“…Here, we report that TMEM16F Q559K current persisted with prolonged exposure to high intracellular Ca 2+ . Previous studies have indicated that Q559 faces the ionic permeation pathway and that lysine substitution reduces TMEM16F cation selectivity (6,9), and we now further show that this mutant displays different Na + /Clselectivity in different Ca 2+ concentrations. The difference in ion selectivity did not correlate with alterations of the open states, but instead was regulated by the electrostatic change along the permeation pathway, on which divalent ions (such as Ca 2+ and Zn 2+ ) had more significant impact than monovalent ions.…”

“…This process, known as lipid scrambling, initiates many physiological processes such as recruitment of thrombin to the platelet surface to trigger blood coagulation and induction of immune responses in T lymphocytes (5)(6)(7)(8). TMEM16F is also a Ca 2+ -activated small-conductance ion channel, yielding non-selective cation current in excised patch recordings (6,9). Consistently, activation of TMEM16F increases cytoplasmic Ca 2+ concentration in cells that heterologously express this channel (10).…”

“…We asked whether the two phases of ion selectivity of the Q559K mutant channel corresponds to multiple open states that may correlate with different extent of occupancy of the Ca 2+ binding sites; For example, the channel may be in an intermediate state in 15 µM Ca 2+ but a fully open state in 1 mM Ca 2+ . To test this possibility, we made use of the Ca 2+ -binding-site mutant, E667Q, whose EC50 of Ca 2+ -activation is shifted to the right, to dissociate the effect of Ca 2+ concentration from those attributable to different open states of the channel (6,9). The EC50 of the double-mutant, Q559K_E667Q, was 0.88 ± 0.06 mM, suggesting that the double mutant should not be in the fully open state in 1 mM Ca 2+ (Figure 2A, B).…”

“…There is discrepancy regarding TMEM16F ion selectivity reported by different labs. Recorded with excised-patch inside-out configuration, TMEM16F channels are quickly activated by micromolar Ca 2+ and they are more permeable to cations (mainly physiological cations such as Na + , K + and Ca 2+ ) than Cl -, while prolonged treatment by Ca 2+ leads to current inactivation (6,9). Surprisingly, many groups reported that TMEM16F whole-cell current is activated several minutes after Ca 2+ elevation, and it displays less cation-selectivity (12) or even greater selectivity for Clthan Na + (13)(14)(15)(16).…”