Lipids and Ions Traverse the Membrane by the Same Physical Pathway in the nhTMEM16 Scramblase

Abstract

From bacteria to mammals, different phospholipid species are segregated between the inner and outer leaflets of the plasma membrane by ATP-dependent lipid transporters. Disruption of this asymmetry by ATP-independent phospholipid scrambling is important in cellular signaling, but its mechanism remains incompletely understood. Using MD simulations coupled with experimental assays, we show that the surface hydrophilic transmembrane cavity exposed to the lipid bilayer on the fungal scramblase nhTMEM16 serves as the pathway for both lipid translocation and ion conduction across the membrane. Ca 2+ binding stimulates its open conformation by altering the structure of transmembrane helices that line the cavity. We have identified key amino acids necessary for phospholipid scrambling and validated the idea that ions permeate TMEM16 Cl -channels via a structurally homologous pathway by showing that mutation of two residues in the pore region of the TMEM16A Ca 2+ -activated Cl -channel convert it into a robust scramblase.

Out-of-the-groove transport of lipids by TMEM16 and GPCR scramblases

“…We envisage that out-of-the-groove lipid transport is enabled by groove-dependent local distortions and thinning of the membrane in the vicinity of the protein. This hypothesis is consistent with the finding that the nhTMEM16 scramblase induces a pronounced membrane thinning in the proximity of the groove in molecular dynamics calculations (18,33). An alternative interpretation of these results is that the groove could dilate to accommodate these very large headgroups, which would require a major rearrangement induced by the permeating PEGylated lipids.…”

“…The cryoelectron microscopy structures of the TMEM16A channel homolog revealed that Ca 2+ gating involves the movement of the intracellular portion of the pore-lining TM6 helix (37,38). We and others identified a network of polar residues located at the extracellular entryway of the nhTMEM16 groove that is important for lipid recruitment (18,33,39) and that undergoes a conformational rearrangement to allow the groove to open, enabling penetration of the lipid headgroups and scrambling (39). We reasoned that if all lipids permeate through the groove, then impairing either conformational change should affect their scrambling rates to the same extent.…”

“…First, do TMEM16 proteins translocate lipids strictly via the hydrophilic cavity? For example, it has been proposed that these proteins deform and thin the membrane in the vicinity of the groove (18,32,33), potentially allowing lipid movement outside the cavity. Second, can such an out-of-the-cavity scrambling mechanism also apply to other scramblases?…”

Regulation of TMEM16A/ANO1 and TMEM16F/ANO6 ion currents and phospholipid scrambling by Ca2+ and plasma membrane lipid

“…We therefore suggest that phospholipids and Cl − ions share the same intramolecular pathway, as reported recently for nhTMEM16 by Jiang et al . (). Taken together, the present data suggest regulation of TMEM16A/ANO1 and TMEM16F/ANO6 being more complex than thought initially.…”

Lipids surf the groove in scramblases

“…1, right protomer). Indeed, molecular dynamic simulations on TMEM16 and opsin scramblases suggest that these proteins bend and thin the membrane at the protein-lipid interface (8,13,17,23).…”