The nhTMEM16 Scramblase Is Also a Nonselective Ion Channel

Abstract

The TMEM16 family comprises Ca²⁺-activated Cl^- channels and phospholipid scramblases. The crystal structure of a fungal homolog, nhTMEM16, revealed an important architectural feature of this protein family in the form of a bilayer-spanning hydrophilic groove that is directly exposed to the membrane. This groove likely provides a pathway for lipid translocation. As mutations that alter ion channel activity of the TMEM16 proteins localize around the groove, it was suggested that the ion and lipid pathways coincide such that the ion pore is partly lined by phospholipids. However, this proposal was not supported by the observation that nhTMEM16 does not mediate ion transport. Here we show that nhTMEM16 mediates both ion and lipid transport and that its properties closely resemble those of a previously characterized fungal homolog, afTMEM16. We show that the reported lack of ion transport activity of nhTMEM16 is due to the lipid composition of the reconstitution membranes and to the presence of a GFP tag. Thus, nhTMEM16, like afTMEM16 and the mammalian TMEM16F, mediates simultaneous lipid scrambling and nonspecific ion transport. This supports the hypothesis that these two processes are tightly correlated and likely to be a general functional feature of the TMEM16 scramblases and therefore of general importance in understanding their biological roles.

Figure 1

nhTMEM16 mediates ion and lipid transport. (A and B) Representative traces of phospholipid scrambling mediated by afTMEM16 (A) and nhTMEM16-SBP (B). (C) Quantification of the scrambling activity by afTMEM16 and nhTMEM16-SBP in the presence and absence of Ca²⁺. (D and E) Representative traces of the ion transport activity of afTMEM16 (D) and nhTMEM16-SBP (E) assayed with the flux assay. Scale bar is 10 s. (F) Average percentage of proteoliposomes containing at least one active afTMEM16 and nhTMEM16-SBP channel. In all panels, green indicates protein-free liposomes with 0.5 mM Ca²⁺; red, proteoliposomes with 0.5 mM Ca²⁺; black, proteoliposomes with 2 mM EGTA. The symbols ^∗ and ˆ, respectively, denote the addition of dithionite to initiate the scrambling reaction and of detergent to solubilize liposomes. Data in (C) and (F) represent the mean ± SE of greater than six independent experiments.

Figure 2

Lipid composition affects the activity of afTMEM16 and nhTMEM16-SBP. (A) Average percentage of proteoliposomes containing at least one active afTMEM16 and nhTMEM16-SBP channel. Vesicles were prepared from PE/PG mixed membranes. (B and C) Representative traces of phospholipid scrambling mediated by afTMEM16 (B) and nhTMEM16-SBP (C) in PE/PG liposomes. (D) Quantification of the scrambling activity by afTMEM16 and nhTMEM16-SBP in the presence and absence of Ca²⁺ in PE/PG liposomes. (E) Reconstitution efficiency of afTMEM16 (cyan) and nhTMEM16-SBP (dark blue) in PE/PG liposomes. Color-coding and symbols in (A–D) as in Fig. 1. Each data point represents the mean ± SE of 6+ independent experiments (n = 3 for the data in E).

Figure 3

Terminal tags modulate the functions of afTMEM16 and nhTMEM16. (A–F) Representative traces of phospholipid scrambling mediated by afTMEM16-GFP (A and B), GFP-nhTMEM16 (C and D), and nhTMEM16-SBP (E and F) before and after tag cleavage in PE/PG/PC vesicles. (G) Average scrambling of the constructs in (A–F). (H) Average fraction of liposomes with at least one active TMEM16 channel corresponding to the constructs in (A–F). Color-coding: protein free (green); protein with tag in 0 Ca²⁺ (gray), in 0.5 mM Ca²⁺ (yellow); protein after tag cleavage in 0 Ca²⁺ (black) and in 0.5 mM Ca²⁺ (red). (G and H) Each data point represents the mean ± SE of 6+ independent experiments.