ABSTRACT
The calcium-activated TMEM16 proteins and the mechanosensitive/osmolarity-activated OSCA/TMEM63 proteins belong to the Transmembrane Channel/Scramblase (TCS) superfamily. Within the superfamily, OSCA/TMEM63 proteins, as well as TMEM16A and TMEM16B, are thought to function solely as ion channels. However, most TMEM16 members, including TMEM16F, maintain an additional function as scramblases, rapidly exchanging phospholipids between leaflets of the membrane. Although recent studies have advanced our understanding of TCS structure-function relationships, the molecular determinants of TCS ion and lipid permeation remain unclear. Here we show that single mutations along the transmembrane helix (TM) 4/6 interface allow non-scrambling TCS members to permeate phospholipids. In particular, this study highlights the key role of TM 4 in controlling TCS ion and lipid permeation and offers novel insights into the evolution of the TCS superfamily, suggesting that, like TMEM16s, the OSCA/TMEM63 family maintains a conserved potential to permeate ions and phospholipids.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
1.Tested more mutations and justified the choice of the mutations: 1)We extended our lysine mutation scanning from the conserved inner activation gate towards the C-terminus of TM 4 for TMEM16F (Fig. 1), TMEM16A (Fig. 3), OSCA1.2 (Fig. 4) and TMEM63A (Fig. 6). 2)In our previous study, we have reported the side chain properties of the activation gate residues are important in controlling gating and ion/lipid permeation (Nat. Commun. 2019, https://doi.org/10.1038/s41467-019-09778-7). Our new experiments (Supplementary Fig. 2) further demonstrated that charged amino acids are much more efficient in promoting lipid scrambling than alanine mutation. 3)We also characterized a corresponding lysine mutation in TM 6 to demonstrate that the charges destabilize the TM 4/6 interface and that the subsequent enhancement in lipid scrambling is not just restricted to TM 4 (Fig. 2). The TM 6 mutation has a similar effect on controlling lipid scrambling. 2.Blockers on mutant channels: We used a TMEM16A specific inhibitor, Ani9, to demonstrate that the lysine mutations are indeed responsible for the spontaneous lipid scrambling activity and enhanced channel activity (Supplementary Fig. 3). 3.Osmolarity activation of OSCA1.2 and TMEM63A: We recorded hypotonicity-induced activation of whole cell OSCA1.2 current (Supplementary Fig. 6). Together with hypotonicity-induced lipid scrambling through OSCA1.2 A439K (Fig. 5), our findings demonstrate that cell swelling-induced membrane stretch can activate OSCA1.2 channel and the mutant lipid scramblase. In contrast, hypotonicity was insufficient to induce significant scramblase activity from the analogous mutation in TMEM63A, A476K (Supplementary Fig. 7). 4.Added experimental details for lipid scrambling assay: We have added this detail in our Methods section - Fluorescence imaging of scramblase-mediated PS Exposure.
