TMEM16 proteins are found in all eukaryotes, with the family consisting of ten genes (TMEM16A-K, missing out I). Following TMEM16A and B being discovered as the elusive calcium activated chloride channels (CaCC) in 2008 by 3 independent groups (1, 2, 3) it was fully expected that all the other members of the family would also be CaCC’s because of their high sequence similarity. However, this was not the case and TMEM16C-K have been shown to be phospholipid scramblases (4). So how do TMEM16 proteins on the one hand scramble plasma membrane phospholipids, and on the other hand operate as anion channels?
In an attempt to answer this question Whitlock and Hartzell in their review (5) put forward a novel hypothesis: –
- TMEM16A and B evolved from an ancestral scramblase.
- TMEM16A pore shares structural similarity to an ancestral TMEM16 lipid channel.
- The TMEM16A Cl– selective pore is formed not of pure protein, as ion channel dogma is conceived at the moment, but is composed partly of lipids.
The authors propose that, TMEM16A protein stabilizes a non-bilayer phase in the membrane so that the 2 leaflets are continuous where they interact with the protein. The lipid head groups would then provide a hydrophilic environment forming half of the pore and ions could move across the membrane in the ‘aqueous channel’ formed between the protein and the lipid head groups. In putting forward this idea they have extrapolated Pomorki and Menon’s proposed mechanism for scramblases (6), in which a hydrophobic furrow would allow the phospholipid head groups to translocate from one side of the membrane to another while the acyl chains remain in the hydrophobic phase of the membrane, and suggest TMEM16A has evolved to conduct Cl– ions but not lipids using the same conduction pathway.
They draw their evidence from the recent X-ray crystal structure of fungal nhTMEM16 provided by Brunner et al. (4) and then using their homology models show TMEM16A has a hydrophilic furrow very similar to that of nhTMEM16 with the exception of a patch of hydrophobic amino acids at the cytoplasm end of the furrow. They suggest this patch might explain why TMEM16A is not a scramblase but an ion channel, because it forms a barrier to the movement of the hydrophilic head groups of phospholipids entering the furrow.
This is strengthened with reference to mutagenesis experiments where a chimeric protein was made with 35 amino acids from the lipid scrambling domain of TMEM16F and substituted into TMEM16A which them became a scramblase (7). Suggesting this hypothesis might well be plausible, rather than as the title of Whitlock and Hartzell’s review suggests a poor idea, however, it needs further investigation to prove it right or wrong.
However, functional details remain obscure for most TMEM16 paralogs, making it unclear if they produce ion channels or not. If this novel hypothesis is proven, it will have a major impact on our perception of the dogma of proteinaceous ion channels and the discovery of further TMEM16A inhibitors. It might also explain why TMEM16A inhibitors are, as the authors suggest, somewhat weird and not the ‘classical’ channel blockers, as it is unclear whether the current TMEM16 inhibitors block by acting in the permeation pathway or allosterically.
Blog written by Roy Fox
- Caputo A, Caci E, Ferrera L, Pedemonte N, Barsanti C, Sondo E, Pfeffer U, Ravazzolo R, Zegarra-Moran O, Galietta LJV (2008) TMEM16A, a membrane protein associated with calcium dependent chloride channel activity. Science 322:590–
- Schroeder BC, Cheng T, Jan YN, Jan LY (2008) Expression cloning of TMEM16A as a calcium-activated chloride channel subunit. Cell 134:1019–
- Yang YD, Cho H, Koo JY, Tak MH, Cho Y, Shim WS, Park SP, Lee J, Lee B, Kim BM, Raouf R, Shin YK, Oh U (2008) TMEM16A confers receptor-activated calcium-dependent chloride conductance. Nature 455:1210–
- Brunner JD, Lim NK, Schenck S, Duerst A, Dutzler R (2014) Xray structure of a calcium-activated TMEM16 lipid scramblase. Nature 516:207–
- Whitlock JM and Hartzell HC (2016) A pore idea: the ion conduction pathway of TMEM16/ANO proteins is composed partly of lipid. Eur. J. Physiol. 468: 455-473.
- Pomorski T, Menon AK (2006) Lipid flippases and their biological functions. Cell Mol Life Sci 63:2908–
- Yu K, Whitlock JM, Lee K, Ortlund EA, Cui YY, Hartzell HC (2015) Identification of a lipid scrambling domain in ANO6/TMEM16F. Elife 4:e06901.