Phospholipid scrambling (PLS) is a ubiquitous cellular mechanism involving the regulated bidirectional transport of phospholipids down their concentration gradient between membrane leaflets. ANO6/TMEM16F has been shown to be essential for Ca2+-dependent PLS, but controversy surrounds whether ANO6 is a phospholipid scramblase or an ion channel like other ANO/TMEM16 family members. Combining patch clamp recording with measurement of PLS, we show that ANO6 elicits robust Ca2+-dependent PLS coinciding with ionic currents that are explained by ionic leak during phospholipid translocation. By analyzing ANO1-ANO6 chimeric proteins, we identify a domain in ANO6 necessary for PLS and sufficient to confer this function on ANO1, which normally does not scramble. Homology modeling shows that the scramblase domain forms an unusual hydrophilic cleft that faces the lipid bilayer and may function to facilitate translocation of phospholipid between membrane leaflets. These findings provide a mechanistic framework for understanding PLS and how ANO6 functions in this process.DOI: http://dx.doi.org/10.7554/eLife.06901.001

Anoctamin/TMEM16 proteins exhibit diverse functions in cells throughout the body and are implicated in several human diseases. Although the founding members ANO1 (TMEM16A) and ANO2 (TMEM16B) are Ca2+-activated Cl− channels, most ANO paralogs are Ca2+-dependent phospholipid scramblases that serve as channels that facilitate the movement (“scrambling”) of phospholipids between leaflets of the membrane bilayer. Phospholipid scrambling significantly alters the physical properties of the membrane and its landscape and has vast downstream signaling consequences. In particular, phosphatidylserine exposed on the external leaflet of the plasma membrane functions as a ligand for receptors vital for cell-cell communication. A major consequence of Ca2+-dependent scrambling is the release of extracellular vesicles that function as intercellular messengers by delivering signaling proteins and non-coding RNAs to alter target cell function. We discuss the physiological implications of Ca2+-dependent phospholipid scrambling, the extracellular vesicles associated with this activity, and the roles of ANOs in these processes.

Limb-girdle muscular dystrophies are a genetically diverse group of diseases characterized by chronic muscle wasting and weakness. Recessive mutations in ANO5 (TMEM16E) have been directly linked to several clinical phenotypes including limb-girdle muscular dystrophy type 2L and Miyoshi myopathy type 3, although the pathogenic mechanism has remained elusive. ANO5 is a member of the Anoctamin/TMEM16 superfamily that encodes both ion channels and regulators of membrane phospholipid scrambling. The phenotypic overlap of ANO5 myopathies with dysferlin-associated muscular dystrophies has inspired the hypothesis that ANO5, like dysferlin, may be involved in the repair of muscle membranes following injury. Here we show that Ano5-deficient mice have reduced capacity to repair the sarcolemma following laser-induced damage, exhibit delayed regeneration after cardiotoxin injury and suffer from defective myoblast fusion necessary for the proper repair and regeneration of multinucleated myotubes. Together, these data suggest that ANO5 plays an important role in sarcolemmal membrane dynamics. Genbank Mouse Genome Informatics accession no. 3576659.

Limb-girdle muscular dystrophy type 2L (LGMD2L) is a myopathy arising from mutations in ANO5; however, information about the contribution of ANO5 to muscle physiology is lacking. To explain the role of ANO5 in LGMD2L, we previously hypothesized that ANO5-mediated phospholipid scrambling facilitates cell–cell fusion of mononucleated muscle progenitor cells (MPCs), which is required for muscle repair. Here, we show that heterologous overexpression of ANO5 confers Ca2+-dependent phospholipid scrambling to HEK-293 cells and that scrambling is associated with the simultaneous development of a nonselective ionic current. MPCs isolated from adult Ano5−/− mice exhibit defective cell fusion in culture and produce muscle fibers with significantly fewer nuclei compared with controls. This defective fusion is associated with a decrease of Ca2+-dependent phosphatidylserine exposure on the surface of Ano5−/− MPCs and a decrease in the amplitude of Ca2+-dependent outwardly rectifying ionic currents. Viral introduction of ANO5 in Ano5−/− MPCs restores MPC fusion competence, ANO5-dependent phospholipid scrambling, and Ca2+-dependent outwardly rectifying ionic currents. ANO5-rescued MPCs produce myotubes having numbers of nuclei similar to wild-type controls. These data suggest that ANO5-mediated phospholipid scrambling or ionic currents play an important role in muscle repair.

Since their first descriptions, ion channels have been conceived as proteinaceous conduits that facilitate the passage of ionic cargo between segregated environments. This concept is reinforced by crystallographic structures of cation channels depicting ion conductance pathways completely lined by protein. Although lipids are sometimes present in fenestrations near the pore or may be involved in channel gating, there is little or no evidence that lipids inhabit the ion conduction pathway. Indeed, the presence of lipid acyl chains in the conductance pathway would curse the design of the channel’s aqueous pore. Here, we make a speculative proposal that anion channels in the TMEM16/ANO superfamily have ion conductance pathways composed partly of lipids. Our reasoning is based on the idea that TMEM16 ion channels evolved from a kind of lipid transporter that scrambles lipids between leaflets of the membrane bilayer and the modeled structural similarity between TMEM16 lipid scramblases and TMEM16 anion channels. This novel view of the TMEM16 pore offers explanation for the biophysical and pharmacological oddness of TMEM16A. We build upon the recent X-ray structure of nhTMEM16 and develop models of both TMEM16 ion channels and lipid scramblases to bolster our proposal. It is our hope that this model of the TMEM16 pore will foster innovative investigation into TMEM16 function.