Eukaryotic cells are separated from their environment by a 30-Å-thick delimitation of lipid and protein—the plasma membrane (PM). Although a greater measure of affection has historically been garnered by their protein machines, cells have devoted ∼5% of their genes to building lipids. In this pursuit of lipidic diversity, cells have developed repertoires of thousands of lipid species (van Meer et al., 2008), many of which fall into the category of phospholipids (PLs), which are largely responsible for the PM’s bilayer arrangement. The PLs within the PM bilayer are actively arranged in an asymmetric orientation rendering the exofacial surface largely composed of cylindrical species (e.g., phosphatidylcholine), while the cytofacial leaflet retains most conical and charged species (e.g., phosphatidylethanolamine and phosphatidylserine [PS]). Despite this organization, many of our cells maintain the ability to rapidly abolish PM lipid asymmetry, exposing cytofacial PLs to the exofacial surface as a means of coordinating intercellular processes (Bevers and Williamson, 2016). Bevers et al. (1982) first described this PL “scrambling” in platelet activation, where PS exposure on the exofacial surface propagates coagulation. A few years after Bevers’ description, several others noted that platelet activation was accompanied by a rapid alkalization of intracellular pH (pHi), and that blocking this alkalization inhibited PS exposure (Bucki et al., 2006; Zavoico et al., 1986). In an earlier issue of the Journal of General Physiology, Liang and Yang (2021) show us why. In fact, these platelets, and likely other cells that use nonapoptotic scrambling as a signaling module, have tamed their scramblases to fit a distinct biological niche via pHi.

In an earlier issue of the Journal of General Physiology, Liang and Yang (2021) offer an insightful peek into the mechanism by which the TMEM16F scramblase is regulated by pHi. Using simultaneous monitoring of fluorescent PS probe binding and electrophysiological recordings, Liang and Yang (2021) assess how alterations in pHi impact both TMEM16F-dependent lipid rearrangements and the ionic current associated with PM scrambling. Under physiological Ca2+ concentrations, Liang and Yang (2021) find that TMEM16F is bidirectionally “tamed” by pHi, both entirely inhibited by cytosolic acidification and potentiated by alkalization. However, the inhibition of TMEM16F via low pHi can be overcome with super-physiological Ca2+ concentrations. pHi appears to exert its effect on TMEM16F by altering the protonation state of the scramblase’s Ca2+-binding sites. To this point, reducing TMEM16F Ca2+ affinity also proportionately reduces the sensitivity of TMEM16F to pHi. Either “reined” in by an acidic cytosol or spurred into a “gallop” through alkaline potentiation, these data demonstrate that pHi bidirectionally tames the TMEM16 scramblase.

The mechanism described by Liang and Yang (2021) finally illuminates a likely mechanism by which the Na+/H+ antiporter NHEI regulates the activation of platelets. Inhibition of NHEI function acidifies platelet pHi and inhibits the PS exposure and microvesicle release observed in response to Ca2+, as alluded to above; however, the mechanism by which NHEI regulates these platelet functions has remained a mystery for decades (Bucki et al., 2006). A vital tool used to study PS exposure and microvesicle release in platelets has historically been the use of cells from Scott’s syndrome patients, which lack both of these activities in platelets and red cells. Previous work has demonstrated that the loss of these platelet/red cell activities stems from a loss of their scramblase, TMEM16F (Suzuki et al., 2010). Based on the work of Liang and Yang (2021), it would appear that physiological alkalization of the platelet cytosol may very well be a mechanism platelets employ to potentiate their scramblase and achieve rapid PS exposure and microvesicle release in response to injury. Moreover, the loss of platelet activation in response to NHEI inhibition likely stems from the acidic environment of the cytosol reining in the platelet’s scramblase, TMEM16F.

For the last 10 yr, we have appreciated that much of the nonapoptotic scrambling that cells employ in intercellular signaling is performed by the TMEM16 family of Ca2+-activated phospholipid scramblases (Ca2+-PLSases). The most abundant PLSase in mammals is TMEM16F (reviewed in Whitlock and Hartzell, 2017). Of note, this family also includes two dearly loved ion channel members that do not scramble PLs but pass ions through the PM (i.e., TMEM16s A and B). Thanks to the work of many, we now know a great deal about which TMEM16s are scramblases, how they ferry lipids between PM leaflets, how they are activated by Ca2+, and their structures. Moreover, we also appreciate that these scramblases are nonselective in respect to which lipids they move between leaflets, and that some TMEM16s even scramble lipids in membranes other than the PM (Tsuji et al., 2019). What we are just beginning to appreciate are the mechanisms that cells use to manage these potent remodelers of the PM beyond Ca2+ activation.

In an earlier issue of the Journal of General Physiology, Liang and Yang (2021) identify a novel mechanism by which cells can tame TMEM16F activities to fit their biological purpose using pHi. This pHi regulatory mechanism is one of several newly discovered ways that cells can tame TMEM16 scramblases to fit a particular biological system. Moving forward, it will be vital to better elucidate how pHi and other means of taming TMEM16 scramblases fit into the ever-growing list of biological processes that are activated by scrambling.

A TMEM16 scramblase module is coming into focus

TMEM16s are unequivocally activated by intracellular Ca2+, but how these Ca2+-activated machines are adapted to fit distinct biological contexts moves beyond cytosolic Ca2+. Because their ion channel cousins were discovered first, our ideas of how TMEM16 scramblases are regulated has largely been colored by the electrophysiological characterization of the Cl channel TMEM16A. TMEM16A is activated by Ca2+, but this activity is regulated by membrane voltage, PIP2, cholesterol, extracellular pH, and pHi (Yu et al., 2019; Le et al., 2019; De Jesús-Pérez et al., 2018; Xiao et al., 2011; Cruz-Rangel et al., 2017; Chun et al., 2015). Although biological functions differ dramatically between TMEM16 ion channels and scramblases, it appears that many routes of regulation are conserved family traits. In particular, TMEM16F is also regulated by membrane voltage, PIP2, and, as we learn from Liang and Yang, pHi (Scudieri et al., 2015; Ye et al., 2018; Liang and Yang, 2021). Moreover, the mechanism by which Liang and Yang (2021) propose pHi regulates TMEM16F (through the protonation of its Ca2+ binding sites) is shared with TMEM16A (Chun et al., 2015). Together, this shared pHi regulatory mechanism, observed in TMEM16s of differing activities, suggests that most TMEM16s may be regulated by pHi. However, is it reasonable to assume that TMEM16 sensitivity to pHi is similar between family members? All TMEM16s are activated by Ca2+ binding, and the residues coordinating Ca2+ in TMEM16s are highly conserved across the family. However, despite this conservation, TMEM16s differ drastically in their sensitivity to Ca2+ (e.g., Ca2+ sensitivity differs ∼1,000-fold between TMEM16s A and F). If the mechanism by which pHi regulates TMEM16s involves these same Ca2+-coordinating residues, how can we trust that pHi sensitivity would be the same between relatives when Ca2+ sensitivity is not? Moreover, Liang and Yang (2021) demonstrate that increased Ca2+ can overcome pHi inhibition. If some TMEM16s are ∼1,000-fold more sensitive to Ca2+, is it reasonable to expect that all would escape pHi regulation at the same Ca2+ concentration? Determining the pHi sensitivity of different TMEM16s may help further resolve how the same cell can regulate the activities of several TMEM16s at once. Perhaps evolution’s differential tuning of these shared TMEM16 regulatory mechanisms has ensured that some TMEM16s never “see” the activating signals of their relatives, even though they are within the same membrane of the same cell.

Although TMEM16F and other TMEM16 scramblases are expressed in a diverse array of cell types and tissues, we have now begun to appreciate several unique mechanisms that regulate the location, magnitude, and duration of their activities. Together, these disparate regulatory factors come together to create a “TMEM16 scramblase module” employed by many cells and used to uniquely tame, or tune, TMEM16-dependent scrambling to fit a cell’s individual biological niche (Fig. 1).

It should be noted that there is an independent PL scrambling pathway associated with apoptotic caspase activity and the Xk-related protein family (Suzuki et al., 2014). Interestingly, this apoptotic scrambling is also modulated by pHi but in the opposite direction described above, with lower pHi promoting exofacial PS exposure. Importantly, this response is observed in cells from healthy donors and those from Scott’s syndrome patients lacking TMEM16F, demonstrating that this is indeed an alternative scrambling response to that described by Liang and Yang (Stout et al., 1997). Caspase- and Ca2+-activated scrambling pathways are parallel yet independent processes and differ substantially in a number of ways, including the amount of time between activation of the pathway and PS exposure (hours versus minutes), the distribution of the PS each expose in native cells (whole membrane versus patches), and likely the reversibility of PS exposure (caspases cleave PS transporters while Ca2+ reversibly inhibits them), and here Liang and Yang (2021) demonstrate that Ca2+-activated PM scrambling differs in its response to pHi compared with caspase-activated PM scrambling. Moving forward, it will be important to consider the differences between these scramblase pathways that both result in the exposure of cytofacial lipids and altered PM physical properties.

And now for the most important question: how do cells, tissues, and systems employ TMEM16 scramblase regulation for biological purposes (Fig. 2)?

How does the taming of a scramblase affect biological processes?

As the list of methods available to cells for taming TMEM16 scramblase function grow, we are left with a major question: how are these regulatory mechanisms employed in the local domestication of scramblases in biologically relevant contexts? In light of this latest contribution by Liang and Yang (2021), a few biological processes come to mind.

In addition to platelet activation, nonapoptotic PS exposure is a reoccurring signal employed in the coordination of cell–cell fusion processes (Whitlock and Chernomordik, 2021). The role of TMEM16 scramblases has been linked to the fusion of gametes, skeletal muscle, osteoclasts, placental trophoblasts, and virus–cell fusion. These could be ideal systems to dissect the mechanistic contribution of pHi in the exposure of PS required for these fusion events. In particular, during capacitation and/or the acrosome reaction, PS is exposed on the heads of sperm and is required for fertilization (Rival et al., 2019). Interestingly, during capacitation and/or the acrosome reaction, sperm both alkalinize their cytosol and increase its Ca2+ concentration simultaneously (Chávez et al., 2018). TMEM16E, a close scramblase relative of TMEM16F, is highly expressed in sperm, and its loss perturbs murine fertility. Characterization of pHi in fertilization and its impact on TMEM16E function may offer insight into how mammalian gametes prepare for fusion and better inform our understanding of the regulatory processes surrounding human fertility.

Platelets, red cells, and sperm each exhibit high steady-state levels of a particular TMEM16 scramblase, but many cell types exhibit abundant levels of several TMEM16 scramblases simultaneously. For example, we previously demonstrated that skeletal muscle exhibits high levels of both TMEM16s E and F (Whitlock et al., 2018). What advantage would one cell gain by maintaining multiple scramblases that are each activated by a single, shared signal? Upon increases in cytosolic Ca2+, each of these TMEM16 scramblases should be activated simultaneously. However, we found that loss of TMEM16E perturbed Ca2+-activated PS exposure in primary muscle cells despite high levels of TMEM16F, suggesting that the two play distinct roles in the same cell type. At the time, we assumed TMEM16s E and F were functionally separated in muscle by differences in Ca2+ sensitivity and/or trafficking, but could the function of these scramblase cousins be separated by pHi? pHi is rather distinct in skeletal muscle as compared with other, nonexcitable cell types with a pHi of ∼6 at resting membrane potential (Carter et al., 1967). Depending on the Ca2+ concentration, the observations of Liang and Yang (2021) would suggest that TMEM16F could be entirely inhibited in resting skeletal muscle fibers. This idea is of course speculative but serves to illustrate how nodes within the TMEM16 scramblase module (Fig. 1) may serve to functionally separate TMEM16s in the same cell. One can easily assert that because the Ca2+ binding sites of TMEM16s F and E are so highly conserved, their pHi sensitivity should be similar. After all, these are the same residues where pHi likely exerts its regulatory effect in both. However, we found that TMEM16E appears to be much more sensitive to Ca2+ than TMEM16F (∼50% of cells expressing TMEM16E scramble in response to 1 µM Ca2+, while >100 µM Ca2+ was required to achieve a similar response in TMEM16F expressing cells; Whitlock et al., 2018). If these relatives, with highly conserved Ca2+ binding sites, can differ dramatically in their sensitivity to Ca2+, how can we assume they will not differ in their sensitivity to pHi? By moving past Ca2+, TMEM16 machines may have afforded themselves greater utility and promoted the diversity of their family in many cells.

Apart from healthy, physiological processes, pathogens may also use pHi to tame TMEM16 scramblase function. PD-1 is an immunoregulatory protein that, when exposed on the surface of a variety of different cell types, dampens T cell activation and prevents self-reactive, pathogenic T cells. Perturbed, excessive exposure of PD-1 at the cell surface is a major contributor to T cell exhaustion in chronic HIV-1 infection (Porichis and Kaufmann, 2012). Interestingly, mice lacking TMEM16F also experience T cell exhaustion, and TMEM16F activity appears to play an important role in regulating the trafficking of PD-1 to the PM (Hu et al., 2016; Bricogne et al., 2019). Our laboratory has demonstrated that HIV activates TMEM16F and utilizes exposed PS as a cofactor in the fusion of the viral envelope with the PM (Zaitseva et al., 2017). After HIV gains entry into the cell, the HIV protein Vpr specifically down-regulates the expression of NHEI and acidifies the cytosol of infected cells (Janket et al., 2007). If the reader will permit some additional speculation, acidification may be a mechanism by which HIV inhibits TMEM16F in infected cells by downregulating NHEI and acidifying the cytosol. Inhibiting TMEM16F would likely contribute to T cell exhaustion, just as observed in the TMEM16F knockout model, and facilitate the ability of HIV-infected cells to avoid detection by the immune system, likely through increased trafficking of PD-1 to the PM. Perhaps HIV is a master scramblase tamer, both activating TMEM16F to gain cell entry and silencing the scramblase once in the cell?

In this reader’s view, Liang and Yang’s observations in the earlier issue of the Journal of General Physiology are tightly focused, but the consequences of these observations are broadly reaching (Liang and Yang, 2021). For what purpose do we tame something if not to leverage it for our goals? Over the last decade, we have learned much about the business of taming a scramblase. In the next, let us resolve what that taming is for (Fig. 2).

Crina M. Nimigean served as editor.

The author thanks the work of dozens of laboratories whose contributions were not appropriately acknowledged here due to length restrictions. In spite of the many open questions facing our field, we have entered a golden age of the scramblase. Bethany G. Whitlock contributed mightily to the construction of the figures herein. Moreover, Dr. Criss Hartzell offered much appreciated critical feedback that greatly improved the final manuscript.

The research of J.M. Whitlock is supported by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health.

The author declares no competing financial interests.

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