Ca2+-Activated Chloride Channels and Phospholipid Scramblases

The functional characterization of the TMEM16 protein family unexpectedly broughttogether two different research fields in membrane biology: anion channel and membranelipid organization. Almost 40 years ago, Miledi described for the first time the presence ofion channels allowing the permeation of chloride ions activated by intracellular Ca2+ onthe plasma membrane of Xenopus laevis oocytes [ 1]. In the same year, the investigation ofplatelets activation revealed the presence of Ca2+-dependent mechanisms mediating theexposure phosphatidylserine on the outer leaflet of the membrane dissipating the lipidasymmetry [2, 3]. Later, the term "scramblase" was proposed for the proteins mediatingthis process.Further investigations shows that both Ca2+-activated Cl ? channels (CaCCs) andscramblases are expressed in various tissues playing important physiological roles. Inparticular, CaCCs are involved in the secretion of different types of exocrine glands; regulatethe contraction of vascular smooth muscle cells relevant to the modulation of blood pressure;control the chloride secretion in different epithelia functionally interacting with the cysticfibrosis transmembrane regulator (CFTR); and modulate the neuronal firing activity andthe sensory transduction in olfactory systems [ 4, 5]. Similarly, phospholipid scramblase hasa pivotal role in the blood coagulation and in the removal of apoptotic cells [6].In 2008 and 2010, the molecular identities of Ca2+-activated Cl ? channels and phospho-lipid scramblases were discovered [ 7- 10]. Different expression cloning approaches revealedthat two members of the "transmembrane proteins with unknown function 16", TMEM16,encode for CaCCs, and at least one member of the same family forms the phospholipidscramblase [ 7 -10 ]. These seminal discoveries opened the possibility of investigation at themolecular level of CaCCs and phospholipid scramblases.Today, we know that in mammals, the TMEM16 family, also known as the anoc-tamin family, is composed of 10 members with different functions and physiological roles(Figure 1) [11,12]. TMEM16A and B are classical CaCCs expressed mainly in epithelial andneuronal cell types, respectively [ 13]. TMEME16C, D, E, F, G, J and K are phospholipidscramblases [ 14 - 16]. Moreover, TMEM16D, E and F are also Ca2+-activated ion chan-nels [17-19 ], and TMEM16J is an ion channel activated by the cAMP/PKA pathway [ 20 ].Finally, TMEM16C modulates the activity of Na+-activated K+ channels [21]. Many of theTMEM16 proteins are involved in human diseases. In particular, mutations in TMEM16Ccause craniocervical dystonia [22], TMEM16E cause gnatodiaphyseal dysplasia [23 ] andmuscular dystrophy [24 ], mutations in TMEM16F are responsible for Scott syndrome [ 16],while a form of spinocerebellar ataxia is due to mutations in TMEM16K [ 25]. Moreover,many data show that TMEM16 is involved in cell proliferation and is overexpressed inseveral types of cancer [26 ]. TMEM16A can also contributes to pathogenesis of cysticfibrosis by a complex functional interplay with CFTR [ 27 ]. Finally, TMEM16E, G and J arealso involved in several types of cancer [28-31].In the Special Issue of International Journal of Molecular Sciences "Ca2+-Activated Chlo-ride Channels and Phospholipid Scramblases", we edited several papers bringing newlight on the function of this interesting protein family.Choi et al. reported that TMEM16A could be involved in psoriasis pathogenesis [32].Psoriasis, affecting about 2% of the human population, is a multifactorial skin diseasecausing erythematous plaques, papules and pruritus [ 33]. Psoriatic skin shows bothhyperplasia of the epidermis caused by over-proliferation of keratinocytes, and alterationin the proinflammatory response [33 ]. Choi et al. find that TMEM16A is overexpressedin psoriatic skin from human subjects. Pharmacological blockage and gene silencing ofTMEM16A reduces the proliferation of the human keratocytes cell line HaCaT. Moreover,the inhibition TMEM16A decreased the psoriatic symptoms in a pharmacological-inducedpsoriasis mouse model. This effect could be partially due to a reduction of proinflammatorycytokines production and inhibition of AKT/ERK pathways [ 32]. These results confirm therelevant role on TMEM16A in cell proliferation and can be important to find new targetsfor psoriasis treatment.Centerio et al. find that the CLCA1 protein controls the airway mucus productionby modulating TMEM16A [34 ]. CLCA1 is a secreted protein that stabilizes and increasesthe membrane expression of TMEM16A in several tissues [ 35 ]. Therefore, the interplaybetween CLCA1 and TMEM16A represents a novel and interesting approach to modulatethe CaCCs activity. Centerio et al. report that in mice, the application of CLCA in theairway does not increase the membrane expression of TMEM16A, however it provokes asignificant increase in mucus production. Interestingly, mucus production mediated byCLCA1 application is further increased in the mouse model of asthma. Moreover, with anin vitro model of human airway epithelium, they show that mucus production induced byInt. J. Mol. Sci. 2022, 23, 2158 3 of 6CLCA1 is dependent on TMEM16A expression without an increase of ion secretion. Finally,the proinflammatory cytokine IL-13 upregulates the expression of CLCA1, enhancingmucus production. These data provide a foundation for future work investigating theprecise functional interaction between TMEM16A and CLCA1 in airway epithelia.Seo et al. identified a new blocker for TMEM16A showing a proapoptic effect onlung cancer cells [36 ]. The pharmacology of the TMEM16 protein is still rudimental; veryfew specific blockers or agonists have been reported, and for most of them, the molecularmechanisms of blockage or activation are still unknown [37,38].Using a high-throughput approach using the halide sensitive YFP, Seo et al. identified anew blocker of TMEM16A, diethelstilbestrol (DES). Unfortunately, DES also partially blocksTMEM16B. Considering that TMEM16A is overexpressed in some types of lung cancers [ 39 ],they screened several cell lines derived from human lung cancer for TMEM16A expression.PC9 cells show a high level of TMEM16A. DES significantly reduces the proliferation andmigration of PC9 cells, whereas a smaller response is observed in H1975 cells lackingthe TMEM16A expression. Interestingly, DES does not only block the current mediatedby TMEM16A, but it also reduces the protein expression after chronic application for 72h. Moreover, DES inhibits the EGFR and ERK pathway that are involved in TMEM16A-mediated cell proliferation [40 ,41 ]. Finally, they find that DES is able to induce apoptoticcell death in PC9 cells. These results show the possibility to pharmacologically control theTMEM16A expression and could be important to finding new targets for cancer treatment.Ko and Suh investigated the role of membrane PI(4,5)P2 in controlling the TMEM16Agating [ 42]. Membrane lipids, and particularly the phosphoinositides, play a complexrole in regulation of the TMEM16 proteins [ 43 -46 ]. Here, Ko and Suh show that PI(4,5)P2depletion inhibits TMEM16A depending on the splice variant. In particular, they find thatonly the isoform containing the exon c, coding a short stretch of four amino acids (EAVK), isinhibited by PI(4,5)P2 hydrolysis. This effect is specific for PI(4,5)P2, since it is not observedby reducing the concentration of PI(3,4,5)P3 and PI4P. Activation of PLC mediated cascadethrough the M1 muscarinic receptor induced the same effect of the PI(4,5)P2 depletion,indicating that this modulation could be physiologically relevant. This study again showsthe intricated pathway controlling the gating of TMEM16A.The complexity of the mechanisms controlling the opening of TMEM16A and otherTMEM16 proteins is fully explored in the comprehensive review by Agostinelli and Tam-maro [ 47]. They highlight how different stimuli, such as Ca2+, voltage, low extracellularpH, heat, membrane lipids, etc., regulate the gating of TMEM16 proteins [ 47]. Theyalso review the recent structures of some TMEM16 proteins starting to build a model ofTMEM16 gating.The remaining two papers of this Special Issue deal with TMEM16F, a phospho-lipid scramblase that also mediates ion channel activity [48 ,49 ]. A still debated aspect ofTMEM16F function as an ion channel is its ionic selectivity [ 50 ,51 ]. Indeed, even if allstudies generally agree that TMEM16F is a poorly selective channel, some results obtainedin whole-cell recordings from TMEM16F heterologously expressed in HEK-293 cells showa higher permeability to Cl ? than Na+ [ 52- 54 ]. In contrast, inside-out experiments indicatethat TMEM16F is more permeable to cations than anions [19,55,56].Stabilini et al. performed a detailed side-by-side comparison of electrophysiologicalproperties of TMEM16F recorded in inside-out and whole-cell configuration [ 49]. Theyfound that TMEM16F shows different behaviors depending on the recording method. Inparticular, in both conditions, TMEM16F is activated by ?M of intracellular Ca2+, but inwhole-cell configuration, TMEM16-meditated current develops with several seconds ofdelay that is not observed in inside-out. Moreover, they found that in whole-cell recordings,TMEM16F has a slight preference for anions; indeed, the permeability ration betweenNa+ and Cl (PNa/PCl) is 0.4, whereas in inside-out, PNa/PCl is 3.7, indicating a higherNa+ permeability.These results could, at least partially, be explained by the role of Ca2+ investigated byNguyen et al. [48 ]. They found that Ca2+ and other divalents in the millimolar range canInt. J. Mol. Sci. 2022, 23, 2158 4 of 6modulate TMEM16A and 16F-mediated current. This effect depends on the membrane con-centration of PI(4,5)P2. Interestingly, in the Q559W mutant of TMEM16F, the intracellularapplication of millimoles of Ca2+ significantly reduced the permeability to Na+. Based onstructural data, the author proposes that the gating of TMEM16A and 16F creates a groovein the protein big enough for the entry of PI(4,5)P2, where divalents can also enter, partiallyshielding the negative charges. The alteration of the local electrical field affects the ionselectivity. The bigger effects observed in TMEM16F could be due to its intrinsic lowerselectivity with respect to TMEM16A. Further experiments will clarify this mechanism andthe relevance for the function of other TMEM16 proteins.All these new studies clearly show the complexity and versatility of the cellularprocesses mediated by Ca2+-activated chloride channels and phospholipid scramblases.We hope that in the near future we can gain a better understanding of TMEM16 physiologynecessary to help treat the human diseases caused by TMEM16 mutation or mis-regulation.