The functional characterization of the TMEM16 protein family unexpectedly brought together two different research fields in membrane biology: anion channel and membrane lipid organization. Almost 40 years ago, Miledi described for the first time the presence of ion channels allowing the permeation of chloride ions activated by intracellular Ca2+ on the plasma membrane of Xenopus laevis oocytes [ 1]. In the same year, the investigation of platelets activation revealed the presence of Ca2+-dependent mechanisms mediating the exposure phosphatidylserine on the outer leaflet of the membrane dissipating the lipid asymmetry [2, 3]. Later, the term "scramblase" was proposed for the proteins mediating this process. Further investigations shows that both Ca2+-activated Cl ? channels (CaCCs) and scramblases are expressed in various tissues playing important physiological roles. In particular, CaCCs are involved in the secretion of different types of exocrine glands; regulate the contraction of vascular smooth muscle cells relevant to the modulation of blood pressure; control the chloride secretion in different epithelia functionally interacting with the cystic fibrosis transmembrane regulator (CFTR); and modulate the neuronal firing activity and the sensory transduction in olfactory systems [ 4, 5]. Similarly, phospholipid scramblase has a 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 revealed that 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 phospholipid scramblase [ 7 -10 ]. These seminal discoveries opened the possibility of investigation at the molecular 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 and neuronal cell types, respectively [ 13]. TMEME16C, D, E, F, G, J and K are phospholipid scramblases [ 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 the TMEM16 proteins are involved in human diseases. In particular, mutations in TMEM16C cause craniocervical dystonia [22], TMEM16E cause gnatodiaphyseal dysplasia [23 ] and muscular 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 in several types of cancer [26 ]. TMEM16A can also contributes to pathogenesis of cystic fibrosis by a complex functional interplay with CFTR [ 27 ]. Finally, TMEM16E, G and J are also 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 new light 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 disease causing erythematous plaques, papules and pruritus [ 33]. Psoriatic skin shows both hyperplasia of the epidermis caused by over-proliferation of keratinocytes, and alteration in the proinflammatory response [33 ]. Choi et al. find that TMEM16A is overexpressed in psoriatic skin from human subjects. Pharmacological blockage and gene silencing of TMEM16A reduces the proliferation of the human keratocytes cell line HaCaT. Moreover, the inhibition TMEM16A decreased the psoriatic symptoms in a pharmacological-induced psoriasis mouse model. This effect could be partially due to a reduction of proinflammatory cytokines production and inhibition of AKT/ERK pathways [ 32]. These results confirm the relevant role on TMEM16A in cell proliferation and can be important to find new targets for psoriasis treatment. Centerio et al. find that the CLCA1 protein controls the airway mucus production by modulating TMEM16A [34 ]. CLCA1 is a secreted protein that stabilizes and increases the membrane expression of TMEM16A in several tissues [ 35 ]. Therefore, the interplay between CLCA1 and TMEM16A represents a novel and interesting approach to modulate the CaCCs activity. Centerio et al. report that in mice, the application of CLCA in the airway does not increase the membrane expression of TMEM16A, however it provokes a significant increase in mucus production. Interestingly, mucus production mediated by CLCA1 application is further increased in the mouse model of asthma. Moreover, with an in vitro model of human airway epithelium, they show that mucus production induced by Int. J. Mol. Sci. 2022, 23, 2158 3 of 6 CLCA1 is dependent on TMEM16A expression without an increase of ion secretion. Finally, the proinflammatory cytokine IL-13 upregulates the expression of CLCA1, enhancing mucus production. These data provide a foundation for future work investigating the precise functional interaction between TMEM16A and CLCA1 in airway epithelia. Seo et al. identified a new blocker for TMEM16A showing a proapoptic effect on lung cancer cells [36 ]. The pharmacology of the TMEM16 protein is still rudimental; very few specific blockers or agonists have been reported, and for most of them, the molecular mechanisms of blockage or activation are still unknown [37,38]. Using a high-throughput approach using the halide sensitive YFP, Seo et al. identified a new blocker of TMEM16A, diethelstilbestrol (DES). Unfortunately, DES also partially blocks TMEM16B. 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 and migration of PC9 cells, whereas a smaller response is observed in H1975 cells lacking the TMEM16A expression. Interestingly, DES does not only block the current mediated by TMEM16A, but it also reduces the protein expression after chronic application for 72 h. 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 apoptotic cell death in PC9 cells. These results show the possibility to pharmacologically control the TMEM16A 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 TMEM16A gating [ 42]. Membrane lipids, and particularly the phosphoinositides, play a complex role in regulation of the TMEM16 proteins [ 43 -46 ]. Here, Ko and Suh show that PI(4,5)P2 depletion inhibits TMEM16A depending on the splice variant. In particular, they find that only the isoform containing the exon c, coding a short stretch of four amino acids (EAVK), is inhibited by PI(4,5)P2 hydrolysis. This effect is specific for PI(4,5)P2, since it is not observed by reducing the concentration of PI(3,4,5)P3 and PI4P. Activation of PLC mediated cascade through 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 shows the intricated pathway controlling the gating of TMEM16A. The complexity of the mechanisms controlling the opening of TMEM16A and other TMEM16 proteins is fully explored in the comprehensive review by Agostinelli and Tam- maro [ 47]. They highlight how different stimuli, such as Ca2+, voltage, low extracellular pH, heat, membrane lipids, etc., regulate the gating of TMEM16 proteins [ 47]. They also review the recent structures of some TMEM16 proteins starting to build a model of TMEM16 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 of TMEM16F function as an ion channel is its ionic selectivity [ 50 ,51 ]. Indeed, even if all studies generally agree that TMEM16F is a poorly selective channel, some results obtained in whole-cell recordings from TMEM16F heterologously expressed in HEK-293 cells show a higher permeability to Cl ? than Na+ [ 52- 54 ]. In contrast, inside-out experiments indicate that TMEM16F is more permeable to cations than anions [19,55,56]. Stabilini et al. performed a detailed side-by-side comparison of electrophysiological properties of TMEM16F recorded in inside-out and whole-cell configuration [ 49]. They found that TMEM16F shows different behaviors depending on the recording method. In particular, in both conditions, TMEM16F is activated by ?M of intracellular Ca2+, but in whole-cell configuration, TMEM16-meditated current develops with several seconds of delay 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 between Na+ and Cl (PNa/PCl) is 0.4, whereas in inside-out, PNa/PCl is 3.7, indicating a higher Na+ permeability. These results could, at least partially, be explained by the role of Ca2+ investigated by Nguyen et al. [48 ]. They found that Ca2+ and other divalents in the millimolar range can Int. J. Mol. Sci. 2022, 23, 2158 4 of 6 modulate 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 intracellular application of millimoles of Ca2+ significantly reduced the permeability to Na+. Based on structural data, the author proposes that the gating of TMEM16A and 16F creates a groove in the protein big enough for the entry of PI(4,5)P2, where divalents can also enter, partially shielding the negative charges. The alteration of the local electrical field affects the ion selectivity. The bigger effects observed in TMEM16F could be due to its intrinsic lower selectivity with respect to TMEM16A. Further experiments will clarify this mechanism and the relevance for the function of other TMEM16 proteins. All these new studies clearly show the complexity and versatility of the cellular processes mediated by Ca2+-activated chloride channels and phospholipid scramblases. We hope that in the near future we can gain a better understanding of TMEM16 physiology necessary to help treat the human diseases caused by TMEM16 mutation or mis-regulation.

The brain is a complex organ composed of billions of neurons connected through excitatory and inhibitory synapses. Its structure reveals a modular topological organization, where neurons are arranged in interconnected assemblies. The generated patterns of electrophysiological activity are shaped by two main factors: network heterogeneity and the topological properties of the underlying connectivity that strongly push the dynamics toward different brain-states. In this work, we exploited an innovative polymeric structure coupled to Micro-Electrode Arrays (MEAs) to recreate in vitro heterogeneous interconnected (modular) neuronal networks made up of cortical and hippocampal neurons. We investigated the propagation of spike sequences between the two interconnected subpopulations during the networks' development, correlating functional and structural connectivity to dynamics. The simultaneous presence of two neuronal types shaped the features of the functional connections (excitation vs. inhibition), orchestrating the emerging patterns of electrophysiological activity. In particular, we found that hippocampal neurons mostly project inhibitory connections toward the cortical counterpart modulating the temporal scale of the population events (network bursts). In contrast, cortical neurons establish a larger amount of intrapopulation connections. Moreover, we proved topological properties such as small-worldness, degree distribution, and modularity of neuronal assemblies were favored by the physical environment where networks developed and matured.

Aims: To elucidate the mechanism by which (-)-epigallocatechin-3-gallate (EGCG) mediates intracellular Ca2+ increase in androgen-independent prostate cancer (PCa) cells. Main methods: Following exposure to different doses of EGCG, viability of DU145 and PC3 PCa cells was evaluated by MTT assay and the intracellular Ca2+ dynamics by the fluorescent Ca2+ chelator Fura-2. The expression of different channels was investigated by qPCR analysis and sulfhydryl bonds by Ellman's assay. Key findings: EGCG inhibited DU145 and PC3 proliferation with IC50, = 46 and 56 mu M, respectively, and induced dose-dependent peaks of internal Ca2+ that were dependent on extracellular Ca2+. The expression of TRPC4 and TRPC6 channels was revealed by qPCR in PC3 cells, but lack of effect by modulators and blockers ruled out an exclusive role for these, as well as for voltage-dependent T-type Ca2+ channels. Application of dithiothreitol and catalase and sulfhydryl (SH) measurements showed that EGCG-induced Ca2+ rise depends on SH oxidation, while the effect of EGTA, dantrolene, and the PLC inhibitor U73122 suggested that EGCG-induced Ca2+ influx acts as a trigger for Ca2+-induced Ca2+ release, involving both ryanodine and IP3 receptors. Different from EGCG, ATP caused a rapid Ca2+ increase, which was independent of external Ca2+, but sensitive to U73122. Significance: EGCG induces an internal Ca2+ increase in PCa cells by a multi-step mechanism. As dysregulation of cytosolic Ca2+ is directly linked to apoptosis in PCa cells, these data confirm the possibility of using EGCG as a synergistic adjuvant in combined therapies for recalcitrant malignancies like androgen-independent PCa.

The double photoionization of Mg has been studied experimentally and theoretically in a kinematic where the two photoelectrons equally share the excess energy. The observation of a symmetrized gerade amplitude, which strongly deviates from the Gaussian ansatz, is explained by a two-electron interference predicted theoretically, but never before observed experimentally. Similar to the Cooper minima in the single photoionization cross section, the effect finds its origin in the radial extent and oscillation of the target wave function.

A Ca(2+)-activated Cl(-) current constitutes a large part of the transduction current in olfactory sensory neurons. The binding of odorants to olfactory receptors in the cilia produces an increase in cAMP concentration; Ca(2+) enters into the cilia through CNG channels and activates a Cl(-) current. In intact mouse olfactory sensory neurons little is known about the kinetics of the Ca(2+)-activated Cl(-) current. Here, we directly activated CNG channels by flash photolysis of caged cAMP or 8-Br-cAMP and measured the current response with the whole cell voltage-clamp technique in mouse neurons. We measured multiphasic currents in the rising phase of the response at -50 mV. The current rising phase became monophasic in the absence of extracellular Ca(2+), at +50 mV, or when most of the intracellular Cl(-) was replaced by gluconate to shift the equilibrium potential for Cl(-) to -50 mV. These results show that the second phase of the current in mouse intact neurons is attributed to a Cl(-) current activated by Ca(2+), similarly to previous results on isolated frog cilia. The percentage of the total saturating current carried by Cl(-) was estimated in two ways: 1) by measuring the maximum secondary current and 2) by blocking the Cl(-) channel with niflumic acid. We estimated that in the presence of 1 mM extracellular Ca(2+) and in symmetrical Cl(-) concentrations the Cl(-) component can constitute up to 90% of the total current response. These data show how to unravel the CNG and Ca(2+)-activated Cl(-) component of the current rising phase.

Vertebrate olfactory sensory neurons rapidly adapt to repetitive odorant stimuli. Previous studies have shown that the principal molecular mechanisms for odorant adaptation take place after the odorant-induced production of cAMP, and that one important mechanism is the negative feedback modulation by Ca2+-calmodulin (Ca2+-CaM) of the cyclic nucleotide-gated (CNG) channel. However, the physiological role of the Ca2+-dependent activity of phosphodiesterase (PDE) in adaptation has not been investigated yet. We used the whole-cell voltage-clamp technique to record currents in mouse olfactory sensory neurons elicited by photorelease of 8-Br-cAMP, an analogue of cAMP commonly used as a hydrolysis-resistant compound and known to be a potent agonist of the olfactory CNG channel. We measured currents in response to repetitive photoreleases of cAMP or of 8-Br-cAMP and we observed similar adaptation in response to the second stimulus. Control experiments were conducted in the presence of the PDE inhibitor IBMX, confirming that an increase in PDE activity was not involved in the response decrease. Since the total current activated by 8-Br-cAMP, as well as that physiologically induced by odorants, is composed not only of current carried by Na+ and Ca2+ through CNG channels, but also by a Ca2+-activated Cl- current, we performed control experiments in which the reversal potential of Cl- was set, by ion substitution, at the same value of the holding potential, -50 mV. Adaptation was measured also in these conditions of diminished Ca2+-activated Cl- current. Furthermore, by producing repetitive increases of ciliary's Ca2+ with flash photolysis of caged Ca2+, we showed that Ca2+-activated Cl- channels do not adapt and that there is no Cl- depletion in the cilia. All together, these results indicate that the activity of ciliary PDE is not required for fast adaptation to repetitive stimuli in mouse olfactory sensory neurons.

Sorcin, a 21.6 kDa two-domain penta-EF-hand (PEF) protein, when activated by Ca2+ binding, interacts with target proteins in a largely uncharacterized process. The two physiological EF-hands EF3 and EF2 do not belong to a structural pair but are connected by the D helix. To establish whether this helix is instrumental in sorcin activation, two D helix residues were mutated: W105, located near EF3 and involved in a network of interactions, and W99, located near EF2 and facing solvent, were substituted with glycine. Neither mutation alters calcium affinity. The interaction of the W105G and W99G mutants with annexin VII and the cardiac ryanodine receptor (RyR2), requiring the sorcin N- terminal and C-terminal domain, respectively, was studied. Surface plasmon resonance experiments show that binding of annexin VII to W99G occurs at the same Ca2+ concentration as that of the wild type, whereas W105G requires a significantly higher Ca2+ concentration. Ca2+ spark activity of isolated heart cells monitors the sorcin-RyR2 interaction and is unaltered by W105G but is reduced equally by W99G and the wild type. Thus, substitution of W105, via disruption of the network of D helix interactions, affects the capacity of sorcin to recognize and interact with either target at physiological Ca2+ concentrations, while mutation of solvent-facing W99 has little effect. The D helix appears to amplify the localized structural changes that occur at EF3 upon Ca2+ binding and thereby trigger a structural rearrangement that enables interaction of sorcin with its molecular targets. The same activation process may apply to other PEF proteins in view of the D helix conservation.

X-ray diffraction and infrared absorption analyses have been carried out on zinc-substituted and strontium-substituted beta-tricalcium phosphate prepared by solid-state reaction. Zinc can substitute calcium up to 20 atom %, inducing a nonlinear variation of the lattice constants and an increase in degeneracy of the PO43- infrared absorption bands. On the other hand, up to 80 atom % of strontium can enter into the crystal structure of beta-tricalcium phosphate, causing a linear enlargement of the unit cell, in agreement with its greater ionic radius compared to that of calcium. Furthermore, strontium incorporation provokes the shift of the PO43- absorption bands toward lower frequencies. On the basis of the data previously obtained on magnesium-substituted beta-tricalcium phosphate, the different behaviours exhibited by zinc and strontium could be attributed to a different distribution into the cationic sites of the beta-tricalcium phosphate structure. The results allow us to relate the effect of bivalent ions on the structure and relative stability of calcium phosphates with their ionic radius, and can be utilized to interpret the role of ionic composition on the properties of biological phosphates. (C) 1997 Elsevier Science Inc.

Anandamide (AnNH, N-arachidonoyl-ethanolamine) has been recently proposed as the endogenous ligand for mammalian brain cannabinoid receptor. Non-cannabinoid receptor-mediated, intracellular actions have been also found for this novel mediator. Here we present evidence for the modulation by anandamide of rat brain protein kinase C (PKC) activity in vitro. The ethanolamide of arachidonic acid (AA) was more active than the free acid in increasing phosphatidylserine (PS)-induced PKC activation (EC50 = 40 microM), but inhibited dioleylglycerol-induced potentiation of both Ca(2+)- and Ca2+/PS-induced PKC activation (IC50 = 8 microM and 30 microM, respectively). A dual modulatory action of anandamide on PKC, exerted by binding to the diacylglycerol regulatory site, is hypothesized in rat brain.