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It is important for medical students to understand the relationship between nutrition, obesity, and diabetes to educate their patients in the future. However, medical training does not always include nutritional education. An experiential learning project was incorporated into the medical school curriculum as an effort to implement nutrition in the physiology course. First-year medical students ( n = 140) received lectures on the regulation of blood glucose levels and their relationship to carbohydrates with different glycemic indexes (GI), obesity, and diabetes. Lectures were followed by a laboratory exercise where students calculated their body mass index (BMI), percentage body fat, and percentage muscle using a Bioelectrical Impedance Commercial Scale. While 63% of students had normal BMI, 31% were overweight or obese and 5% were underweight. A subgroup of 54 students tested different types of breakfasts with varying GI and provided blood samples at 0, 30, 60, 90, and 120 min. Their glucose responses were plotted based on the breakfast GI. Pre- and posttests were conducted to assess the teaching intervention where the Wilcoxon signed ranks test indicated that posttest ranks were significantly higher than pretest ranks (Z = −6.6, P < 0.001), suggesting the intervention was beneficial to students.

Various human tissues express the calcium-activated chloride channel Anoctamin 1 (ANO1), also known as TMEM16A. ANO1 allows the passive chloride flux that controls different physiological functions ranging from muscle contraction, fluid and hormone secretion, gastrointestinal motility, and electrical excitability. Overexpression of ANO1 is associated with pathological conditions such as hypertension and cancer. The molecular cloning of ANO1 has led to a surge in structural, functional, and physiological studies of the channel in several tissues. ANO1 is a homodimer channel harboring two pores - one in each monomer - that work independently. Each pore is activated by voltage-dependent binding of two intracellular calcium ions to a high-affinity-binding site. In addition, the binding of phosphatidylinositol 4,5-bisphosphate to sites scattered throughout the cytosolic side of the protein aids the calcium activation process. Furthermore, many pharmacological studies have established ANO1 as a target of promising compounds that could treat several illnesses. This chapter describes our current understanding of the physiological roles of ANO1 and its regulation under physiological conditions as well as new pharmacological compounds with potential therapeutic applications.

Anoctamin-1 (ANO1 or TMEM16A) is a homo-dimeric Ca2+-activated Cl− channel responsible for essential physiological processes. Each monomer harbours a pore and a Ca2+-binding pocket; the voltage-dependent binding of two intracellular Ca2+ ions to the pocket gates the pore. However, in the absence of intracellular Ca2+ voltage activates TMEM16A by an unknown mechanism. Here we show voltage-activated anion currents that are outwardly rectifying, time-independent with fast or absent tail currents that are inhibited by tannic and anthracene-9-carboxylic acids. Since intracellular protons compete with Ca2+ for binding sites in the pocket, we hypothesized that voltage-dependent titration of these sites would induce gating. Indeed intracellular acidification enabled activation of TMEM16A by voltage-dependent protonation, which enhanced the open probability of the channel. Mutating Glu/Asp residues in the Ca2+-binding pocket to glutamine (to resemble a permanent protonated Glu) yielded channels that were easier to activate at physiological pH. Notably, the response of these mutants to intracellular acidification was diminished and became voltage-independent. Thus, voltage-dependent protonation of glutamate/aspartate residues (Glu/Asp) located in the Ca2+-binding pocket underlines TMEM16A activation in the absence of intracellular Ca2+.

Numerous essential physiological processes depend on the TMEM16A-mediated Ca2+-activated chloride fluxes. Extensive structure–function studies have helped to elucidate the Ca2+ gating mechanism of TMEM16A, revealing a Ca2+-sensing element close to the anion pore that alters conduction. However, substrate selection and the substrate–gating relationship in TMEM16A remain less explored. Here, we study the gating–permeant anion relationship on mouse TMEM16A expressed in HEK 293 cells using electrophysiological recordings coupled with site-directed mutagenesis. We show that the apparent Ca2+ sensitivity of TMEM16A increased with highly permeant anions and SCN− mole fractions, likely by stabilizing bound Ca2+. Conversely, mutations at crucial gating elements, including the Ca2+-binding site 1, the transmembrane helix 6 (TM6), and the hydrophobic gate, impaired the anion permeability and selectivity of TMEM16A. Finally, we found that, unlike anion-selective wild-type channels, the voltage dependence of unselective TMEM16A mutant channels was less sensitive to SCN−. Therefore, our work identifies structural determinants of selectivity at the Ca2+ site, TM6, and hydrophobic gate and reveals a reciprocal regulation of gating and selectivity. We suggest that this regulation is essential to set ionic selectivity and the Ca2+ and voltage sensitivities in TMEM16A.

Chloride fluxes through the calcium-gated chloride channel Anoctamin-1 (TMEM16A) control blood pressure, secretion of saliva, mucin, insulin, and melatonin, gastrointestinal motility, sperm capacitation and motility, and pain sensation. Calcium activates a myriad of regulatory proteins but how these proteins affect TMEM16A activity is unresolved. Here we show by co-immunoprecipitation that increasing intracellular calcium with ionomycin or by activating sphingosine-1-phosphate receptors, induces coupling of calcium/calmodulin-dependent phosphatase calcineurin and prolyl isomerase FK506-binding protein 12 (FKBP12) to TMEM16A in HEK-293 cells. Application of drugs that target either calcineurin (cyclosporine A) or FKBP12 (tacrolimus known as FK506 and sirolimus known as rapamycin) caused a decrease in TMEM16A activity. In addition, FK506 and BAPTA-AM prevented co-immunoprecipitation between FKBP12 and TMEM16A. FK506 rendered the channel insensitive to cyclosporine A without altering its apparent calcium sensitivity whereas zero intracellular calcium blocked the effect of FK506. Rapamycin decreased TMEM16A activity in cells pre-treated with cyclosporine A or FK506. These results suggest the formation of a TMEM16A-FKBP12-calcineurin complex that regulates channel function. We conclude that upon a cytosolic calcium increase the TMEM16A-FKPB12-calcineurin trimers are assembled. Such hetero-oligomerization enhances TMEM16A channel activity but is not mandatory for activation by calcium.

TMEM16A (ANO1), the pore forming subunit of a Ca2+-dependent Cl− channel (CaCC), is activated by direct, voltage-dependent, binding of intracellular Ca2+. Endogenous CaCCs are regulated by extracellular protons; however, the molecular basis of such regulation remains unidentified. Here, we evaluated the effects of different extracellular proton concentrations ([H+]o) on mouse TMEM16A expressed in HEK-293 cells using whole-cell and inside-out patch clamp recordings. We found that increasing the [H+]o from 10−10 to 10−5.5 M caused a progressive increase in the chloride current (ICl) that is described by titration of a protonatable site with pK = 7.3. Protons regulate TMEM16A in a voltage-independent manner, regardless of channel state (open or closed), and without altering its apparent Ca2+ sensitivity. Noise analysis showed that protons regulate TMEM16A by tuning its open probability without modifying the single channel current. We found a robust reduction of the proton effect at high [Ca2+]i. To identify protonation targets we mutated all extracellular glutamate and histidine residues and four of eleven aspartates. Most mutants were sensitive to protons. However, E623Q displayed a titration curve shifted to the left relative to WT channels and the ICl was nearly insensitive to proton concentrations between 10−5.5 and 10−9.0 M. Additionally, ICl of the D405N mutant was partially inhibited by a proton concentration of 10−5.5 M, but 10−9.0 M produced the same effect as in WT. Based on our findings we propose that external protons titrate glutamic acid 623, which enables voltage activation of TMEM16A at non-saturating [Ca2+]i. This article is protected by copyright. All rights reserved

TMEM16A (ANO1), the pore-forming subunit of calcium-activated chloride channels, regulates several physiological and pathophysiological processes such as smooth muscle contraction, cardiac and neuronal excitability, salivary secretion, tumour growth and cancer progression. Gating of TMEM16A is complex because it involves the interplay between increases in intracellular calcium concentration ([Ca(2+)]i), membrane depolarization, extracellular Cl(-) or permeant anions and intracellular protons. Our goal here was to understand how these variables regulate TMEM16A gating and to explain four observations. (a) TMEM16A is activated by voltage in the absence of intracellular Ca(2+). (b) The Cl(-) conductance is decreased after reducing extracellular Cl(-) concentration ([Cl(-)]o). (c) ICl is regulated by physiological concentrations of [Cl(-)]o. (d) In cells dialyzed with 0.2 μM [Ca(2+)]i, Cl(-) has a bimodal effect: at [Cl(-)]o

Chloride fluxes through the calcium-gated chloride channel Anoctamin-1 (TMEM16A) control blood pressure, secretion of saliva, mucin, insulin, and melatonin, gastrointestinal motility, sperm capacitation and motility, and pain sensation. Calcium activates a myriad of regulatory proteins but how these proteins affect TMEM16A activity is unresolved. Here we show by co-immunoprecipitation that increasing intracellular calcium with ionomycin or by activating sphingosine-1-phosphate receptors, induces coupling of calcium/calmodulin-dependent phosphatase calcineurin and prolyl isomerase FK506-binding protein 12 (FKBP12) to TMEM16A in HEK-293 cells. Application of drugs that target either calcineurin (cyclosporine A) or FKBP12 (tacrolimus known as FK506 and sirolimus known as rapamycin) caused a decrease in TMEM16A activity. In addition, FK506 and BAPTA-AM prevented co-immunoprecipitation between FKBP12 and TMEM16A. FK506 rendered the channel insensitive to cyclosporine A without altering its apparent calcium sensitivity whereas zero intracellular calcium blocked the effect of FK506. Rapamycin decreased TMEM16A activity in cells pre-treated with cyclosporine A or FK506. These results suggest the formation of a TMEM16A-FKBP12-calcineurin complex that regulates channel function. We conclude that upon a cytosolic calcium increase the TMEM16A-FKPB12-calcineurin trimers are assembled. Such hetero-oligomerization enhances TMEM16A channel activity but is not mandatory for activation by calcium.

Key points Calcium-activated chloride channels TMEM16A and TMEM16B support important physiological processes such as fast block of polyspermy, fluid secretion, control of blood pressure and sensory transduction. Given the physiological importance of TMEM16 channels, it is important to study how incoming stimuli activate these channels. Here we study how channels open and close and how the process of gating is regulated. We show that TMEM16A and TMEM16B display fast and slow gating. These gating modes are regulated by voltage and external chloride. Dual gating explains the complex time course of the anion current. Residues within the first intracellular loop of the channel influence the slow gating mode. Dual gating is an intrinsic property observed in endogenous calcium-activated chloride channels and could be relevant to physiological processes that require sustained chloride ion movement. Abstract TMEM16A and TMEM16B are molecular components of the physiologically relevant calcium-activated chloride channels (CaCCs) present in many tissues. Their gating is dictated by membrane voltage (Vm), intracellular calcium concentrations ([Ca2+]i) and external permeant anions. As a consequence, the chloride current (ICl) kinetics is complex. For example, TMEM16A ICl activates slowly with a non-mono-exponential time course while TMEM16B ICl activates rapidly following a mono-exponential behaviour. To understand the underlying mechanism responsible for the complex activation kinetics, we recorded ICl from HEK-293 cells transiently transfected with either TMEM16A or TMEM16B as well as from mouse parotid acinar cells. Two distinct Vm-dependent gating modes were uncovered: a fast-mode on the millisecond time scale followed by a slow mode on the second time scale. Using long (20 s) depolarizing pulses both gating modes were activated, and a slowly rising ICl was recorded in whole-cell and inside-out patches. The amplitude of ICl at the end of the long pulse nearly doubled and was blocked by 100 μm tannic acid. The slow gating mode was strongly reduced by decreasing the [Cl−]o from 140 to 30 mm and by altering the sequence of the first intracellular loop. Mutating 480RSQ482 to AVK in the first intracellular loop of TMEM16B nearly abolished slow gating, but, mutating 448AVK451 to RSQ in TMEM16A has little effect. Deleting 448EAVK451 residues in TMEM16A reduced slow gating. We conclude that TMEM16 CaCCs have intrinsic Vm- and Cl−-sensitive dual gating that elicits complex ICl kinetics.

The ATP-gated P2X4 and P2X7 receptors are cation channels, co-expressed in excitable and non-excitable cells and play important roles in pain, bone development, cytokine release and cell death. Although these receptors interact the interacting domains are unknown and the functional consequences of this interaction remain unclear. Here we show by co-immunoprecipitation that P2X4 interacts with the C-terminus of P2X7 and by fluorescence resonance energy transfer experiments that this receptor-receptor interaction is driven by ATP. Furthermore, disrupting the ATP-driven interaction by knocking-out P2X4R provoked an attenuation of P2X7-induced cell death, dye uptake and IL-1β release in macrophages. Thus, P2X7 interacts with P2X4 via its C-terminus and disrupting the P2X7/P2X4 interaction hinders physiological responses in immune cells.

Candida glabrata has emerged as an important opportunistic pathogen in both mucosal and bloodstream infections. C. glabrata contains 67 adhesin-like glycosylphosphatidylinositol-cell-wall proteins (GPI-CWPs), which are classified into seven groups and the largest is the Epa family. Epa proteins are very diverse and their expression is differentially regulated. Like many of the EPA genes, EPA2 is localized in a subtelomeric region where it is subject to chromatin-based transcriptional silencing and its role remains largely unexplored. In this study, we show that EPA2 gene is induced specifically in vitro in the presence of oxidative stress generated by H2O2. This induction is dependent on both Yap1 and Skn7, whereas Msn4 represses EPA2 expression. Interestingly, EPA2 is not induced during phagocytosis, but its expression can be identified in the liver in a murine model of systemic infection. Epa2 has no effect on the virulence of C. glabrata. The work presented herein provides a foundation for future studies to dissect the molecular mechanism(s) by which EPA2 of C. glabrata can be induced in the presence of oxidative stress in a region subject to subtelomeric silencing.

The TMEM16A-mediated Ca(2+)-activated Cl(−) current drives several important physiological functions. Membrane lipids regulate ion channels and transporters but their influence on members of the TMEM16 family is poorly understood. Here we have studied the regulation of TMEM16A by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), cholesterol, and fatty acids using patch clamp, biochemistry and fluorescence microscopy. We found that depletion of membrane PI(4,5)P2 causes a decline in TMEM16A current that is independent of cytoskeleton, but is partially prevented by removing intracellular Ca(2+). On the other hand, supplying PI(4,5)P2 to inside-out patches attenuated channel rundown and/or partially rescued activity after channel rundown. Also, depletion (with methyl-β-cyclodextrin M-βCD) or restoration (with M-βCD + cholesterol) of membrane cholesterol slows down the current decay observed after reduction of PI(4,5)P2. Neither depletion nor restoration of cholesterol change PI(4,5)P2 content. However, M-βCD alone transiently increases TMEM16A activity and dampens rundown whereas M-βCD + cholesterol increases channel rundown. Thus, PI(4,5)P2 is required for TMEM16A function while cholesterol directly and indirectly via a PI(4,5)P2-independent mechanism regulate channel function. Stearic, arachidonic, oleic, docosahexaenoic, and eicosapentaenoic fatty acids as well as methyl stearate inhibit TMEM16A in a dose- and voltage-dependent manner. Phosphatidylserine, a phospholipid whose hydrocarbon tails contain stearic and oleic acids also inhibits TMEM16A. Finally, we show that TMEM16A remains in the plasma membrane after treatment with M-βCD, M-βCD + cholesterol, oleic, or docosahexaenoic acids. Thus, we propose that lipids and fatty acids regulate TMEM16A channels through a membrane-delimited protein-lipid interaction.

We studied the effects of extracellular ATP and Ca2+ on uptake of bacteria (Staphylococcus aureus or Escherichia coli) and live yeast (Candida glabrata) by J774 macrophages to determine the role of endogenous P2X7 receptors in phagocytosis. Our findings show that phagocytosis of bio-particles coated with S. aureus or E. coli was blocked by ATP and the P2X7 receptor agonist BzATP, while yeast phagocytosis was not. A438079, an antagonist of P2X7 receptors, partially reverted the effects of ATP on bacterial phagocytosis. To determine if P2X7-mediated Ca2+ entry into macrophages was blocking the engulfment of bacteria, we measured phagocytic activity in the absence or presence of 2 mM extracellular Ca2+ with or without ATP. Ca2+, in the absence of ATP, was required for engulfment of E. coli and C. glabrata but not S. aureus. Adding ATP inhibited phagocytosis of S. aureus and E. coli regardless of Ca2+, suggesting that Ca2+ entry was not important for inhibiting phagocytosis. On the other hand, phagocytosis of normal or hyper-adherent C. glabrata mutants had an absolute requirement for extracellular Ca2+ due to yeast adhesion to macrophages mediated by Ca2+-dependent adhesion proteins. We conclude that unstimulated P2X7 from J774 cells act as scavenger receptor for the uptake of S. aureus and E. coli but not of yeast; Ca2+ entry via P2X7 receptors play no role in phagocytosis of S. aureus and E. coli; while the effect of Ca2+ on C. glabrata phagocytosis was mediated by the adhesins Epa1, Epa6 and Epa7.

There are numerous experimental approaches to identify the interaction networks of soluble proteins, but strategies for the identification of membrane protein interactomes remain limited. We discuss in detail the logic of an experimental design that led us to identify the interactome of a membrane protein of complex membrane topology, the calcium activated chloride channel Anoctamin 1/Tmem16a (Ano1). We used covalent chemical stabilizers of protein-protein interactions combined with magnetic bead immuno-affinity chromatography, quantitative SILAC mass-spectrometry and in silico network construction. This strategy led us to define a putative Ano1 interactome from which we selected key components for functional testing. We propose a combination of procedures to narrow down candidate proteins interacting with a membrane protein of interest for further functional studies.

The newly discovered Ca 2+ -activated Cl − channel (CaCC), Anoctamin 1 (Ano1 or TMEM16A), has been implicated in vital physiological functions including epithelial fluid secretion, gut motility, and smooth muscle tone. Overexpression of Ano1 in HEK cells or Xenopus oocytes is sufficient to generate Ca 2+ -activated Cl − currents, but the details of channel composition and the regulatory factors that control channel biology are incompletely understood. We used a highly sensitive quantitative SILAC proteomics approach to obtain insights into stoichiometric protein networks associated with the Ano1 channel. These studies provide a comprehensive footprint of putative Ano1 regulatory networks. We find that Ano1 associates with the signaling/scaffolding proteins ezrin, radixin, moesin, and RhoA, which link the plasma membrane to the cytoskeleton with very high stoichiometry. Ano1, ezrin, and moesin/radixin colocalize apically in salivary gland epithelial cells, and overexpression of moesin and Ano1 in HEK cells alters the subcellular localization of both proteins. Moreover, interfering RNA for moesin modifies Ano1 current without affecting its surface expression level. Another network associated with Ano1 includes the SNARE and SM proteins VAMP3, syntaxins 2 and -4, and syntaxin-binding proteins munc18b and munc18c, which are integral to translocation of vesicles to the plasma membrane. A number of other regulatory proteins, including GTPases, Ca 2+ -binding proteins, kinases, and lipid-interacting proteins are enriched in the Ano1 complex. These data provide stoichiometrically prioritized information about mechanisms regulating Ano1 function and trafficking to polarized domains of the plasma membrane.

The volume-sensitive chloride current (I(ClVol)) exhibit a time-dependent decay presumably due to channel inactivation. In this work, we studied the effects of chloride ions (Cl(-)) and H(+) ions on I(ClVol) decay recorded in HEK-293 and HL-60 cells using the whole-cell patch clamp technique. Under control conditions ([Cl(-)](e) = [Cl(-)](i) = 140 mM and pH(i) = pH(e) = 7.3), I(ClVol) in HEK cells shows a large decay at positive voltages but in HL-60 cells I(ClVol) remained constant independently of time. In HEK-293 cells, simultaneously raising the [Cl(-)](e) and [Cl(-)](i) from 25 to 140 mM (with pH(e) = pH(i) = 7.3) increased the fraction of inactivated channels (FIC). This effect was reproduced by elevating [Cl(-)](i) while keeping the [Cl(-)](e) constant. Furthermore, a decrease in pH(e) from 7.3 to 5.5 accelerated current decay and increased FIC when [Cl(-)] was 140 mM but not 25 mM. In HL-60 cells, a slight I(ClVol) decay was seen when the pH(e) was reduced from 7.3 to 5.5. Our data show that inactivation of I(ClVol) can be controlled by changing either the Cl(-) or H(+) concentration or both. Based on our results and previously published data, we have built a model that explains VRAC inactivation. In the model the H(+) binding site is located outside the electrical field near the extracellular entry whilst the Cl(-) binding site is intracellular. The model depicts inactivation as a pore constriction that happens by simultaneous binding of H(+) and Cl(-) ions to the channel followed by a voltage-dependent conformational change that ultimately causes inactivation.

Mouse parotid acinar cells express P2X4 and P2X7 receptors (mP2X4R and mP2X7R) whose physiological function remains undetermined. Here we show that mP2X4R expressed in HEK-293 cells do not allow the passage of tetraethylammonium (TEA+) and promote little, if any, ethidium bromide (EtBr) uptake when stimulated with ATP or BzATP. In contrast, mP2X7R generates slowly decaying TEA+ current, sustained Na+ current and promotes robust EtBr uptake. However, ATP-activated TEA+ current from acinar cells was unlike that generated by mP2X7R or mP2X4R. Functional interactions between mP2X4R and mP2X7R were investigated in HEK cells co-transfected with different mP2X4 : mP2X7 cDNA ratios and using solutions containing either TEA+ or Na+ ions. Co-expressed channels generated a TEA+ current that displayed faster decay during ATP stimulation than mP2X7R alone. Moreover, cells transfected with a 2 : 1 cDNA ratio displayed decaying kinetics similar to those observed in acinar cells. Concentration–response curves in Na+-containing solutions were constructed for heterologously expressed mP2X4R, mP2X7R and mP2X4R:mP2X7R co-expressions as well as acinar cells. The EC50 values determined were 11, 220, 434 and 442 μm, respectively. Na+ currents generated by expressing mP2X4R or mP2X7R alone were potentiated by ivermectin (IVM). In contrast, IVM potentiation in acinar cells and HEK cells co-expressing P2X4 and P2X7 (1 : 1 or 2 : 1 cDNA ratios) was seen only when the ATP concentration was lowered from 5 to 0.03 mm. Taken together our observations indicate a functional interaction between murine P2X7 and P2X4 receptors. Such interaction might occur in acinar cells to shape the response to extracellular ATP in salivary epithelia.

We previously reported that mouse parotid acinar cells display anion conductance (I(ATPCl)) when stimulated by external ATP in Na+-free extracellular solutions. It has been suggested that the P2X7 receptor channel (P2X7R) might underlie I(ATPCl). In this work we show that I (ATPCl) can be activated by ATP, ADP, AMP-PNP, ATPgammaS and CTP. This is consistent with the nucleotide sensitivity of P2X7R. Accordingly, acinar cells isolated from P2X7R( -/- ) mice lacked I(ATPCl). Experiments with P2X7R heterologously expressed resulted in ATP-activated currents (I(ATP-P2X7)) partially carried by anions. In Na(+)-free solutions, I (ATP-P2X7) had an apparent anion permeability sequence of SCN(-) > I(-) congruent with NO3(-) > Br(-) > Cl(-) > acetate, comparable to that reported for I(ATPCl) under the same conditions. However, in the presence of physiologically relevant concentrations of external Na+, the Cl(-) permeability of I(ATP-P2X7) was negligible, although permeation of Br(-) or SCN(-) was clearly resolved. Relative anion permeabilities were not modified by addition of 1 mM: carbenoxolone, a blocker of Pannexin-1. Moreover, cibacron blue 3GA, which blocks the Na(+) current activated by ATP in acinar cells but not I(ATPCl), blocked I(ATP-P2X7) in a dose-dependent manner when Na+ was present but failed to do so in tetraethylammonium containing solutions. Thus, our data indicate that P2X7R is fundamental for I(ATPCl) generation in acinar cells and that external Na+ modulates ion permeability and conductivity, as well as drug affinity, in P2X7R.

TMEM16A, the pore forming subunit of the Ca2+-dependent Cl- channel (CaCCs) plays an important role in physiological processes such as fluid secretion, muscular contractility and thermal nociception. This channel is activated in response to increments in intracellular [Ca2+] and membrane depolarization. Previous studies established that endogenous CaCCs are regulated by extracellular protons ([H+]o). In this work the effect of [H+]o on the TMEM16A activity was evaluated using the whole-cell patch clamp technique. Stably transfected HEK293 cells were dialyzed with a solution containing 140 mM Cl- and 0.2 µM Ca2+. We found that [H+]o regulates the chloride current (ICl) in a bimodal manner. Decreasing [H+]o from 10−7.3 to 10−10 M decreased ICl without changing the activation kinetics. In contrast, increasing the [H+]o from 10−7.3 to 10−6 M increased ICl; but a further increase of [H+]o to 10−5, 10−4.5 or 10−4 M reduced ICl to 37% respect to 10−7.3 M. The effect of protons on ICl peaked at 10−5.5 M, were independent of the conformational state (closed or open) of the channel as well as of membrane voltage. The bimodal regulation suggests the existence of two titrable sites with pK values of 7.1 and 5.2, respectively. To verify this we generated the following mutants E362Q, E368Q, E623Q, E624Q, H402Y, H802Y, and H807Y, but none of the mutations prevented the effects of [H+]o. Then to investigate if the effect of [H+]o depended on the degree of TMEM16A activation we dialyzed cells with different [Ca2+]i. Surprisingly we found that increasing the [Ca2+]i produces a strong reduction of the H+ effect, suggesting that extracellular protons regulate TMEM16A by altering the open probability of the channel. Supported by grant 219949 from CONACyT.

The strategies available for treating salivary gland hypofunction are limited because relatively little is known about the secretion process in humans. An initial microarray screen detected ion transport proteins generally accepted to be critically involved in salivation. We tested for the activity of some of these proteins, as well as for specific cell properties required to support fluid secretion. The resting membrane potential of human acinar cells was near −51 mV, while the intracellular [Cl−] was ∼62 mM, about fourfold higher than expected if Cl ions were passively distributed. Active Cl−uptake mechanisms included a bumetanide-sensitive Na+-K+-2Cl−cotransporter and paired DIDS-sensitive Cl−/HCO3−and EIPA-sensitive Na+/H+exchangers that correlated with expression of NKCC1, AE2, and NHE1 transcripts, respectively. Intracellular Ca2+stimulated a niflumic acid-sensitive Cl−current with properties similar to the Ca2+-gated Cl channel BEST2. In addition, intracellular Ca2+stimulated a paxilline-sensitive and voltage-dependent, large-conductance K channel and a clotrimazole-sensitive, intermediate-conductance K channel, consistent with the detection of transcripts for KCNMA1 and KCNN4, respectively. Our results demonstrate that the ion transport mechanisms in human parotid glands are equivalent to those in the mouse, confirming that animal models provide valuable systems for testing therapies to prevent salivary gland dysfunction.

Many agents that activate neutrophils, enabling them to adhere to venular walls at sites of inflammation, cause a rapid Cl− efflux. This Cl− efflux and the increase in the number and affinity of β2 integrin surface adhesion molecules (up-regulation) are all inhibited by ethacrynic acid and certain aminomethyl phenols. The effectiveness of the latter compounds correlates with their inhibition of swelling-activated Cl− channels (IClvol), suggesting that IClvol mediates the activator-induced Cl− efflux. To test this hypothesis, we used whole-cell patch clamp in hypotonic media to examine the effects of inhibitors of up-regulation on IClvol in neutrophils and promyelocytic leukemic HL-60 cells. Both the channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid and [3-methyl-1-p-sulfophenyl-5-pyrazolone-(4)]-[1,3-dibutylbarbituric acid]-pentamethine oxonol (WW781), a nonpenetrating oxonol, inhibited IClvol at concentrations similar to those that inhibit β2 integrin up-regulation. However, ethacrynic acid, at the same concentration that inhibits activator-induced Cl− efflux and up-regulation, had no effect on IClvol and swelling-activated Cl− efflux, providing evidence against the involvement of IClvol in the activator-induced Cl− efflux.

We measured unidirectional K+ in- and efflux through an inward rectifier K channel (IRK1) expressed in Xenopus oocytes. The ratio of these unidirectional fluxes differed significantly from expectations based on independent ion movement. In an extracellular solution with a K+ concentration of 25 mM, the data were described by a Ussing flux-ratio exponent, n′, of ∼2.2 and was constant over a voltage range from −50 to −25 mV. This result indicates that the pore of IRK1 channels may be simultaneously occupied by at least three ions. The IRK1 n′ value of 2.2 is significantly smaller than the value of 3.5 obtained for Shaker K channels under identical conditions. To determine if other permeation properties that reflect multi-ion behavior differed between these two channel types, we measured the conductance (at 0 mV) of single IRK1 channels as a function of symmetrical K+ concentration. The conductance could be fit by a saturating hyperbola with a half-saturation K+ activity of 40 mM, substantially less than the reported value of 300 mM for Shaker K channels. We investigated the ability of simple permeation models based on absolute reaction rate theory to simulate IRK1 current–voltage, conductance, and flux-ratio data. Certain classes of four-barrier, three-site permeation models are inconsistent with the data, but models with high lateral barriers and a deep central well were able to account for the flux-ratio and single channel data. We conclude that while the pore in IRK1 and Shaker channels share important similarities, including K+ selectivity and multi-ion occupancy, they differ in other properties, including the sensitivity of pore conductance to K+ concentration, and may differ in the number of K+ ions that can simultaneously occupy the pore: IRK1 channels may contain three ions, but the pore in Shaker channels can accommodate four or more ions.

Background Activation of ATP-gated P2X7 receptors (P2X7R) in macrophages leads to production of reactive oxygen species (ROS) by a mechanism that is partially characterized. Here we used J774 cells to identify the signaling cascade that couples ROS production to receptor stimulation. Methods J774 cells and mP2X7-transfected HEK293 cells were stimulated with Bz-ATP in the presence and absence of extracellular calcium. Protein inhibitors were used to evaluate the physiological role of various kinases in ROS production. In addition, phospho-antibodies against ERK1/2 and Pyk2 were used to determine activation of these two kinases. Results ROS generation in either J774 or HEK293 cells (expressing P2X7, NOX2, Rac1, p47phox and p67phox) was strictly dependent on calcium entry via P2X7R. Stimulation of P2X7R activated Pyk2 but not calmodulin. Inhibitors of MEK1/2 and c-Src abolished ERK1/2 activation and ROS production but inhibitors of PI3K and p38 MAPK had no effect on ROS generation. PKC inhibitors abolished ERK1/2 activation but barely reduced the amount of ROS produced by Bz-ATP. In agreement, the amount of ROS produced by PMA was about half of that produced by Bz-ATP. Conclusions Purinergic stimulation resulted in calcium entry via P2X7R and subsequent activation of the PKC/c-Src/Pyk2/ERK1/2 pathway to produce ROS. This signaling mechanism did not require PI3K, p38 MAPK or calmodulin. General significance ROS is generated in order to kill invading pathogens, thus elucidating the mechanism of ROS production in macrophages and other immune cells allow us to understand how our body copes with microbial infections.

When I had my first research experience, as a college student in a campus laboratory, I found that I loved collecting data to understand intriguing biological phenomena. I enjoyed exploring new ideas every day in the lab, and I appreciated the freedom I had to independently plan my experiments. So I

The effects of α 1 -adrenoceptor and muscarinic acetylcholine receptor stimulation on action potential and tension were studied in guinea pig papillary muscles obtained from both right and left ventricles. Stimulation of muscarinic acetylcholine receptors with carbachol produced a reduction of the action potential duration and a positive inotropic effect in papillary muscles from both ventricles. Both effects were concentration dependent and atropine sensitive. However, differential responsiveness was found upon α 1 -adrenoceptor activation in muscles obtained from left and right ventricles. In right side papillary muscles, the α 1 -adreneceptor agonist, methoxamine, decreased the action potentail duration and produced a positive inotropic effect. In contrast, methoxamine decreased the action potential duration but failed to produce a positive inotropic effect in left side papillary muscles. All methoxamine effects were antagonized by prazosin. Responses to maximum concentration of carbachol and methoxamine on the action potential duration and contractility were additive in right side papillary muscles. Phorbol 12,13-dibutyrate (PDB), a direct protein kinase C activator, also decreases the action potential duration in a manner that was additive to both carbachol and methoxamine. However, PDB reversed the positive inotropic effect of carbachol indomethacin and nordihydroguaiaretic acid, blockers of arachidonic acid metabolism, but not by the protein kinase C antagonist, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7). These results demonstrate (1) a side-dependent effect of methoxamine, and not carbachol, on muscle contraction, (2) additivity of the effects of α 1 -adrenoceptor and muscarinic acetylcholine receptor activation on the action potential duration and tension, and (3) that these receptor-mediated effects may involve different second messenger system and/or distinct substrate pools.

We have investigated some of the permeation properties of the pore in Shaker K channels. We determined the apparent permeability ratio of K+, Rb+, and NH4+ ions and block of the pore by external Cs+ ions. Shaker channels were expressed with the baculovirus/Sf9 expression system and the channel currents measured with the whole-cell variant of the patch clamp technique. The apparent permeability ratio, PRb/PK, determined in biionic conditions with internal K+, was a function of external Rb+ concentration. A large change in PRb/PK occurred with reversed ionic conditions (internal Rb+ and external K+). These changes in apparent permeability were not due to differences in membrane potential. With internal K+, PNH4/PK was not a function of external NH4+ concentration (at least over the range 50-120 mM). We also investigated block of the pore by external Cs+ ions. At a concentration of 20 mM, Cs+ block had a voltage dependence equivalent to that of an ion with a valence of 0.91; this increased to 1.3 at 40 mM Cs+. We show that a 4-barrier, 3-site permeation model can simulate these and many of the other known properties of ion permeation in Shaker channels.

It has been reported that in human neutrophils, external ATP activates plasma membrane purinergic P2X(7) receptors (P2X(7)R) to elicit Ca(2+) entry, production of reactive oxygen species (ROS), processing and release of pro-inflammatory cytokines, shedding of adhesion molecules and uptake of large molecules. However, the expression of P2X(7)R at the plasma membrane of neutrophils has also been questioned since these putative responses are not always reproduced. In this work, we used electrophysiological recordings to measure functional responses associated with the activation of membrane receptors, spectrofluorometric measurements of ROS production and ethidium bromide uptake to asses coupling of P2X(7)R activation to downstream effectors, immune-labelling of P2X(7)R using a fluorescein isothiocyanate-conjugated antibody to detect the receptors at the plasma membrane, RT-PCR to determine mRNA expression of P2X(7)R and Western blot to determine protein expression in neutrophils and HL-60 cells. None of these assays reported the presence of P2X(7)R in the plasma membrane of neutrophils and non-differentiated or differentiated HL-60 cells-a model cell for human neutrophils. We concluded that P2X(7)R are not present at plasma membrane of human neutrophils and that the putative physiological responses triggered by external ATP should be reconsidered.

We investigated whether pannexin-1, a carbenoxolone-sensitive hemichannel activated in erythrocytes by swelling, could be activated by swelling stress and contribute to swelling-activated chloride currents (I(Cl,swell)) in HEK-293 cells. We used ethidium bromide uptake as an index of pannexin-1 activation and I(C,swell) activation as an index of plasma membrane stretching. I(Cl,swell) activated by a hypotonic solution was reversible inhibited by carbenoxolone (IC(50) 98+/-5 microM). However, the hypotonic solution that activated I(Cl,swell) did not induce ethidium bromide uptake indicating that pannexin-1 was not activated by cell swelling. The mimetic peptide (10)panx1, a pannexin-1 antagonist, did not affect I(Cl,swell) activation but completely inhibited the ATP-induced ethidium bromide uptake coupled to P2X(7) receptors activation. We conclude that carbenoxolone directly inhibited I(Cl,swell) independent of pannexin-1 and that pannexin-1 hemichannels are not activated by swelling in HEK-293 cells.

We describe a novel, strongly outwardly rectifying anion current in Xenopus laevis oocytes, that we have named I(Cl,Or)- The properties of I(Cl,Or) are different from those of any other anion conductance previously described in these cells. Typically, I(Cl,Or) amplitude was small when extracellular Cl- (Cle) was the permeant anion. However, when Cle was replaced by lyotropic anions I(Cl,Or) became evident as a time-independent current. (ICl,Or) was voltage dependent and showed a remarkable outwards rectification with little or no inwards tail current. The relative selectivity sequence determined from current amplitudes was: SCN-or = ClO4-I-Br-or = NO3-Cl- x I(Cl,Or) was insensitive to Gd3+ but was blocked by micromolar concentrations of niflumic acid, DIDS or Zn2+. Furthermore, I(Cl,Or) was not affected by buffering intracellular Ca2+ with BAPTA. Low extracellular pH inhibited I(Cl,Or) with a pK of 5.8. We propose that I(Cl,Or) might result from activation of endogenous ClC-5-like Cl- channels present in Xenopus oocytes.

The effect of intracellular cAMP and cystic fibrosis conductance regulator (CFTR) protein on the calcium-activated chloride current (ICaCl) present in parotid acinar cells was studied using the patch clamp technique. Application of 1 mM of 8-(4-chlorophenylthio)adenosine 3′:5′-cyclic monophosphate (CPT-cAMP), a permeable analog of cAMP, inhibited ICaCl only at positive potentials. This inhibition was partially abolished in cells dialyzed with 20 nM PKI 6–22 amide, a potent peptide that specifically inhibits PKA. Because cAMP is an activator of the CFTR Cl− channel, a known regulator of ICaCl, we also investigated if the inhibition of ICaCl was mediated by activation of CFTR. To test this idea, we added 1 mM CPT-cAMP to acinar cells isolated from knockout animals that do not express the CFTR channel. In these cells the cAMP effect was totally abolished. Thus, our data provide evidence that cAMP regulates ICaCl by a dual mechanism involving PKA and CFTR.

The participation of an extracellular loop in C-type inactivation of voltage-gated K+ channels was investigated. A wild-type phenylalanine (at position 425) between the fifth putative transmembrane segment (S5) and the pore region of the Shaker K+ channel was mutated to a histidine and the functional consequences of protonating the imidizole group of the histidine were examined. C-type inactivation of both wild-type and mutant channels was sensitive to external pH over the range of 5.2-8. The pH dependence of wild-type channels was characterized by an apparent pK value of 5. 0. The inactivation kinetics of F425H mutant channels had a pH dependence with a pK of 5.8 - in addition to the pH dependence of the wild-type channels. Moreover, at pH 7 and 8 the voltage dependence of C-type inactivation kinetics was manifest only in the F425H mutant channels. C-type inactivation in wild-type channels involves a chemical group with a low pK. Taken together, these results suggest that residues located in the extracellular S5-pore loop of the Shaker K+ channel participate in C-type inactivation.

Recent reports indicate that the long known ethidium bromide uptake resulting from prolong stimulation of cell surface P2X7 receptors is due to Pannexin-1 (Panx-1) hemichannels, which are probably activated by the carboxy terminus of the receptor itself. It has also been proposed that Panx-1 is activated by exposing the cells to hypotonic solutions without the need for P2X7 receptor activation and that this maneuver results in ATP release from cells probably via Panx-1. However, other groups have previously proposed that the hypotonic-induced ATP release is via cell swelling-activated chloride channels. In this work we explored activation of endogenous Panx-1 in HEK 293 cells (untransfected or transfected with P2X7 receptors) exposed to hypotonic solutions, while simultaneously measuring whole cell chloride current and fluorescence signals resulting from ethidium bromide uptake (an index of Panx-1 activation). When HEK cells were exposed to hypotonic solutions that induced activation of cell swelling-activated chloride current (ICl,swell) no ethidium bromide uptake was detected. Treatment of the cells with the Panx-1 inhibitor mimetic peptide 10panx1 resulted in complete inhibition of ethidum bromide uptake induced by ATP activation of P2X7 receptors without affecting ICl,swell. Pretreatment with carbenoxolone (CBX), another blocker of Panx-1, inhibited ethidium bromide uptake induced by stimulation of HEK cells expressing P2X7 receptors and at the same time inhibited ICl,swell in a dose-dependent manner. We conclude that hypotonic conditions most likely induce ATP release by via activation of CBX-sensitive volume-sensitive chloride channels without participation of Panx-1 hemichannels.Supported by CONACyT grants 45895 and 79897.

In silico simulation based on Markov chains is a powerful way to describe and predict the activity of many transport proteins including ion channels. However, modeling and simulation using realistic models of voltage- or ligand-gated ion channels exposed to a wide range of experimental conditions require building complex kinetic schemes and solving complicated differential equations. To circumvent these problems, we developed IonChannelLab a software tool that includes a user-friendly Graphical User Interface and a simulation library. This program supports channels with Ohmic or Goldman-Hodgkin-Katz behavior and can simulate the time-course of ionic and gating currents, single channel behavior and steady-state conditions. The program allows the simulation of experiments where voltage, ligand and ionic concentration are varied independently or simultaneously.

The gating mechanism of TMEM16A, an essential subunit of the Ca2+-activated Cl- channel, remains incompletely understood. Gating of TMEM16A channels is complex because elevation of intracellular calcium concentration ([Ca2+]i), strong depolarizations (Vm), and high extracellular Cl or highly permeant anions seem to contribute. In the present work we show that in TMEM16A no gating currents can be detected, however, external Cl ions might play a role in voltage sensing because the kinetics and magnitude of current activation were dependent on [Cl-]e. The onset and offset of Cl- currents produced by 0.5 s depolarisations of cells bathed in 137 mM [Cl-]e followed a mono-exponential behaviour; a subsequent reduction of [Cl-]e to 10 mM diminished the conductance ∼2-fold. These data suggest a single gating process regulated by extracellular Cl-. In contrast, when cells were exposed to 137 mM [Cl-]e the onset and offset currents elicited by 20s depolarisations exhibited fast and slow kinetics. Activation of the slow component resulted in large onset Cl- currents that did not reach steady-state at the end of the 20s pulse. This behaviour was abolished after activating TMEM16A with 5 µM [Ca2+]i or after deleting four residues (448EAVK451) located in the first intracellular loop. Deleting four additional residues (444EEEEEAVK451) resulted in Cl- currents that decayed at the end of the 20 s depolarization. To explain our data we developed a 12-state gating model assuming that TMEM16A is activated by a direct, Vm-dependent binding of two Ca2+ ions. In this model external Cl- increases channel open probability by promoting stable Vm-dependent Ca2+ binding. Our model reproduced the gating behaviour of the TMEM16A currents in response to voltage, Ca2+, and [Cl-]e. Thus, we conclude that external Cl stabilises Ca binding and promotes two gating modes in TMEM16A. Supported by grant 219949 from CONACyT.