Patricia Pérez-Cornejo

Anoctamin 1 (Tmem16A) Ca2+-activated chloride channel stoichiometrically interacts with an ezrin–radixin–moesin network

The newly discovered Ca2+-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 Ca2+-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, Ca2+-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.

Permeant anions control gating of calcium-dependent chloride channels.

The effects of external anions (SCN−, NO3−, I−, Br−, F−, glutamate, and aspartate) on gating of Ca2+-dependent Cl− channels from rat parotid acinar cells were studied using the whole-cell configuration of the patch-clamp technique. Shifts in the reversal potential of the current induced by replacement of external Cl− with foreign anions, gave the following selectivity sequence based on permeability ratios (Px/PCl): SCN−>I−>NO3−>Br−>Cl−>F−>aspartate>glutamate. Using a continuum electrostatic model we calculated that this lyotropic sequence resulted from the interaction between anions and a polarizable tunnel with an effective dielectric constant of ∼23. Our data revealed that anions with Px/PCl > 1 accelerated activation kinetics in a voltage-independent manner and slowed deactivation kinetics. Moreover, permeant anions enhanced whole-cell conductance (g, an index of the apparent open probability) in a voltage-dependent manner, and shifted leftward the membrane potential-g curves. All of these effects were produced by the anions with an effectiveness that followed the selectivity sequence. To explain the effects of permeant anions on activation kinetics and gCl we propose that there are 2 different anion-binding sites in the channel. One site is located outside the electrical field and controls channel activation kinetics, while a second site is located within the pore and controls whole-cell conductance. Thus, interactions of permeant anions with these two sites hinder the closing mechanism and stabilize the channel in the open state.

Oxidative stress induced by P2X7 receptor stimulation in murine macrophages is mediated by c-Src/Pyk2 and ERK1/2.

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.

Simulating complex ion channel kinetics with IonChannelLab

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 P2X7/P2X4 interaction shapes the purinergic response in murine macrophages.

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.

Human neutrophils do not express purinergic P2X7 receptors

It has been reported that in human neutrophils, external ATP activates plasma membrane purinergic P2X7 receptors (P2X7R) to elicit Ca2+ 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 P2X7R 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 P2X7R activation to downstream effectors, immune-labelling of P2X7R using a fluorescein isothiocyanate-conjugated antibody to detect the receptors at the plasma membrane, RT-PCR to determine mRNA expression of P2X7R and Western blot to determine protein expression in neutrophils and HL-60 cells. None of these assays reported the presence of P2X7R in the plasma membrane of neutrophils and non-differentiated or differentiated HL-60 cells—a model cell for human neutrophils. We concluded that P2X7R are not present at plasma membrane of human neutrophils and that the putative physiological responses triggered by external ATP should be reconsidered.

Lack of coupling between membrane stretching and pannexin-1 hemichannels.

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.

Na+ Modulates Anion Permeation and Block of P2X7 Receptors from Mouse Parotid Glands

We previously reported that mouse parotid acinar cells display anion conductance (IATPCl) when stimulated by external ATP in Na+-free extracellular solutions. It has been suggested that the P2X7 receptor channel (P2X7R) might underlie IATPCl. In this work we show that IATPCl can be activated by ATP, ADP, AMP-PNP, ATPγS and CTP. This is consistent with the nucleotide sensitivity of P2X7R. Accordingly, acinar cells isolated from P2X7R−/− mice lacked IATPCl. Experiments with P2X7R heterologously expressed resulted in ATP-activated currents (IATP-P2X7) partially carried by anions. In Na+-free solutions, IATP-P2X7 had an apparent anion permeability sequence of SCN− > I− ≅ NO3− > Br− > Cl− > acetate, comparable to that reported for IATPCl under the same conditions. However, in the presence of physiologically relevant concentrations of external Na+, the Cl− permeability of IATP-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 IATPCl, blocked IATP-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 IATPCl generation in acinar cells and that external Na+ modulates ion permeability and conductivity, as well as drug affinity, in P2X7R.

Gating modes of calcium‐activated chloride channels TMEM16A and TMEM16B

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.

Extracellular protons enable activation of the calcium‐dependent chloride channel TMEM16A

The calcium‐activated chloride channel TMEM16A provides a pathway for chloride ion movements that are key in preventing polyspermy, allowing fluid secretion, controlling blood pressure, and enabling gastrointestinal activity. TMEM16A is opened by voltage‐dependent calcium binding and regulated by permeant anions and intracellular protons. Here we show that a low proton concentration reduces TMEM16A activity while maximum activation is obtained when the external proton concentration is high. In addition, protonation conditions determine the open probability of TMEM16A without changing its calcium sensitivity. External glutamic acid 623 (E623) is key for TMEM16As ability to respond to external protons. At physiological pH, E623 is un‐protonated and TMEM16A is activated when intracellular calcium increases; however, under acidic conditions E623 is partially protonated and works synergistically with intracellular calcium to activate the channel. These findings are critical for understanding physiological and pathological processes that involve changes in pH and chloride flux via TMEM16A.