P. aeruginosa Induced Lipid Peroxidation Causes Ferroptotic Cell Death in Airways

BACKGROUND/AIMS: Oxidative stress and infections by Pseudomonas aeruginosa (P. aeruginosa) are prominent in lungs of patients suffering from cystic fibrosis (CF). METHODS: The present study examines effects of P. aeruginosa on lipid peroxidation in human and mouse lungs, and cell death induced by P. aeruginosa in human airway epithelial cells. The role of the Ca2+ activated Cl- channel TMEM16A, the phospholipid scramblase TMEM16F, and the CFTR Cl- channel for ferroptotic cell death is examined. RESULTS: Lipid peroxidation was detected in human CF lungs, which correlated with bacterial infection. In vivo inoculation with P. aeruginosa or Staphylococcus aureus (S. aureus) induced lipid peroxidation in lungs of mice lacking expression of CFTR, and in lungs of wild type animals. Incubation of CFBE human airway epithelial cells with P. aeruginosa induced an increase in reactive oxygen species (ROS), causing lipid peroxidation and cell death independent of expression of wt-CFTR or F508del-CFTR. Knockdown of TMEM16A attenuated P. aeruginosa induced cell death. Antioxidants such as coenzyme Q10 and idebenone as well as the inhibitor of ferroptosis, ferrostatin-1, inhibited P. aeruginosa-induced cell death. CFBE cells expressing wtCFTR, but not F508del-CFTR, activated a basal Cl- conductance upon exposure to P. aeruginosa, which was caused by an increase in intracellular basal Ca2+ concentrations and activation of Ca2+-dependent adenylate cyclase. CONCLUSION: The data suggest an intrinsic pro-inflammatory phenotype in CF epithelial cells, while ferroptosis is observed in both non-CF and CF epithelial cells upon infection with P. aeruginosa. CF cells fail to activate fluid secretion in response to infection with P. aeruginosa. The use of antioxidants and inhibitors of ferroptosis is proposed as a treatment of pneumonia caused by infection with P. aeruginosa.

Ataxia-linked SLC1A3 mutations alter EAAT1 chloride channel activity and glial regulation of CNS function

Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system (CNS). Excitatory Amino Acid Transporters (EAATs) regulate extracellular glutamate by transporting it into cells, mostly glia, to terminate neurotransmission and to avoid neurotoxicity. EAATs are also chloride (Cl-) channels, but the physiological role of Cl- conductance through EAATs is poorly understood. Mutations of human EAAT1 (hEAAT1) have been identified in patients with episodic ataxia type 6 (EA6). One mutation showed increased Cl- channel activity and decreased glutamate transport, but the relative contributions of each function of hEAAT1 to mechanisms underlying the pathology of EA6 remain unclear. Here we investigated the effects of five additional EA6-related mutations on hEAAT1 function in Xenopus laevis oocytes, and on CNS function in a Drosophila melanogaster model of locomotor behavior. Our results indicate that mutations with decreased hEAAT1 Cl- channel activity and functional glutamate transport can also contribute to the pathology of EA6, highlighting the importance of Cl- homeostasis in glial cells for proper CNS function. We also identified a novel mechanism involving an ectopic sodium (Na+) leak conductance in glial cells. Together, these results strongly support the idea that EA6 is primarily an ion channelopathy of CNS glia.

Calmodulin-Dependent Regulation of Overexpressed but Not Endogenous TMEM16A Expressed in Airway Epithelial Cells

Regulation of the Ca2+-activated Cl− channel TMEM16A by Ca2+/calmodulin (CAM) is discussed controversially. In the present study, we compared regulation of TMEM16A by Ca2+/calmodulin (holo-CAM), CAM-dependent kinase (CAMKII), and CAM-dependent phosphatase calcineurin in TMEM16A-overexpressing HEK293 cells and TMEM16A expressed endogenously in airway and colonic epithelial cells. The activator of the Ca2+/CAM-regulated K+ channel KCNN4, 1-EBIO, activated TMEM16A in overexpressing cells, but not in cells with endogenous expression of TMEM16A. Evidence is provided that CAM-interaction with TMEM16A modulates the Ca2+ sensitivity of the Cl− channel. Enhanced Ca2+ sensitivity of overexpressed TMEM16A explains its activity at basal (non-elevated) intracellular Ca2+ levels. The present results correspond well to a recent report that demonstrates a Ca2+-unbound form of CAM (apo-CAM) that is pre-associated with TMEM16A and mediates a Ca2+-dependent sensitization of activation (and inactivation). However, when using activators or inhibitors for holo-CAM, CAMKII, or calcineurin, we were unable to detect a significant impact of CAM, and limit evidence for regulation by CAM-dependent regulatory proteins on receptor-mediated activation of endogenous TMEM16A in airway or colonic epithelial cells. We propose that regulatory properties of TMEM16A and and other members of the TMEM16 family as detected in overexpression studies, should be validated for endogenous TMEM16A and physiological stimuli such as activation of phospholipase C (PLC)-coupled receptors.

The Cl- channel TMEM16A controls the generation of cochlear Ca2+ waves and promotes the refinement of auditory brainstem networks

Before hearing onset (postnatal day 12 in mice), inner hair cells (IHC) spontaneously fire action potentials thereby driving pre-sensory activity in the ascending auditory pathway. The rate of IHC action potential bursts is modulated by inner supporting cells (ISC) of Kölliker's organ through the activity of the Ca2+ activated Cl- channel TMEM16A. Here we show that conditional deletion of Tmem16a in mice disrupts the generation of Ca2+ waves within Köllike's organ, reduces the burst firing activity and the frequency-selectivity of auditory brainstem neurons in the medial nucleus of the trapezoid body (MNTB), and also impairs the refinement of MNTB projections to the lateral superior olive (LSO). These results reveal the importance of the activity of Köllike's organ for the refinement of central auditory connectivity. In addition, our study suggests a mechanism for the generation of Ca2+ waves, which may also apply to other tissues expressing TMEM16A.

The ATP-Releasing Maxi-Cl Channel: Its Identity, Molecular Partners, and Physiological/Pathophysiological Implications

The Maxi-Cl phenotype accounts for the majority (app. 60%) of reports on the large-conductance maxi-anion channels (MACs) and has been detected in almost every type of cell, including placenta, endothelium, lymphocyte, cardiac myocyte, neuron, and glial cells, and in cells originating from humans to frogs. A unitary conductance of 300–400 pS, linear current-to-voltage relationship, relatively high anion-to-cation selectivity, bell-shaped voltage dependency, and sensitivity to extracellular gadolinium are biophysical and pharmacological hallmarks of the Maxi-Cl channel. Its identification as a complex with SLCO2A1 as a core pore-forming component and two auxiliary regulatory proteins, annexin A2 and S100A10 (p11), explains the activation mechanism as Tyr23 dephosphorylation at ANXA2 in parallel with calcium binding at S100A10. In the resting state, SLCO2A1 functions as a prostaglandin transporter whereas upon activation it turns to an anion channel. As an efficient pathway for chloride, Maxi-Cl is implicated in a number of physiologically and pathophysiologically important processes, such as cell volume regulation, fluid secretion, apoptosis, and charge transfer. Maxi-Cl is permeable for ATP and other small signaling molecules serving as an electrogenic pathway in cell-to-cell signal transduction. Mutations at the SLCO2A1 gene cause inherited bone and gut pathologies and malignancies, signifying the Maxi-Cl channel as a perspective pharmacological target.

Differential Regulation of Ca2+-Activated Cl− Channel TMEM16A Splice Variants by Membrane PI(4,5)P2

TMEM16A is a Ca2+-activated Cl− channel that controls broad cellular processes ranging from mucus secretion to signal transduction and neuronal excitability. Recent studies have reported that membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is an important cofactor that allosterically regulates TMEM16A channel activity. However, the detailed regulatory actions of PIP2 in splice variants of TMEM16A remain unclear. Here, we demonstrated that the attenuation of membrane phosphoinositide levels selectively inhibited the current amplitude of the TMEM16A(ac) isoform by decreasing the slow, but not instantaneous, Cl− currents, which are independent of the membrane potential and specific to PI(4,5)P2 depletion. The attenuation of endogenous PI(4,5)P2 levels by the activation of Danio rerio voltage-sensitive phosphatase (Dr-VSP) decreased the Cl− currents of TMEM16A(ac) but not the TMEM16A(a) isoform, which was abolished by the co-expression of PIP 5-kinase type-1γ (PIPKIγ). Using the rapamycin-inducible dimerization of exogenous phosphoinositide phosphatases, we further revealed that the stimulatory effects of phosphoinositide on TMEM16A(ac) channels were similar in various membrane potentials and specific to PI(4,5)P2, not PI4P and PI(3,4,5)P3. Finally, we also confirmed that PI(4,5)P2 resynthesis is essential for TMEM16A(ac) recovery from Dr-VSP-induced current inhibition. Our data demonstrate that membrane PI(4,5)P2 selectively modulates the gating of the TMEM16A(ac) channel in an agonistic manner, which leads to the upregulation of TMEM16A(ac) functions in physiological conditions.

Positive Inotropic Effects of ATP Released via the Maxi-Anion Channel in Langendorff-Perfused Mouse Hearts Subjected to Ischemia-Reperfusion

The organic anion transporter SLCO2A1 constitutes an essential core component of the ATP-conductive large-conductance anion (Maxi-Cl) channel. Our previous experiments using Langendorff-perfused mouse hearts showed that the Maxi-Cl channel contributes largely to the release of ATP into the coronary effluent observed during 10-min reperfusion following a short period (6 min) of oxygen-glucose deprivation. The present study examined the effect of endogenous ATP released via Maxi-Cl channels on the left ventricular contractile function of Langendorff-perfused mouse hearts, using a fluid-filled balloon connected to a pressure transducer. After the initial 30-min stabilization period, the heart was then perfused with oxygen-glucose-deprived Tyrode solution for 6 min, which was followed by a 10-min perfusion with oxygenated normal Tyrode solution in the absence and presence of an ATP-hydrolyzing enzyme, apyrase, and/or an adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). In the absence of apyrase and DPCPX, the left ventricular developed pressure (LVDP) decreased from a baseline value of 72.3 ± 7.1 to 57.5 ± 5.5 mmHg (n = 4) at the end of 6-min perfusion with oxygen-glucose-deprived Tyrode solution, which was followed by a transient increase to 108.5 ± 16.5 mmHg during subsequent perfusion with oxygenated normal Tyrode solution. However, in the presence of apyrase and DPCPX, the LVDP decreased to the same degree during 6-min perfusion with oxygen-glucose-deprived Tyrode solution, but failed to exhibit a transient increase during a subsequent perfusion with oxygenated normal Tyrode solution. These results strongly suggest that endogenous ATP released through Maxi-Cl channels contributes to the development of transient positive inotropy observed during reperfusion after short-period hypoxia/ischemia in the heart.