Membrane Anion Channel Research Articles

Ferroptosis inducers – erastin and analogues (review)

Introduction. Improving the efficacy of chemotherapy is a non-trivial task of modern oncology. Its successful solution requires knowledge in many fields, including physiology, pathology, clinical oncology, pharmacology and others. The search for small molecules that selectively kill tumor cells led to the accidental discovery of erastin.Text. Erastin is a unique molecule that has a quinazoline fragment in its structure. Not so long ago it became known that the antitumour effect of this compound is due to the induction of ferroptosis – an iron-dependent form of cell death caused by lipid peroxidation. Erastin is able to induce ferroptosis through various biochemical pathways, including blocking of cystine-glutamate transport channel of cell membrane and potential-dependent anion channel of mitochondria, as well as activation of p53 protein.Conclusion. Pharmacological induction of ferroptosis by erastin and its analogues represents a promising direction in cancer chemotherapy. In addition, erastin and its analogues are able to increase sensitivity to chemotherapy and radiation therapy, which allows us to talk about the possibility of their use in the combined treatment of malignant neoplasms.

Hexokinase dissociation from mitochondria promotes oligomerization of VDAC that facilitates NLRP3 inflammasome assembly and activation.

NLRP3 inflammasome activation is a highly regulated process for controlling secretion of the potent inflammatory cytokines IL-1β and IL-18 that are essential during bacterial infection, sterile inflammation, and disease, including colitis, diabetes, Alzheimer's disease, and atherosclerosis. Diverse stimuli activate the NLRP3 inflammasome, and unifying upstream signals has been challenging to identify. Here, we report that a common upstream step in NLRP3 inflammasome activation is the dissociation of the glycolytic enzyme hexokinase 2 from the voltage-dependent anion channel (VDAC) in the outer membrane of mitochondria. Hexokinase 2 dissociation from VDAC triggers activation of inositol triphosphate receptors, leading to release of calcium from the ER, which is taken up by mitochondria. This influx of calcium into mitochondria leads to oligomerization of VDAC, which is known to form a macromolecule-sized pore in the outer membranes of mitochondria that allows proteins and mitochondrial DNA (mtDNA), often associated with apoptosis and inflammation, respectively, to exit the mitochondria. We observe that VDAC oligomers aggregate with NLRP3 during initial assembly of the multiprotein oligomeric NLRP3 inflammasome complex. We also find that mtDNA is necessary for NLRP3 association with VDAC oligomers. These data, together with other recent work, help to paint a more complete picture of the pathway leading to NLRP3 inflammasome activation.

The cyclic lipopeptide micafungin induces rupture of isolated mitochondria by reprograming the mitochondrial inner membrane anion channel

The antifungal activity of the drug micafungin, a cyclic lipopeptide that interacts with membrane proteins, may involve inhibition of fungal mitochondria. In humans, mitochondria are spared by the inability of micafungin to cross the cytoplasmic membrane. Using isolated mitochondria, we find that micafungin initiates the uptake of salts, causing rapid swelling and rupture of mitochondria with release of cytochrome c. The inner membrane anion channel (IMAC) is altered by micafungin to transfer both cations and anions. We propose that binding of anionic micafungin to IMAC attracts cations into the ion pore for the rapid transfer of ion pairs.

Identification of LRRC8/VRAC channel structural elements required for regulation by cell volume increase and intracellular ionic strength.

VRACs mediate volume regulatory Cl- and organic solute efflux and are encoded by Lrrc8a-e genes. Native LRRC8/VRACs are heteromers with unknown stoichiometry comprising the essential LRRC8A subunit and another paralog. We utilize a chimeric protein approach to define LRRC8/VRAC structure-function relationships. Functional chimeric channels with physiologically relevant properties require the presence of the first intracellular loop (IL1) of LRRC8A and the first extracellular loop of another paralog. Replacement of the C-terminus of LRRC8C or LRRC8E with the C-terminus of LRRC8A (8A(C-term)) gives rise to robust plasma membrane anion channel activity. 8C-8A(C-term) and 8E-8A(C-term) chimeric channels exhibit greatly reduced sensitivity to reduced intracellular ionic strength (IS) and cannot be activated by hypotonicity-induced cell volume increases of up to 4-fold. However, both channels could be activated by rapid, stepwise volume increases of 2-2.5-fold induced by positive pressure applied to the patch pipette. Addition of the LRRC8A IL1 to both chimeras restored normal sensitivity to hypotonicity-induced volume increases and IS. A cryo-EM structure of a LRRC8 chimeric channel comprising LRRC8C with 25 amino acids unique to the LRRC8A IL1, 8C-8A(IL125), suggested a possible functional interaction between 8A(IL125) and leucine-rich repeat motif 5, LRR5. Deletion of 9 cytoplasm-facing LRR5 amino acids or substitution with alanine greatly reduced volume and IS sensitivity of 8C-8A(IL125) channels. Point mutations in the 8A(IL125) region identified two unique lysine residues required for channel regulation. We conclude that the LRRC8 C-terminus is 1) required for channel volume and IS sensing, 2) that channel regulation requires a functional interaction between the unique 8A(IL125) region and LRR5 and 3) that LRR5 and the 8A(IL125) region are key components of the LRRC8/VRAC volume and IS sensing machinery.

Bone Marrow Mononuclear Cells Restore Normal Mitochondrial Ca2+ Handling and Ca2+-Induced Depolarization of the Internal Mitochondrial Membrane by Inhibiting the Permeability Transition Pore After Ischemia/Reperfusion

Acute kidney injury due to ischemia followed by reperfusion (IR) is a severe clinical condition with high death rates. IR affects the proximal tubule segments due to their predominantly oxidative metabolism and profoundly altered mitochondrial functions. We previously described the impact of IR on oxygen consumption, the generation of membrane potential (ΔΨ), and formation of reactive oxygen species, together with inflammatory and structural alterations. We also demonstrated the benefits of bone marrow mononuclear cells (BMMC) administration in these alterations. The objective of the present study has been to investigate the effect of IR and the influence of BMMC on the mechanisms of Ca2+ handling in mitochondria of the proximal tubule cells. IR inhibited the rapid accumulation of Ca2+ (Ca2+ green fluorescence assays) and induced the opening of the cyclosporine A-sensitive permeability transition pore (PTP), alterations prevented by BMMC. IR accelerated Ca2+-induced decrease of ΔΨ (Safranin O fluorescence assays), as evidenced by decreased requirement for Ca2+ load and t1/2 for complete depolarization. Addition of BMMC and ADP recovered the normal depolarization profile, suggesting that stabilization of the adenine nucleotide translocase (ANT) in a conformation that inhibits PTP opening offers a partial defense mechanism against IR injury. Moreover, as ANT forms a complex with the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane, it is possible that this complex is also a target for IR injury—thus favoring Ca2+ release, as well as the supramolecular structure that BMMC protects. These beneficial effects are accompanied by a stimulus of the citric acid cycle—which feed the mitochondrial complexes with the electrons removed from different substrates—as the result of accentuated stimulus of citrate synthase activity by BMMC.

Modulation of frequency and height of cytosolic calcium spikes by plasma membrane anion channels in guard cells.

Cytosolic calcium ([Ca2+]cyt) elevation activates plasma membrane anion channels in guard cells, which is required for stomatal closure. However, involvement of the anion channels in the [Ca2+]cyt elevation remains unclear. We investigated the involvement using Arabidopsis thaliana anion channel mutants, slac1-4 slah3-3 and slac1-4 almt12-1. Extracellular calcium induced stomatal closure in the wild-type plants but not in the anion channel mutant plants whereas extracellular calcium induced [Ca2+]cyt elevation both in the wild-type guard cells and in the mutant guard cells. The peak height and the number of the [Ca2+]cyt spike were lower and larger in the slac1-4 slah3-3 than in the wild type and the height and the number in the slac1-4 almt12-1 were much lower and much larger than in the wild type. These results suggest that the anion channels are involved in the regulation of [Ca2+]cyt elevation in guard cells.

Combined action of guard cell plasma membrane rapid- and slow-type anion channels in stomatal regulation

Initiation of stomatal closure by various stimuli requires activation of guard cell plasma membrane anion channels, which are defined as rapid (R)- and slow (S)-type. The single-gene loss-of-function mutants of these proteins are well characterized. However, the impact of suppressing both the S- and R-type channels has not been studied. Here, by generating and studying double and triple Arabidopsis thaliana mutants of SLOW ANION CHANNEL1 (SLAC1), SLAC1 HOMOLOG3 (SLAH3), and ALUMINUM-ACTIVATED MALATE TRANSPORTER 12/QUICK-ACTIVATING ANION CHANNEL 1 (QUAC1), we show that impairment of R- and S-type channels gradually increased whole-plant steady-state stomatal conductance. Ozone-induced cell death also increased gradually in higher-order mutants with the highest levels observed in the quac1 slac1 slah3 triple mutant. Strikingly, while single mutants retained stomatal responsiveness to abscisic acid, darkness, reduced air humidity, and elevated CO2, the double mutant lacking SLAC1 and QUAC1 was nearly insensitive to these stimuli, indicating the need for coordinated activation of both R- and S-type anion channels in stomatal closure.

Adaptable and Multifunctional Ion-Conducting Aquaporins.

Aquaporins function as water and neutral solute channels, signaling hubs, disease virulence factors, and metabolon components. We consider plant aquaporins that transport ions compared to some animal counterparts. These are candidates for important, as yet unidentified, cation and anion channels in plasma, tonoplast, and symbiotic membranes. For those individual isoforms that transport ions, water, and gases, the permeability spans 12 orders of magnitude. This requires tight regulation of selectivity via protein interactions and posttranslational modifications. A phosphorylation-dependent switch between ion and water permeation in AtPIP2;1 might be explained by coupling between the gates of the four monomer water channels and the central pore of the tetramer. We consider the potential for coupling between ion and water fluxes that could form the basis of an electroosmotic transducer. A grand challenge in understanding the roles of ion transporting aquaporins is their multifunctional modes that are dependent on location, stress, time, and development.

Fluorescence assay for simultaneous quantification of CFTR ion-channel function and plasma membrane proximity

The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma membrane anion channel that plays a key role in controlling transepithelial fluid movement. Excessive activation results in intestinal fluid loss during secretory diarrheas, whereas CFTR mutations underlie cystic fibrosis (CF). Anion permeability depends both on how well CFTR channels work (permeation/gating) and on how many are present at the membrane. Recently, treatments with two drug classes targeting CFTR—one boosting ion-channel function (potentiators) and the other increasing plasma membrane density (correctors)—have provided significant health benefits to CF patients. Here, we present an image-based fluorescence assay that can rapidly and simultaneously estimate both CFTR ion-channel function and the protein's proximity to the membrane. We monitor F508del-CFTR, the most common CF-causing variant, and confirm rescue by low temperature, CFTR-targeting drugs and second-site revertant mutation R1070W. In addition, we characterize a panel of 62 CF-causing mutations. Our measurements correlate well with published data (electrophysiology and biochemistry), further confirming validity of the assay. Finally, we profile effects of acute treatment with approved potentiator drug VX-770 on the rare-mutation panel. Mapping the potentiation profile on CFTR structures raises mechanistic hypotheses on drug action, suggesting that VX-770 might allow an open-channel conformation with an alternative arrangement of domain interfaces. The assay is a valuable tool for investigation of CFTR molecular mechanisms, allowing accurate inferences on gating/permeation. In addition, by providing a two-dimensional characterization of the CFTR protein, it could better inform development of single-drug and precision therapies addressing the root cause of CF disease.

GM1 as Adjuvant of Innovative Therapies for Cystic Fibrosis Disease.

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein is expressed at the apical plasma membrane (PM) of different epithelial cells. The most common mutation responsible for the onset of cystic fibrosis (CF), F508del, inhibits the biosynthesis and transport of the protein at PM, and also presents gating and stability defects of the membrane anion channel upon its rescue by the use of correctors and potentiators. This prompted a multiple drug strategy for F508delCFTR aimed simultaneously at its rescue, functional potentiation and PM stabilization. Since ganglioside GM1 is involved in the functional stabilization of transmembrane proteins, we investigated its role as an adjuvant to increase the effectiveness of CFTR modulators. According to our results, we found that GM1 resides in the same PM microenvironment as CFTR. In CF cells, the expression of the mutated channel is accompanied by a decrease in the PM GM1 content. Interestingly, by the exogenous administration of GM1, it becomes a component of the PM, reducing the destabilizing effect of the potentiator VX-770 on rescued CFTR protein expression/function and improving its stabilization. This evidence could represent a starting point for developing innovative therapeutic strategies based on the co-administration of GM1, correctors and potentiators, with the aim of improving F508del CFTR function.

The inverse relation between mitochondrial transmembrane potential and proteins α-helix in neuronal-like cells under static magnetic field and the role of VDAC

ABSTRACTIn this study, a correlation between cell channel α-helices displacement and the mitochondrial transmembrane potential after exposure of 3, 7, 15 and 24 h of neuronal-like cells to a uniform magnetic field at the intensity of 2 mT was shown. Fourier Transform Infrared (FTIR) Spectroscopy and fluorescence techniques were used to analyze the secondary structure of protein content and mitochondrial transmembrane potential, respectively. The main result of this study was represented by a significant inverse relation between the mitochondrial transmembrane potential and the intensity of the Amide I band that can be associated with time exposure. Given that mitochondrial transmembrane potential should be related to the gating state of voltage-dependent anion channel (VDAC) in mitochondrial membrane, this result could have a relevant role in medicine. Indeed, VDAC's irregular behavior can be associated with several varieties of mitochondria-associated pathologies and various forms of cancer and neurodegeneration.

Cigarette Smoke Exposure Induces Retrograde Trafficking of CFTR to the Endoplasmic Reticulum

Chronic obstructive pulmonary disease (COPD), which is most commonly caused by cigarette smoke (CS) exposure, is the third leading cause of death worldwide. The cystic fibrosis transmembrane conductance regulator (CFTR) is an apical membrane anion channel that is widely expressed in epithelia throughout the body. In the airways, CFTR plays an important role in fluid homeostasis and helps flush mucus and inhaled pathogens/toxicants out of the lung. Inhibition of CFTR leads to mucus stasis and severe airway disease. CS exposure also inhibits CFTR, leading to the decreased anion secretion/hydration seen in COPD patients. However, the underlying mechanism is poorly understood. Here, we report that CS causes CFTR to be internalized in a clathrin/dynamin-dependent fashion. This internalization is followed by retrograde trafficking of CFTR to the endoplasmic reticulum. Although this internalization pathway has been described for bacterial toxins and cargo machinery, it has never been reported for mammalian ion channels. Furthermore, the rapid internalization of CFTR is dependent on CFTR dephosphorylation by calcineurin, a protein phosphatase that is upregulated by CS. These results provide new insights into the mechanism of CFTR internalization, and may help in the development of new therapies for CFTR correction and lung rehydration in patients with debilitating airway diseases such as COPD.

Dual Role of Mitochondrial Porin in Metabolite Transport across the Outer Membrane and Protein Transfer to the Inner Membrane

The mitochondrial inner membrane harbors a large number of metabolite carriers. The precursors of carrier proteins are synthesized in the cytosol and imported into mitochondria by the translocase of the outer membrane (TOM) and the carrier translocase of the inner membrane (TIM22). Molecular chaperones in the cytosol and intermembrane space bind to the hydrophobic precursors to prevent their aggregation. We report that the major metabolite channel of the outer membrane, termed porin or voltage-dependent anion channel (VDAC), promotes efficient import of carrier precursors. Porin interacts with carrier precursors arriving in the intermembrane space and recruits TIM22 complexes, thus ensuring an efficient transfer of the precursors to the inner membrane translocase. Porin channel mutants impaired in metabolite transport are not disturbed in carrier import into mitochondria. We conclude that porin serves distinct functions as outer membrane channel for metabolites and as coupling factor for protein translocation into the inner membrane.

Effects of Chromogranin A and B Association on the Anion Channel Function in the Regulated Secretory Pathway

Chromogranins A and B (CHGA and CHGB), of the granin superfamily, are pivotal for the biogenesis, maturation and release of secretory granules in the regulated secretory pathway. They have been used as biomarkers for neuroendocrine tumors, cardiovascular diseases, and are genetically associated with Type 2 diabetes and psychiatric disorders. Although their roles in regulated secretion are conserved within the kingdom of eukaryotes, the molecular mechanisms underlying their functions inside the cells are unclear, which makes it difficult to target them for specific diseases. Chromogranin B was demonstrated to function as an anion channel in membrane. It is proposed to serve the long-sought anion conductance that is needed to shunt positive potential developed by H+-ATPase-drive proton pumping into dense-core secretory granules and maintain a more stable acidic intra-vesicular environment at pH ∼5.5. CHGA, on the other hand, was found to be in both a membrane-bound state and a soluble state. The membrane-bound CHGA alone does not conduct ions. Because CHGA and CHGB are usually present together in the secretory granules, we are studying the interactions between CHGA and CHGB in membrane, and investigate the roles of the CHGA/CHGB complex in granule maturation in neuroendocrine cells. Further, we are developing high-through screen assays to search for specific binders to the CHGA/CHGB complex and resolve the structural basis for its function using cryo-electron microscopy.

Structure-guided combination therapy to potently improve the function of mutant CFTRs

Available drugs are unable to effectively rescue the folding defects in vitro and ameliorate the clinical-phenotype of cystic fibrosis (CF), caused by deletion of F508 (ΔF508 or F508del) and some point mutations in the CF transmembrane conductance regulator (CFTR), a plasma membrane (PM) anion channel. To overcome the corrector efficacy ceiling, here we show that compounds targeting distinct structural defects of CFTR can synergistically rescue mutants expression and function at the PM. High throughput cell-based screens and mechanistic analysis identified three small-molecule series that target defects at the nucleotide binding domain (NBD1), NBD2 and their membrane spanning domains (MSDs) interfaces. While individually these compounds marginally improve ΔF508-CFTR folding efficiency, function, and stability, their combinations lead to ~50–100% of wild type-level correction in immortalized and primary human airway epithelia, and in mouse nasal epithelia. Likewise, corrector combinations were effective for rare missense mutations in various CFTR domains, probably acting via structural allostery, suggesting a mechanistic framework for their broad application.

Global Sensitivity Analysis of OnGuard Models Identifies Key Hubs for Transport Interaction in Stomatal Dynamics.

The physical requirement for charge to balance across biological membranes means that the transmembrane transport of each ionic species is interrelated, and manipulating solute flux through any one transporter will affect other transporters at the same membrane, often with unforeseen consequences. The OnGuard systems modeling platform has helped to resolve the mechanics of stomatal movements, uncovering previously unexpected behaviors of stomata. To date, however, the manual approach to exploring model parameter space has captured little formal information about the emergent connections between parameters that define the most interesting properties of the system as a whole. Here, we introduce global sensitivity analysis to identify interacting parameters affecting a number of outputs commonly accessed in experiments in Arabidopsis (Arabidopsis thaliana). The analysis highlights synergies between transporters affecting the balance between Ca2+ sequestration and Ca2+ release pathways, notably those associated with internal Ca2+ stores and their turnover. Other, unexpected synergies appear, including with the plasma membrane anion channels and H+-ATPase and with the tonoplast TPK K+ channel. These emergent synergies, and the core hubs of interaction that they define, identify subsets of transporters associated with free cytosolic Ca2+ concentration that represent key targets to enhance plant performance in the future. They also highlight the importance of interactions between the voltage regulation of the plasma membrane and tonoplast in coordinating transport between the different cellular compartments.

Tubulin on Biomimetic Mitochondrial Membranes: Structural Features and Lipid Discrimination

Dimeric tubulin, an abundant cytosolic water-soluble protein, has emerged as an important regulatory factor of the permeability of the voltage-dependent anion channel (VDAC) in the mitochondrial outer membrane, with implications for mitochondrial energetics as well as the Warburg effect observed in cancer cells. A wealth of recent in vitro and in vivo evidence points to a mechanism of this regulation, in which a charged C-terminal tail of tubulin inserts into the water-filled VDAC pore, thus blocking metabolite fluxes through this passive transport channel. The membrane-binding properties of tubulin have proven difficult to ascertain, as early work generated contradictory results regarding the tubulin-membrane association, including whether it was integral or peripheral in nature. Here we report on a comprehensive biophysical study of tubulin binding to lipid membranes with compositions that mimic the mitochondrial outer membrane. A combination of surface plasmon resonance, bilayer overtone (second harmonics) analysis, and single-channel recordings show that tubulin distinguishes between lamellar and non-lamellar lipid components of the membrane. To obtain the structural features of the tubulin heterodimer on the membrane surface, we have employed neutron reflectometry (NR) of tubulin on a tethered bilayer lipid membrane platform. The NR results definitively show that tubulin binds peripherally, and in combination with molecular dynamics (MD) simulations suggest the binding domain to be a highly conserved amphipathic α-helix on α-tubulin. Thus tubulin joins a short but growing list of amphitropic proteins that target cell and organelle membranes by sensing lipid packing defects via amphipathic α-helices, suggesting a pathway by which lipid homeostasis regulates mitochondrial function.

Reactive Oxygen Species, Endoplasmic Reticulum Stress and Mitochondrial Dysfunction: The Link with Cardiac Arrhythmogenesis.

Background: Cardiac arrhythmias represent a significant problem globally, leading to cerebrovascular accidents, myocardial infarction, and sudden cardiac death. There is increasing evidence to suggest that increased oxidative stress from reactive oxygen species (ROS), which is elevated in conditions such as diabetes and hypertension, can lead to arrhythmogenesis.Method: A literature review was undertaken to screen for articles that investigated the effects of ROS on cardiac ion channel function, remodeling and arrhythmogenesis.Results: Prolonged endoplasmic reticulum stress is observed in heart failure, leading to increased production of ROS. Mitochondrial ROS, which is elevated in diabetes and hypertension, can stimulate its own production in a positive feedback loop, termed ROS-induced ROS release. Together with activation of mitochondrial inner membrane anion channels, it leads to mitochondrial depolarization. Abnormal function of these organelles can then activate downstream signaling pathways, ultimately culminating in altered function or expression of cardiac ion channels responsible for generating the cardiac action potential (AP). Vascular and cardiac endothelial cells become dysfunctional, leading to altered paracrine signaling to influence the electrophysiology of adjacent cardiomyocytes. All of these changes can in turn produce abnormalities in AP repolarization or conduction, thereby increasing likelihood of triggered activity and reentry.Conclusion: ROS plays a significant role in producing arrhythmic substrate. Therapeutic strategies targeting upstream events include production of a strong reducing environment or the use of pharmacological agents that target organelle-specific proteins and ion channels. These may relieve oxidative stress and in turn prevent arrhythmic complications in patients with diabetes, hypertension, and heart failure.

Abstract 441: Role of the Mitochondrial Translocator Protein (TSPO) in the Proarrhythmic Vulnerability of the Diabetic Heart

In structurally normal hearts, inhibition of the mitochondrial translocator protein (TSPO) prevents ROS-mediated proarrhythmia. Whether and how TSPO modulates electrophysiological (EP) function of diabetic hearts, in which classically cardioprotective pathways are impaired, is unknown. We determined the EP effects of TSPO activation & inhibition in a rat model of type 2 diabetes mellitus (t2DM) and studied the mitochondrial pathway underlying TSPO-related proarrhythmia. Methods: TSPO expression & function were determined in Zucker Diabetic Fatty (ZDF) rats with established t2DM (N=16) compared to controls (ctrl, N=15). Optical mapping was performed before & after challenge of hearts with ischemia for 12 min followed by reperfusion. Hearts underwent TSPO activation (FGIN 4.6uM, N=10), blockade (DZP 64uM, N=7) or no treatment (N=14). Dependence of TSPO-related proarrhythmia on ROS and on the PTP a target of TSPO were also determined. Results: t2DM hearts exhibited markedly increased TSPO expression (mRNA & protein) compared to ctrl. TSPO activation did not alter EP properties at baseline in ctrl or t2DM. In contrast TSPO activation accelerated action potential (AP) shortening during ischemia in t2DM but not ctrl hearts. Following 8 min of ischemia, FGIN-mediated AP shortening was 2X greater in t2DM compared to ctrl (p=0.008). FGIN-treated t2DM (5/7) but not ctrl (0/5) hearts exhibited VT by 12min of ischemia (p=0.027). AP shortening and VT in FGIN-treated t2DM hearts were not prevented by the PTP blocker Cyclosporin A but rather by the ROS scavenger EUK implicating the inner membrane anion channel in the proarrhythmic activity of TSPO. Upon reperfusion, TSPO activation with FGIN caused VF in 100% of ctrl & t2DM hearts compared to ~50% of untreated hearts. DZP prolonged the AP in t2DM (by 53% p<0.01) but not ctrl (p=0.14) hearts consistent with heightened sensitivity of t2DM to TSPO ligands. DZP blunted AP shortening during ischemia and prevented VF upon reperfusion in ctrl and t2DM hearts. Conclusion: t2DM hearts exhibit heightened sensitivity to TSPO ligands. TSPO upregulation may be a key factor in the proarrhythmic vulnerability of the diabetic heart. The cardioprotective efficacy of TSPO inhibition against arrhythmias is preserved in t2DM.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Uses its C-Terminus to Regulate the A2B Adenosine Receptor.

CFTR is an apical membrane anion channel that regulates fluid homeostasis in many organs including the airways, colon, pancreas and sweat glands. In cystic fibrosis, CFTR dysfunction causes significant morbidity/mortality. Whilst CFTR’s function as an ion channel has been well described, its ability to regulate other proteins is less understood. We have previously shown that plasma membrane CFTR increases the surface density of the adenosine 2B receptor (A2BR), but not of the β2 adrenergic receptor (β2AR), leading to an enhanced, adenosine-induced cAMP response in the presence of CFTR. In this study, we have found that the C-terminal PDZ-domain of both A2BR and CFTR were crucial for this interaction, and that replacing the C-terminus of A2BR with that of β2AR removed this CFTR-dependency. This observation extended to intact epithelia and disruption of the actin cytoskeleton prevented A2BR-induced but not β2AR-induced airway surface liquid (ASL) secretion. We also found that CFTR expression altered the organization of the actin cytoskeleton and PDZ-binding proteins in both HEK293T cells and in well-differentiated human bronchial epithelia. Furthermore, removal of CFTR’s PDZ binding motif (ΔTRL) prevented actin rearrangement, suggesting that CFTR insertion in the plasma membrane results in local reorganization of actin, PDZ binding proteins and certain GPCRs.