Redox-dependent Cd2+ inhibition of BK-type Ca2+-activated K+ channels.

Large-conductance Ca2+-activated K+ channels (BK channels) are formed by Slo1 subunits as a homotetramer. Besides Ca2+, other divalent cations, such as Cd2+, also activate BK channels when applied intracellularly by shifting the conductance-voltage relation to more negative voltages. However, we found that if the inside-out patch containing BK channels was treated with solution containing reducing agents such as dithiothreitol (DTT), then subsequent Cd2+ application completely inhibited BK currents. The DTT-dependent Cd2+ inhibition could be reversed by treating the patch with solutions containing H2O2, suggesting that a redox reaction regulates the Cd2+ inhibition of BK channels. Similar DTT-dependent Cd2+ inhibition was also observed in a mutant BK channel, Core-MT, in which the cytosolic domain of the channel is deleted, and in the proton-activated Slo3 channels but not observed in the voltage-gated Shaker K+ channels. A possible mechanism for the DTT-dependent Cd2+ inhibition is that DTT treatment breaks one or more disulfide bonds between cysteine pairs in the BK channel protein and the freed thiol groups coordinate with Cd2+ to form an ion bridge that blocks the channel or locks the channel at the closed state. However, surprisingly, none of the mutations of all cysteine residues in Slo1 affect the DTT-dependent Cd2+ inhibition. These results are puzzling, with an apparent contradiction: on one hand, a redox reaction seems to regulate Cd2+ inhibition of the channel, but on the other hand, no cysteine residue in the Slo1 subunit seems to be involved in such inhibition.

Safety Profile of Rapamycin Perfluorocarbon Nanoparticles for Preventing Cisplatin-Induced Kidney Injury.

Cancer treatment-induced toxicities may restrict maximal effective dosing for treatment and cancer survivors' quality of life. It is critical to develop novel strategies that mitigate treatment-induced toxicity without affecting the efficacy of anti-cancer therapies. Rapamycin is a macrolide with anti-cancer properties, but its clinical application has been hindered, partly by unfavorable bioavailability, pharmacokinetics, and side effects. As a result, significant efforts have been undertaken to develop a variety of nano-delivery systems for the effective and safe administration of rapamycin. While the efficacy of nanostructures carrying rapamycin has been studied intensively, the pharmacokinetics, biodistribution, and safety remain to be investigated. In this study, we demonstrate the potential for rapamycin perfluorocarbon (PFC) nanoparticles to mitigate cisplatin-induced acute kidney injury with a single preventative dose. Evaluations of pharmacokinetics and biodistribution suggest that the PFC nanoparticle delivery system improves rapamycin pharmacokinetics. The safety of rapamycin PFC nanoparticles was shown both in vitro and in vivo. After a single dose, no disturbance was observed in blood tests or cardiac functional evaluations. Repeated dosing of rapamycin PFC nanoparticles did not affect overall spleen T cell proliferation and responses to stimulation, although it significantly decreased the number of Foxp3+CD4+ T cells and NK1.1+ cells were observed.

Gating and Regulatory Mechanisms of TMEM16 Ion Channels and Scramblases.

The transmembrane protein 16 (TMEM16) family consists of Ca2+-activated ion channels and Ca2+-activated phospholipid scramblases (CaPLSases) that passively flip-flop phospholipids between the two leaflets of the membrane bilayer. Owing to their diverse functions, TMEM16 proteins have been implicated in various human diseases, including asthma, cancer, bleeding disorders, muscular dystrophy, arthritis, epilepsy, dystonia, ataxia, and viral infection. To understand TMEM16 proteins in health and disease, it is critical to decipher their molecular mechanisms of activation gating and regulation. Structural, biophysical, and computational characterizations over the past decade have greatly advanced the molecular understanding of TMEM16 proteins. In this review, we summarize major structural features of the TMEM16 proteins with a focus on regulatory mechanisms and gating.

Molecular underpinning of intracellular pH regulation on TMEM16F.

TMEM16F, a dual-function phospholipid scramblase and ion channel, is important in blood coagulation, skeleton development, HIV infection, and cell fusion. Despite advances in understanding its structure and activation mechanism, how TMEM16F is regulated by intracellular factors remains largely elusive. Here we report that TMEM16F lipid scrambling and ion channel activities are strongly influenced by intracellular pH (pHi). We found that low pHi attenuates, whereas high pHi potentiates, TMEM16F channel and scramblase activation under physiological concentrations of intracellular Ca2+ ([Ca2+]i). We further demonstrate that TMEM16F pHi sensitivity depends on [Ca2+]i and exhibits a bell-shaped relationship with [Ca2+]i: TMEM16F channel activation becomes increasingly pHi sensitive from resting [Ca2+]i to micromolar [Ca2+]i, but when [Ca2+]i increases beyond 15 µM, pHi sensitivity gradually diminishes. The mutation of a Ca2+-binding residue that markedly reduces TMEM16F Ca2+ sensitivity (E667Q) maintains the bell-shaped relationship between pHi sensitivity and Ca2+ but causes a dramatic shift of the peak [Ca2+]i from 15 µM to 3 mM. Our biophysical characterizations thus pinpoint that the pHi regulatory effects on TMEM16F stem from the competition between Ca2+ and protons for the primary Ca2+-binding residues in the pore. Within the physiological [Ca2+]i range, the protonation state of the primary Ca2+-binding sites influences Ca2+ binding and regulates TMEM16F activation. Our findings thus uncover a regulatory mechanism of TMEM16F by pHi and shine light on our understanding of the pathophysiological roles of TMEM16F in diseases with dysregulated pHi, including cancer.

An Additional Ca2+ Binding Site Allosterically Controls TMEM16A Activation.

Calcium (Ca2+) is the primary stimulus for transmembrane protein 16 (TMEM16) Ca2+-activated chloride channels and phospholipid scramblases, which regulate important physiological processes ranging from smooth muscle contraction to blood coagulation and tumor progression. Binding of intracellular Ca2+ to two highly conserved orthosteric binding sites in transmembrane helices (TMs) 6-8 efficiently opens the permeation pathway formed by TMs 3-7. Recent structures of TMEM16K and TMEM16F scramblases revealed an additional Ca2+ binding site between TM2 and TM10, whose functional relevance remains unknown. Here, we report that Ca2+ binds with high affinity to the equivalent third Ca2+ site in TMEM16A to enhance channel activation. Our cadmium (Cd2+) metal bridging experiments reveal that the third Ca2+ site's conformational states can profoundly influence TMEM16A's opening. Our study thus confirms the existence of a third Ca2+ site in TMEM16A, defines its functional importance in channel gating, and provides insight into a long-range allosteric gating mechanism of TMEM16 channels and scramblases.

Identification of BBOX1 as a Therapeutic Target in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive and highly lethal disease. Because of its heterogeneity and lack of hormone receptors or HER2 expression, targeted therapy is limited. Here, by performing a functional siRNA screening for 2-OG-dependent enzymes, we identified gamma-butyrobetaine hydroxylase 1 (BBOX1) as an essential gene for TNBC tumorigenesis. BBOX1 depletion inhibits TNBC cell growth while not affecting normal breast cells. Mechanistically, BBOX1 binds with the calcium channel inositol-1,4,5-trisphosphate receptor type 3 (IP3R3) in an enzymatic-dependent manner and prevents its ubiquitination and proteasomal degradation. BBOX1 depletion suppresses IP3R3-mediated endoplasmic reticulum calcium release, therefore impairing calcium-dependent energy-generating processes including mitochondrial respiration and mTORC1-mediated glycolysis, which leads to apoptosis and impaired cell-cycle progression in TNBC cells. Therapeutically, genetic depletion or pharmacologic inhibition of BBOX1 inhibits TNBC tumor growth in vitro and in vivo. Our study highlights the importance of targeting the previously uncharacterized BBOX1-IP3R3-calcium oncogenic signaling axis in TNBC. SIGNIFICANCE: We provide evidence from unbiased screens that BBOX1 is a potential therapeutic target in TNBC and that genetic knockdown or pharmacologic inhibition of BBOX1 leads to decreased TNBC cell fitness. This study lays the foundation for developing effective BBOX1 inhibitors for treatment of this lethal disease.This article is highlighted in the In This Issue feature, p. 1611.

Evidence that polyphenols do not inhibit the phospholipid scramblase TMEM16F.

TMEM16 Ca2+-activated phospholipid scramblases (CaPLSases) mediate rapid transmembrane phospholipid flip-flop and as such play essential roles in various physiological and pathological processes such as blood coagulation, skeletal development, viral infection, cell-cell fusion, and ataxia. Pharmacological tools specifically targeting TMEM16 CaPLSases are urgently needed to understand these novel membrane transporters and their contributions to health and disease. Tannic acid (TA) and epigallocatechin gallate (EGCG) were recently reported as promising TMEM16F CaPLSase inhibitors. However, our present study shows that TA and EGCG do not inhibit the phospholipid-scrambling or ion conduction activities of the dual-functional TMEM16F. Instead, we found that TA and EGCG mainly acted as fluorescence quenchers that rapidly suppress the fluorophores conjugated to annexin V, a phosphatidylserine-binding probe commonly used to report on TMEM16 CaPLSase activity. These data demonstrate the false positive effects of TA and EGCG on inhibiting TMEM16F phospholipid scrambling and discourage the use of these polyphenols as CaPLSase inhibitors. Appropriate controls as well as a combination of both fluorescence imaging and electrophysiological validation are necessary in future endeavors to develop TMEM16 CaPLSase inhibitors.

EAG2 potassium channel with evolutionarily conserved function as a brain tumor target.

Over 20% of the drugs for treating human diseases target ion channels, but no cancer drug approved by the US Food and Drug Administration (FDA) is intended to target an ion channel. We found that the EAG2 (Ether-a-go-go 2) potassium channel has an evolutionarily conserved function for promoting brain tumor growth and metastasis, delineate downstream pathways, and uncover a mechanism for different potassium channels to functionally cooperate and regulate mitotic cell volume and tumor progression. EAG2 potassium channel was enriched at the trailing edge of migrating medulloblastoma (MB) cells to regulate local cell volume dynamics, thereby facilitating cell motility. We identified the FDA-approved antipsychotic drug thioridazine as an EAG2 channel blocker that reduces xenografted MB growth and metastasis, and present a case report of repurposing thioridazine for treating a human patient. Our findings illustrate the potential of targeting ion channels in cancer treatment.

A comprehensive search for calcium binding sites critical for TMEM16A calcium-activated chloride channel activity.

TMEM16A forms calcium-activated chloride channels (CaCCs) that regulate physiological processes such as the secretions of airway epithelia and exocrine glands, the contraction of smooth muscles, and the excitability of neurons. Notwithstanding intense interest in the mechanism behind TMEM16A-CaCC calcium-dependent gating, comprehensive surveys to identify and characterize potential calcium sensors of this channel are still lacking. By aligning distantly related calcium-activated ion channels in the TMEM16 family and conducting systematic mutagenesis of all conserved acidic residues thought to be exposed to the cytoplasm, we identify four acidic amino acids as putative calcium-binding residues. Alterations of the charge, polarity, and size of amino acid side chains at these sites alter the ability of different divalent cations to activate the channel. Furthermore, TMEM16A mutant channels containing double cysteine substitutions at these residues are sensitive to the redox potential of the internal solution, providing evidence for their physical proximity and solvent accessibility.

Cardiac BIN1 folds T-tubule membrane, controlling ion flux and limiting arrhythmia.

Cardiomyocyte T tubules are important for regulating ion flux. Bridging integrator 1 (BIN1) is a T-tubule protein associated with calcium channel trafficking that is downregulated in failing hearts. Here we find that cardiac T tubules normally contain dense protective inner membrane folds that are formed by a cardiac isoform of BIN1. In mice with cardiac Bin1 deletion, T-tubule folding is decreased, which does not change overall cardiomyocyte morphology but leads to free diffusion of local extracellular calcium and potassium ions, prolonging action-potential duration and increasing susceptibility to ventricular arrhythmias. We also found that T-tubule inner folds are rescued by expression of the BIN1 isoform BIN1+13+17, which promotes N-WASP-dependent actin polymerization to stabilize the T-tubule membrane at cardiac Z discs. BIN1+13+17 recruits actin to fold the T-tubule membrane, creating a 'fuzzy space' that protectively restricts ion flux. When the amount of the BIN1+13+17 isoform is decreased, as occurs in acquired cardiomyopathy, T-tubule morphology is altered, and arrhythmia can result.

Calcium-activated chloride channel TMEM16A modulates mucin secretion and airway smooth muscle contraction.

Mucous cell hyperplasia and airway smooth muscle (ASM) hyperresponsiveness are hallmark features of inflammatory airway diseases, including asthma. Here, we show that the recently identified calcium-activated chloride channel (CaCC) TMEM16A is expressed in the adult airway surface epithelium and ASM. The epithelial expression is increased in asthmatics, particularly in secretory cells. Based on this and the proposed functions of CaCC, we hypothesized that TMEM16A inhibitors would negatively regulate both epithelial mucin secretion and ASM contraction. We used a high-throughput screen to identify small-molecule blockers of TMEM16A-CaCC channels. We show that inhibition of TMEM16A-CaCC significantly impairs mucus secretion in primary human airway surface epithelial cells. Furthermore, inhibition of TMEM16A-CaCC significantly reduces mouse and human ASM contraction in response to cholinergic agonists. TMEM16A-CaCC blockers, including those identified here, may positively impact multiple causes of asthma symptoms.