What Biologists Want from Their Chloride Reporters

REVIEW SUBJECT COLLECTION: TOOLS IN CELL BIOLOGY What biologists want from their chloride reporters – a conversation between chemists and biologists Matthew Zajac1,2, Kasturi Chakraborty1,2,3, Sonali Saha4,*, Vivek Mahadevan5,*, Daniel T. Infield6, Alessio Accardi7,8,9, Zhaozhu Qiu10,11 and Yamuna Krishnan1,2,‡

ABSTRACT inhibitory synaptic action potential (Kaila et al., 2014; Medina − + − Impaired chloride transport affects diverse processes ranging from et al., 2014). Under normal conditions, [Cl ]i is kept low by a K -Cl SLC12A5 neuron excitability to water secretion, which underlie epilepsy and cotransporter (KCC2, encoded by the gene ), allowing γ cystic fibrosis, respectively. The ability to image chloride fluxes with activation of the -aminobutyric acid (GABA) receptor (GABAAR) fluorescent probes has been essential for the investigation of the roles to drive chloride down the electrochemical gradient into the neuron of chloride channels and transporters in health and disease. Therefore, (Doyon et al., 2016). Improper chloride homeostasis is therefore developing effective fluorescent chloride reporters is critical to associated with several severe neurological disorders and epilepsies characterizing chloride transporters and discovering new ones. (Ben-Ari et al., 2012; Huberfeld et al., 2007; Payne et al., 2003). In However, each chloride channel or transporter has a unique epithelial cells, the chloride channel activity of the cystic fibrosis functional context that demands a suite of chloride probes with transmembrane regulator (CFTR) is associated with transcellular appropriate sensing characteristics. This Review seeks to juxtapose water and salt secretion. When this process is dysfunctional, the fluid the biology of chloride transport with the chemistries underlying layer lining the conducting airways cannot remove inhaled pathogens chloride sensors by exploring the various biological roles of and debris, leading to cystic fibrosis (CF) (Frizzell and Hanrahan, chloride and highlighting the insights delivered by studies using 2012; Saint-Criq and Gray, 2017). Chloride channels have also been chloride reporters. We then delineate the evolution of small-molecule identified that are sensitive to cell volume and extracellular pH, which sensors and genetically encoded chloride reporters. Finally, we are involved in signaling following cell swelling and hypoxia, analyze discussions with chloride biologists to identify the respectively (Qiu et al., 2014; Yang et al., 2019a). Finally, advantages and limitations of sensors in each biological context, as intracellular chloride channels have been implicated in endosomal well as to recognize the key design challenges that must be overcome pH regulation, lysosomal degradation, and endoplasmic reticulum for developing the next generation of chloride sensors. (ER) and mitochondria function (Chakraborty et al., 2017; Jia et al., 2015; Kornak et al., 2001; Mindell, 2012; Novarino et al., 2010; KEY WORDS: Chloride, Channel, Transporter, Fluorescent reporter, Piwon et al., 2000; Ponnalagu and Singh, 2017; Weinert et al., 2010). probe, Neuronal signaling, Epithelial secretion, Lysosome function Identifying and characterizing these channels has relied heavily on fluorescent chloride reporters. In many cases, screens using Introduction genetically encoded halide-sensitive yellow fluorescent protein As the most abundant anion in the body, chloride plays crucial roles in (YFP) variants have identified the chloride channel involved in a physiology across diverse cell types. As such, dysfunctional chloride given physiological process (Ullrich et al., 2019; Voss et al., 2014, homeostasis leads to a number of serious diseases. Correct chloride Qiu et al., 2014; Yang et al., 2019a). However, diverse physiological flux is maintained by diverse and often tissue-specific families of roles for chloride necessitate reporters that can provide information in anion channels, which exhibit low selectivity among other biological various contexts. For example, pH-sensitivity, sensing regime and anions but are referred to as chloride channels because of the level of quantification are all serious considerations when employing predominance of chloride. In mature neurons, the intracellular fluorescent chloride reporters. Because of this, small-molecule − chloride concentration ([Cl ]i) is a primary determinant of sensors, small molecules conjugated to macromolecules, and genetically encoded reporters have all been designed and fine-tuned to fit specific needs (Biwersi and Verkman, 1991; Kuner and 1Department of Chemistry, The University of Chicago, Chicago, IL 60637, USA. 2Grossman Institute of Neuroscience, Quantitative Biology and Human Behavior, Augustine, 2000; Saha et al., 2015). Furthermore, new classes of The University of Chicago, Chicago, IL 60637, USA. 3Ben May Department for chloride-sensitive molecules continue to emerge as biologists Cancer Research, The University of Chicago, Chicago, IL 60637, USA. 4Leibniz- Forschungsinstitut für Molekulare Pharmakologie (FMP), 13125 Berlin, Germany. uncover novel physiological relevance for chloride transport 5Department of Cell & Systems Biology, University of Toronto, Toronto, ON M5S (Amatori et al., 2012; Collins et al., 2013; Kim et al., 2017). 3G5, Canada. 6Department of Molecular Physiology and Biophysics, The University In this Review, we connect chloride physiology to the chemistry of Iowa, Iowa City, IA 52242, USA. 7Department of Anesthesiology, Weill Cornell Medical School, New York, NY 10065, USA. 8Department of Physiology and of chloride reporters. First, we detail key chloride channels and Biophysics, Weill Cornell Medical School, New York, NY 10065, USA. 9Department transporters, selecting those whose physiological relevance has of Biochemistry, Weill Cornell Medical School, New York, NY 10065, USA. 10Department of Physiology, Johns Hopkins University School of Medicine, been detailed by use of fluorescent reporters and those whose Baltimore, MD 21218, USA. 11Solomon H. Snyder Department of Neuroscience, uncertainties may be resolved by use of future fluorescent reporters. Johns Hopkins University School of Medicine, Baltimore, MD 21218, USA. We then present the current repertoire of fluorescent chloride *Present address: Section on Cellular and Synaptic Physiology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, Bethesda, reporters by outlining their development and refinement. Finally, MD 20892, USA. we hold discussions with chloride biologists to understand their experience using sensors in each biological context. By the end, we ‡Author for correspondence ([email protected]) advise readers on key design challenges that must be addressed

A.A., 0000-0002-6584-0102; Y.K., 0000-0001-5282-8852 with current sensors or overcome with novel chloride sensors. Journal of Cell Science

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The diverse roles of Cl− in biology anion channels, that contribute to glutamate release (Kimelberg Neuronal excitability et al., 1990; Zhou and Danbolt, 2014). One example is the Cl− has garnered attention largely due to its physiological role in ubiquitously expressed volume-regulated anion channel (VRAC), synaptic inhibition in the central nervous system (CNS). Presynaptic which upon cell swelling releases Cl− and organic osmolytes, such release of GABA and glycine activate their cognate postsynaptic as glutamate, to mediate a decrease in cell volume (Osei-Owusu receptors (GABAAR and GlyR, respectively), opening the et al., 2018). The leucine-rich repeat-containing protein 8A associated Cl− channel that then drives Cl− down its (LRRC8A; also known as SWELL1, Fig. 1A) and its homologs electrochemical gradient (Fig. 1A) (Doyon et al., 2016; Kaila, were identified as the subunits forming the pore of VRAC (Qiu − 1994; Staley et al., 1995). In mature neurons of the CNS, [Cl ]i is et al., 2014; Voss et al., 2014). SWELL1 is also implicated in maintained at a low concentration, such that receptor activation lymphocyte development, insulin secretion in pancreatic β-cells, causes Cl− influx and hyperpolarization of the neuronal membrane neuron–glia interaction and apoptotic volume decrease (Kang et al., − − (Fig. 2A). The Cl efflux necessary to sustain such low [Cl ]i is 2018; Planells-Cases et al., 2015; Platt et al., 2017; Yang et al., mediated mainly by the K+-Cl− cotransporter KCC2, which couples 2019b). K+ ion movement along its electrochemical gradient to move Cl− against its gradient (Kaila et al., 2014; Medina et al., 2014; Payne Salt and fluid secretion et al., 2003). In turn, the appropriate K+ gradient is maintained by Secretion by epithelial cells is critical to the function of several the Na+/K+ ATPase. Additionally, the Na+-K+-Cl− cotransporter organs and is best characterized in the respiratory tract and in (NKCC1, encoded by the gene SLC12A2) mediates Cl− influx and exocrine glands. A thin film called the airway surface liquid (ASL) + − − the Na -driven Cl /HCO3 exchanger (NCBE, encoded by the gene protects epithelial cells that lie along the conducting airways SLC4A10) mediates Cl− efflux (Blaesse et al., 2009; Doyon et al., leading to the lungs. Particles and pathogens that are inhaled are 2016). Together, these transporters reflect the dynamic ionic trappedintheuppermucuslayerandareremovedbyciliainthe equilibrium necessary to allow proper neuronal signaling (Fig. 1A). lower periciliary layer of the ASL (Saint-Criq and Gray, 2017). KCC2 is posited to be the primary determinant of this dynamic This process of mucociliary clearance requires the transcellular equilibrium because neuronal cells with low levels of KCC2 exhibit secretion of water to tightly regulate ASL hydration. In humans, − − excitatory GABA and glycine currents due to high [Cl ]i (Klein transcellular secretion is regulated by Cl efflux across the apical et al., 2018; Rivera et al., 1999; Szabadics et al., 2006). membrane into the extracellular space predominantly via CFTR, Furthermore, proper balance between NKCC1 and KCC2 during with some involvement of the Ca2+-activated Cl− channel development and mature neuronal KCC2 function are compromised TMEM16A (also known as ANO1) (Fig. 1B). Cl− transport is in numerous neurological disorders, such as autism spectrum the driving force for paracellular passive Na+ movement, and this disorders, neuropathic pain, Down’s syndrome and Huntington’s increased salt concentration generates an osmotic force for water disease (Coull et al., 2003; Dargaei et al., 2018; Deidda et al., 2015; secretion (Fig. 2B). In parallel, CFTR-mediated secretion of − + + + Tyzio et al., 2014). Additionally, numerous disease-causing KCC2 HCO3 counteracts H secretionbytheH/K ATPase subunit variants have been identified in human epilepsies (Moore et al., ATP12A to maintain pH homeostasis of the ASL (Shah et al., 2017). Owing to the centrality of these transporters in neurological 2016). In CF, defective CFTR function therefore compromises disorders, therapeutic strategies targeting KCC2 and NKCC1 at the formation of the periciliary layer, and the diminution of the fluid mRNA and protein levels are fast emerging (Mahadevan and layer leads to the accumulation of mucus and bacteria (Frizzell and Woodin, 2016; Mahadevan et al., 2017; Tang et al., 2019). Hanrahan, 2012). Interestingly, mice with dysfunctional CFTR do Conversely, excitatory currents are predominantly regulated not develop the bacterial infections typical of CF, potentially due through exocytosis of synaptic vesicles containing glutamate, but to expression of other anion channels or decreased expression of there are additional important non-vesicular mechanisms, such as ATP12A (Grubb and Boucher, 1999; Guilbault et al., 2007; Shah

TMEM16A SLC26A CFTR

GABAAR and GlyR Cl− K+ − Cl Secretory ClC-3 KCC2 vesicles Early ClC-4 endosome Cl− PAC

GOLAC ClC-5 − Late Na+ Cl Golgi endosome NCBE Cl− SWELL1 ClC-7 − 2 HCO3 GPHR Lysosome

CLCC1 CFTR BEST1 IMAC K+ 2 K+ Na+ 3 Na+ 2 Cl− CLIC NKCC1 CLIC NA+/K+-ATPase Nucleus

Fig. 1. Cellular location of Cl− transporters discussed in this Review. (A) Neuronal transporters on the plasma membrane that effect Cl− influx and efflux, with − associated counterions. (B) Cl transporters present on membranes of indicated organelles. See main text for further details. Journal of Cell Science

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A Neuronal excitability B Epithelial chloride secretion C Neuronal acidosis + + H H H+ H+ H+ GABA − + + − − Cl H O Na+/H O H − K Cl 2 2 + H O + CO + Cl H 2 2 HCO3+H H+ Lactate Depolarization + H CA IX neuron CFTR KCC2 GABAAR and GlyR MCT PAC Immature − High cytosolic [Cl ] TMEM16A − Cl GABA − Cl Lactic acid NKCC1 AE Hyperpolarization O2 neuron Mature KCC2 GABAAR and GlyR

− Hif-1A Hif-1B CA9 Low cytosolic [Cl ] HRE

D E Lysosomal acidification ER function F Lysosomal function + ATP H ADP+ P − − − i Cl 2+ +/+ / unc/unc V-ATPase Ca + H+ TMEM16A ORAI H V-ATPase

− − TRP3 Cl Cl CIC-7 + V-ATPase CIC-7 K+ Lysosome Ca2+ H BEST1 STIM1 Mature Cation − SERCA Cl channel Immature ER − Absent Cl CIC-7 Ca2+ Degree of ruffled border formation 2H+

− − Fig. 2. Physiology of key Cl transporters. (A) Low KCC2 expression in immature neurons maintains a high cytosolic [Cl ] such that GABAAR and GlyR activation depolarizes neurons through Cl− efflux. As neurons mature and KCC2 expression increases, this trend reverses. (B) CFTR and TMEM16A efflux Cl− on the apical membrane of epithelial cells, thereby causing passive Na+ transport and water secretion. AE, anion exchanger; NKCC1, Na+-K+-Cl− cotransporter. (C) Schematic of important determinants of hypoxia-induced acidosis and subsequent activation of PAC. Under hypoxia, HIF-1A associates with HIF-1B to transcribe target genes with hypoxia-response elements (HRE), such as carbonic anhydrase IX (CA9), which decreases extracellular pH. At the same time, increased lactic acid due to anaerobic glycolysis is exported as lactate through monocarboxylate transporters (MCTs) to decrease extracellular pH. This decreased pH activates PAC to facilitate Cl− influx. (D) Lysosomal pH maintained by ATPase activity, cation efflux and anion influx. (E) BEST1 regulates ER Ca2+ homeostasis through its Cl− channel activity and interaction with Ca2+ modulators. (F) Schematic illustration of the role for lysosomal Cl− import mediated by Clc-7, independent of its function as an exchanger. Osteoclasts from Clc-7unc/unc mice form mature ruffled borders to the same degree as those from wild-type mice (+/+), whereas those from Clc-7−/− mice (−/−) do not. Dysfunction in ruffled border formation is linked to osteopetrosis. Adapted from Weinert et al. (2014). et al., 2016). CFTR also regulates the activity of other anion survival and increased metastasis (Ayoub et al., 2010; West et al., cotransporters in the SLC26A family (Fig. 1B), which when 2004). dysfunctional lead to conditions, such as chondrodysplasias, Cl− diarrhea and deafness through tissue-specific dysregulation of Acidosis anion secretion (Alper and Sharma, 2013; Duran et al., 2010; Acidosis occurs when extracellular pH falls below 7.35 due to Stewartetal.,2009). increased lactic acid production and expression of carbonic anhydrase Identified as the ‘cystic fibrosis gene’ in 1989, CFTR was posited (Fig. 2C) (Jamali et al., 2015). Acidosis accompanies many to encode or regulate a Cl− ion channel because epithelia from CF pathological conditions, such as ischemic stroke and inflammation patients are impermeable to Cl− (Quinton, 1983; Riordan et al., (Capurro et al., 2015; Yingjun and Xun, 2013). Following ischemic 1989; Rommens et al., 1989). In many CF patients, F508 in CFTR is stroke, extracellular pH as low as 6.0 causes neuronal death, while absent, which prevents its proper folding and maturation in the ER during inflammation, it causes pain. Acid-sensing ion channels and leads to its degradation (Du et al., 2005). Thus, current (ASICs) that elicit inwardly rectifying cation currents have long been therapeutic efforts are focused on small molecules that promote implicated in these signal transductions (Wemmie et al., 2013). the proper folding of mutant CFTR. As another example, Ca2+- However, acid-sensitive outwardly rectifying (ASOR) currents have activated Cl− channels (CaCCs) significantly contribute to also been associated with acidosis-induced cell death, albeit by a epithelial anion secretion in all cell types. The first to be less-understood molecular mechanism (Wang et al., 2007). molecularly identified was TMEM16A (Caputo et al., 2008; ASOR currents were first described in rat Sertoli cells and Schroeder et al., 2008; Yang et al., 2008). TMEM16A is a HEK-293 cells, and subsequently in diverse mammalian tissues therapeutic target that could act as a potential compensatory (Auzanneau et al., 2003; Nobles et al., 2004). The biophysical mechanism when CFTR is dysfunctional. Drugs that increase characteristics of the ASOR channel have been established intracellular Ca2+ can indirectly activate TMEM16A to offset low (Lambert and Oberwinkler, 2005; Sato-Numata et al., 2013). CFTR activity (Cuthbert, 2011). Further, TMEM16A is upregulated However, the molecular identity of the channel responsible was only in various cancers, and its overexpression is associated with reduced very recently identified as TMEM206, also known as proton- Journal of Cell Science

3 REVIEW Journal of Cell Science (2020) 133, jcs240390. doi:10.1242/jcs.240390 activated Cl− channel (PAC or PACC1, Fig. 1A), in two acidification, but is yet to be characterized (Fig. 1B). In addition, independent screens (Ullrich et al., 2019; Yang et al., 2019a). a screen for mutant cell lines with delayed protein transport PAC is involved in acid-induced neuronal cell death and PAC- identified the anion channel G protein-coupled receptor 89 knockout mice are partially protected from ischemic brain injury (GPR89), also called the Golgi pH regulator (GPHR, Fig. 1B) as (Yang et al., 2019a). responsible for impaired glycosylation and defective luminal Golgi pH (Maeda et al., 2008). Finally, pancreatic β-cells of ClC-3- Organelle acidification knockout mice displayed defective insulin exocytosis and secretory As the most abundant anion in the body, Cl− also mediates the granule acidification, implicating ClC-3 in the pH regulation of lumenal acidification of organelles. Organelle acidification is well insulin secretory granules (Deriy et al., 2009a). studied in lysosomes and in secretory organelles. Lumenal pH in CFTR has been implicated in pH regulation based on early these organelles is crucial to their functions – post-translational evidence that the Golgi was mildly hypoacidified in epithelial cells modifications along the secretory pathway, ligand trafficking in derived from CF patients (Barasch et al., 1991); this was endosomes and macromolecule degradation in lysosomes are all hypothesized to lower sialyltransferase activity, a known highly dependent on pH (Mindell, 2012). Proper organelle abnormality in CF (Haggie and Verkman, 2009b). Modest acidification has been shown to be compromised in cancer, drug hypoacidification of endosomal pH was also observed in resistance and autism spectrum disorders (Rivinoja et al., 2006; fibroblasts lacking CFTR or expressing ΔF508 CFTR (Biwersi Ullman et al., 2018; Weisz, 2003). The protons needed to establish and Verkman, 1994). Separately, pH measurements in alveolar organelle acidity are actively pumped against their gradient by a macrophages of CFTR-knockout mice revealed defective lysosomal V-ATPase (Ohkuma et al., 1982). Without the movement of ions of acidification, thereby leading to phagolysosomes being too alkaline the appropriate charge (so-called counterions), ATPase action to destroy bacteria (Di et al., 2006). The two most common CFTR would generate a voltage difference that would impede further mutations, ΔF508 and G551D, also hampered bacterial degradation pumping. Thus, regulated anion influx, cation efflux or a and showed defective acidification of lysosomes and phagosomes combination of both are necessary for organelle acidification (Deriy et al., 2009b). However, other studies have since challenged (Fig. 2D). these claims, as the use of more reliable assays revealed that The first evidence for the role of Cl− in organelle acidification organelle acidification is independent of CFTR function (Dunn came from purified lysosomes, which failed to accumulate a weakly et al., 1994; Haggie and Verkman, 2007, 2009a; Lukacs et al., 1992; − 2− basic dye when Cl in the solution was replaced with SO4 Seksek et al., 1996). (Dell’Antone, 1979). The application of a voltage-sensitive dye In short, the role of Cl− in organelle acidification is surprisingly revealed that Cl− and other anions abolished lysosomal membrane still unclear, and much of the evidence is controversial, especially potential more significantly than K+ ions (Harikumar and Reeves, with respect to CFTR. One of the sources of this ambiguity is the 1983). The main candidates posited to be responsible for this Cl− complicated subcellular and tissue distribution of the relevant Cl− flux were members of the ClC (proteins are also known designated channels and transporters. Furthermore, the complete lack of as CLCN) family of Cl− channels and transporters (Fig. 1B). For appropriate Cl− reporters has proved to be a major roadblock in example, ClC-7-knockout mice suffer from osteopetrosis due to clarifying the role of Cl− in organelle acidification. their inability to acidify the ruffled border in osteoclasts, which impairs bone resorption; it is therefore hypothesized that ClC-7 Organelle function facilitates acidification of lysosomes, which in turn fuse with the Cl− plays more subcellular roles beyond its involvement in organelle plasma membrane to form the acidic ruffled border (Kornak acidification. In the ER, the activity of a few Cl− channels is et al., 2001). ClC-7 knockdown reduced lysosomal staining of implicated in Ca2+ homeostasis and thereby ER stress (Fig. 2E). This LysoTracker, implying a hypoacidification of lysosomes (Graves was first shown in the sarcoplasmic reticulum (SR) membrane in et al., 2008). However, more accurate measurements of lysosomal smooth muscle cells, and subsequently also in the ER of epithelial pH in neurons, fibroblasts and macrophages of ClC-7-knockout cells (Hirota et al., 2006; Neussert et al., 2010; Pollock et al., 1998). mice have shown that acidification is unimpaired (Kasper et al., There is compelling evidence that implicates bestrophin-1 (BEST1), a 2005; Kornak et al., 2001; Lange et al., 2006; Steinberg et al., calcium Cl− cotransporter mutated in vitelliform macular dystrophy 2010). It is therefore yet to be established if the Cl− current provided (VMD) or Best disease, as having such a role. Bestrophin-1 was first by ClC-7 directly facilitates lysosomal acidification. Additionally, characterized on the basolateral membrane of retinal pigment ClC-4 knockdown reduces transferrin receptor recycling due to epithelia (RPE) and found to regulate cytosolic Cl− and fluid defective endosome acidification (Mohammad-Panah et al., 2003). secretion, similar to other CaCCs (Kunzelmann et al., 2007; Sun Hepatocyte endosomes from ClC-3-knockout mice showed low et al., 2002). However, it also localizes to the ER, which is the largest [Cl−] and high pH (Hara-Chikuma et al., 2005a). Similarly, intracellular Ca2+ store (Fig. 1B). Ca2+ release from the ER is reduced endosomes in kidney proximal tubule cells of ClC-5-knockout in primary RPE cells lacking bestrophin-1 and mouse models of Best mice failed to acidify and showed reduced [Cl−] (Hara-Chikuma disease (Barro-Soria et al., 2010; Zhang et al., 2010). Another et al., 2005b). putative ER Cl− channel, Cl− channel CLIC-like 1 (CLCC1), is also Organelles on the secretory pathway also undergo progressive implicated in ER stress, potentially through the disruption of Ca2+ acidification, and V-type ATPase activity and counterion flux are homeostasis (Fig. 1B) (Jia et al., 2015). implicated. However, there is a severe lack of knowledge related to Endosomal Cl− levels are crucial for proper ligand trafficking and the underlying transporters here due to the difficulties associated cargo degradation within the lysosome. The two Cl− transporters with purifying intact organelles and distinguishing between implicated in these processes are ClC-5 and ClC-7, which are Cl−/ transiting and resident ion channels (Judah and Thomas, 2006; H+ exchangers that facilitate Cl− influx. ClC-5 is primarily Paroutis et al., 2004). Large anion conductance has been identified expressed in kidney and intestinal epithelia where it resides in in purified rat liver Golgi (Nordeen et al., 2000; Thompson et al., early endosomes. Mutations in ClC-5 cause Dent’s disease, which is

2002). The Golgi anion channel GOLAC may facilitate characterized by proteinuria (Devuyst et al., 1999; Günther et al., Journal of Cell Science

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1998; Lloyd et al., 1996). ClC-7 is ubiquitously expressed and Cl−-sensitive fluorescent probes. The first of these were localizes with its β-subunit Ostm1 to late endosomes and lysosomes synthetic quinoline-based dyes, namely 6-methoxy-N-(3- (Kornak et al., 2001). Mice lacking ClC-7 or Ostm1 exhibit sulfopropyl) quinolinium (SPQ), N-(ethoxycarbonylmethyl)-6- lysosomal storage disorders in neurons and renal cells, as well as methoxyquinolinium (MQAE) and 6-methoxy-N-ethylquinolinium irregular lysosomal morphology (Kasper et al., 2005; Lange et al., (MEQ) (Fig. 3A) (Biwersi and Verkman, 1991; Verkman, 1990). 2006). Evidence that ClC-5 and ClC-7 play physiological roles When these dyes are excited, they collide with Cl− ions in their excited beyond supplying counterions for protons comes from mouse states and return to the ground state through a non-radiative path, models where a single mutation uncouples ion transport and otherwise known as dynamic collisional quenching. A high [Cl−] thus converts the exchangers into unidirectional transporters, designated decreases their fluorescence. These dyes are insensitive to pH and unc unc − as ClC-5 or ClC-7 . Such a transporter retains an endosomal HCO3 and show microsecond response times, yet suffer from some acidification role, but lacks the role provided by exchange. Indeed, critical limitations. First, the dyes are not ratiometric. Thus, the renal endosomes from ClC-5unc mice acidify normally but exhibit fluorescence readout depends on the uptake of the probe, its cellular proteinuria and impaired endocytosis (Novarino et al., 2010). distribution and the optical thickness of the sample (Arosio and Ratto, Similarly, lysosomal pH in ClC-7unc and ClC-7−/− mice is 2014). Second, they suffer from photobleaching (Geddes et al., 2001). unaffected, but the storage disorder or osteopetrosis phenotype of Third, probes like MEQ are cell impermeable, and must be reduced to ClC-7unc mice is as severe as in ClC-7−/− mice, indicating that ClC- the cell-permeable diHMEQ, before being re-oxidized to the Cl−- 7 affects lysosomal function without altering acidification (Fig. 2F) sensitive form (Ashton et al., 2015). Finally, their degree of quenching (Weinert et al., 2010). Interestingly, when mice express a transport- is concentration dependent as they self-quench at high dye deficient mutant of ClC-7, designated as ClC-7td, that is capable of concentrations (Kaneko et al., 2004). In spite of these limitations, undergoing protein–protein interactions, they show normal Cl− indicators have provided useful qualitative information. For pigmentation and less severe neurodegeneration, suggesting a role example, MEQ was used to confirm that Cl− influx into neurons was for ClC-7 interaction partners in those processes (Weinert et al., caused by GABA receptor activation (Inglefield and Schwartz-Bloom, 2014). 1997), and SPQ was used to estimate intracellular Cl− by flow Finally, the nuclear envelope, nucleoplasm and inner mitochondrial cytometry (Pilas and Durack, 1997). In addition, the low-wavelength membrane also host members of the versatile Cl− intracellular channel excitation problem has been addressed with longer wavelength (CLIC) family (Fig. 1B). Interestingly, CLICs are mostly found in the acridinium-based variants, such as N-methylacridinium-9- soluble state, but in response to cues, such as oxidation and pH carboxamide (MACA) and lucigenin (Fig. 3B) (Biwersi et al., 1994; changes, insert into cellular membranes and act as anion channels Kovalchuk and Garaschuk, 2012). under certain conditions (Domingo-Fernández et al., 2017; Shukla Shortly after, YFP was discovered to be halide-sensitive from the et al., 2009). They participate in diverse processes, such as cell cycle observation that its pKa was dependent on the concentration of control, actin remodeling, vesicular pH regulation, membrane halide or nitrate ions (Wachter and Remington, 1999). Recognizing potential regulation and apoptosis, but the pathways are poorly the potential to develop this into a reporter for intracellular Cl−, the defined (Argenzio and Moolenaar, 2016). Five out of the six CLIC protein was engineered for higher halide affinity. The introduction − homologs contain the canonical nuclear localization signal (NLS) of an additional mutation, H148Q, improved the Kd for Cl from KKYR, and have been observed to translocate to the nucleoplasm or 777 mM to 154 mM, and X-ray crystallography revealed a specific outer nuclear membrane (Gururaja Rao et al., 2018). Despite the halide-binding site (Jayaraman et al., 2000; Wachter et al., 2000). absence of mitochondrial targeting sequences, some isoforms localize YFP-H148Q is advantageous over small molecules because of its to the inner mitochondrial membrane (Ponnalagu et al., 2016). higher photostability, longer excitation wavelength, an improved Mitochondrial CLICs are posited to regulate apoptosis, given that Cl− ability for subcellular targeting and cellular retention. It was channel blockers inhibit reactive oxygen species (ROS)-induced and successfully applied to identify novel CFTR agonists in high- p53-mediated apoptosis (Fernández-Salas et al., 2002; Heimlich and throughput screens (Galietta et al., 2001c). Subsequently, further Cidlowski, 2006; Suh et al., 2004). In addition, other anion channels enhancements of its Cl− affinity into physiologically relevant under the umbrella term of the inner mitochondrial anion channel regimes led to the development of the variant YFP-H148Q- − (IMAC) are involved in mitochondrial membrane potential I152L. This variant has a Kd of 88 mM for Cl ,makingit oscillations, yet remain molecularly uncharacterized (Fig. 1B) suitable to measure Cl− in a physiological setting (Galietta et al., (O’Rourke, 2007; Ponnalagu and Singh, 2017; Tomaskova and 2001b). Similar mutants have been used to identify GABA Ondrias, 2010). Thus, our current understanding of mitochondrial and receptor agonists and CaCC inhibitors in high-throughput nuclear Cl− homeostasis is limited, due to the low abundance of these screens (De La Fuente et al., 2008; Kruger et al., 2005; channels or their low probability of being open, as well as the inability Rhoden et al., 2007). to perform unbiased subcellular screens using current reporters. All of the aforementioned Cl− channels and exchangers are described in Second generation – the development of ratiometric quantification Table S1. A major limitation faced by all the aforementioned Cl− sensors is the lack of a second emission wavelength that is insensitive to Cl−, Sensors available to the community which could be used to normalize for reporter distribution. The First generation – the identification of Cl−-sensitive scaffolds absence of ratiometry makes quantitative measurement highly The ability to measure [Cl−] using fluorescent reporters began with the laborious, if not impossible. To overcome this, one must conjugate a identification of Cl−-sensitive dyes and the discovery that YFP is Cl−-sensitive moiety to one that is Cl− insensitive. For small- sensitive to halides. This was a major advance over previous molecule indicators, this was first achieved by chemically linking approaches that relied on electrophysiology, which although highly such two moieties in the synthesis of bis-DMXPQ; here, the Cl−- precise, are not amenable for high-throughput studies or for probing sensitive SPQ is linked to the Cl−-insensitive 6-aminoquinolinium intracellular Cl− channels and transporters. Hence, the ability to (AQ) through a rigid xylyl spacer (Fig. 3A) (Jayaraman et al., 1999). − − study Cl homeostasis was greatly advanced by the discovery of Upon excitation at 365 nm, bis-DMXPQ showed Cl -sensitive Journal of Cell Science

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A HO O O N O O S bis-DMXPQ: R = O SPQ: R = O N COOH N O O R MQ-AF: R = MQAE: R = O N H O MY: R = N N MEQ: R = CH2CH2 MQ-DS: R = O N S N O H O NH2 N B CDE High − HighH R Cl − N N High Cl Low H − Low Cl − Low Cl H O NH 2 Low chloride High chloride Low chloride High chloride N 5 mM 100 mM 5mM 100mM R Low acidity High acidity pH=7 pH=5 Lucigenin: R = H BAC: R = CH2CH2CH2COOH

Cl− pH Cl−

Fig. 3. Structures of first- and second-generation Cl− reporters. (A) Examples of small-molecule sensors based on the quinoline core (blue). Ratiometric reporters conjugate the Cl−-sensitive MQ (blue) to a Cl−-insensitive fluorophore (R). (B) Small-molecule sensors based on an acridinium core (green): MACA (shown on the left), and lucigenin and BAC (right). (C) Clensor uses the stoichiometry between complementary nucleic acid strands conjugated to the Cl−-sensitive BAC (blue diamond) and Cl−-insensitive AF647 (red circle) to obtain a ratiometric readout (AF647/BAC, or red/blue). The calibration curve of Clensor shows a linear fluorescent response across the entire physiological regime. Adapted from Saha et al. (2015). (D) ChloropHore maintains the Cl−-sensing mechanism of Clensor (box), but also features pH-sensitive FRET exchange between AF546 (green) and AF647 (red). At high acidity (low pH), FRET efficiency is increased by a conformational change in the DNA backbone, such that the AF647 emission intensity increases and the ratiometric AF546:AF647 (green/red) ratio is low. The calibration curve of ChloropHore shows a sigmoidal response to pH. This allows ChloropHore to report on Cl− and pH simultaneously and independently. Adapted with from Leung et al. (2019). (E) Clomeleon uses FRET from the Cl− -insensitive CFP (blue circle) to the Cl− -sensitive YFP variant, Topaz (yellow circle) to obtain a ratiometric readout (blue/yellow) of Cl− concentration. As Cl− concentration increases, FRET efficiency decreases and the CFP fluorescence increases relative to the YFP fluorescence. emission at 450 nm and a Cl−-insensitive emission at 565 nm. Other fluorophore on the other in a precise 1:1 stoichiometry (Fig. 3C) similar probes connect the Cl−-sensitive 6-methoxyquinoline (MQ) (Prakash et al., 2016; Saha et al., 2015). The DNA portion also acts to Cl−-insensitive groups, such as 5-aminofluorescein (AF), dansyl as a negatively charged ligand for scavenger receptors, which, after (DS), and 4-amino-1,8-napthalic anhydride (NA), using various binding, traffic the sensor along the endolysosomal pathway, after linkers to generate ratiometric reporters (Fig. 3A) (Li et al., 2012, which it can be directed to other organelles by using aptamers (Dan 2014; Ma et al., 2018). et al., 2019; Modi et al., 2009, 2013, 2014; Narayanaswamy et al., However, these ratiometric reporters suffer from significant self- 2019; Thekkan et al., 2018). This leads to a negligible batch-to- quenching and a low signal-to-noise ratio. One solution is to link batch variation, which enables a quantitative measurement in both moieties to a macromolecule scaffold, which was first achieved different genetic backgrounds with heterogeneity coming only from by conjugating the Cl−-sensitive 10,10′-bis(3-carboxypropyl)-9,9′- the biological system (Chakraborty et al., 2016; Jani et al., 2019; biacridinium dinitrate (BAC, Fig. 3B) and the Cl−-insensitive Krishnan and Simmel, 2011). A Clensor variant that also includes a tetramethylrhodamine (TMR) to dextran (Sonawane et al., 2002). pH-sensitive moiety, called ChloropHore, enables the simultaneous This had the added advantage of a stable localization in endosomes, measurement of lumenal pH and Cl− to resolve populations of and the sensor was used to show the role of Cl− ions in endosome lysosomes in cells derived from patients with lysosome storage acidification (Sonawane et al., 2002). However, this strategy suffers disorders (Fig. 3D) (Leung et al., 2019). Of course, the small- from batch-to-batch variability because the degree of conjugation molecule conjugates still suffer from photobleaching of the cannot be precisely controlled. To improve this, our sensor Clensor photolabile BAC dye, but sequestering the reporters in the small uses a double-stranded DNA backbone to achieve quantitative Cl− lysosomal compartment increases its effective concentration such mapping across the entire physiological regime of Cl− by displaying that the BAC signal is bright enough to take single readings. To − BAC on one strand and a normalizing Alexa Fluor 647 (AF647) perform dynamic imaging with any small-molecule Cl reporter, a Journal of Cell Science

6 REVIEW Journal of Cell Science (2020) 133, jcs240390. doi:10.1242/jcs.240390 correction factor for photobleaching would need to be derived and also been replaced by LSSmKate2, which has a large enough Stokes applied, complicating analysis. shift that it is excited in the same regime as EGFP but emits further In addition to making small-molecule Cl− sensors ratiometric, red (Paredes et al., 2016; Sulis Sato et al., 2017). This allows there has been much effort to modify the halide-sensitive YFP to quantification of pH and Cl− using only two excitation wavelengths, make it quantitative. The first such sensor was Clomeleon, which is thereby improving temporal resolution. comprised of cyan fluorescent protein (CFP) and a YFP variant, Meanwhile, new classes of Cl−-sensitive molecules are connected via a flexible peptide linker (Fig. 3E) (Kuner and emerging, suggesting potential sensing opportunities beyond the Augustine, 2000). Because the emission of CFP at 485 nm overlaps scope of current sensors. For example, the first turn-on Cl− sensors well with YFP excitation and the two proteins are in close proximity, have been realized that use metal complexes. When complexed with they can act as a fluorescence resonance energy transfer (FRET) cadmium(II) (Cd2+), the fluorescence of nitrobenzooxadiazole pair. High Cl− quenches YFP fluorescence, leading to low FRET (NBD) increases with increased Cl−. This fluorescence efficiency. However, obtaining quantitative information from enhancement is due to increased photoinduced charge transfer of Clomeleon is extremely difficult, as its Cl− affinity is highly the NBD-N aromatic amine into NBD, because the halide–metal variable and far from physiological relevance (Arosio and Ratto, interaction weakens the interaction metal ion and the NBD-N − 2014). For example, its Kd for Cl has been reported to be anywhere aromatic amine (Amatori et al., 2012). Based on the hypothesis that from 87 to 167 mM, while physiological cytosolic Cl− ranges from 3 a ligand that forces the metal ion to be closer to the aliphatic amines to 60 mM (Kuner and Augustine, 2000). The variation is likely due to would improve the switch-on sensor, a bis-NBD complex was − the steep dependence of Cl affinity on pH, as the Kd spans two orders realized (Fig. 4A) (Amatori et al., 2014). Another example is the of magnitude within a pH range of 6 to 8 owing to the pH sensitivity interlocked squaraine rotaxane, comprising a deep-red squaraine of FRETefficiency and Cl− binding (Bregestovski and Arosio, 2012). dye coordinated by a tetralactam macrocycle (Fig. 4B) Especially in high Cl−, where fluorescence emission is low, this pH (Collins et al., 2013; Gassensmith et al., 2010). Cl− reversibly dependence leads to significant errors in the readout of Cl− translocates the macrocycle away from the squaraine, increasing its concentration. This has precluded its application in acidic fluorescence. Additionally, turn-on Cl− sensors have been environments or in contexts where Cl− fluxes may be coupled to developed using dyes that exhibit aggregation-induced emission. environmental pH changes. For example, the fluorescence of the dye 1+ increases upon aggregation in the presence of Cl− and is selective over other Third generation – optimizing the old, discovering the new biological anions in water and in acidic conditions (Fig. 4C) (Watt The most recent Cl− reporters are Clomeleon variants with et al., 2015). While none of these probes have been applied in living optimized sensing characteristics, as well as new sensing scaffolds cells, they could be applied in the extracellular space to visualize basedonCl−-sensitive molecules. The first improvement on Cl− secretion in CF. Clomeleon significantly enhanced its affinity for Cl− by replacing Another recently developed class of fluorescent Cl− sensors uses the YFP variant with a mutant of higher affinity (Markova et al., citrate-based biodegradable photoluminescent polymers (BPLPs) 2008). This sensor, called Cl-sensor, has a Kd of 30 mM, and thus that can be processed into polymeric micelles for imaging (Kim is perfectly positioned for the physiological regime (Markova et al., 2017). The Cl−-recognition moiety is a conjugated ring et al., 2008). A cell-free screen of random Clomeleon variants to formed by citrate and L-cysteine that emits blue fluorescence, which study the influence of the residues in the halogen-binding site led is quenched with increased [Cl−] (Fig. 4D). However, the − to a double mutant possessing a Cl Kd of 21.2 mM (Grimley et al., quenching mechanism requires protons and thus it is functional 2013). The improved sensor SuperClomeleon uses this mutation; it only below pH 2.3. Modifications that eliminate the charged also replaces CFP with the brighter donor Cerulean and substituents could abolish this pH dependence. Even so, their incorporates a shorter linker to increase the signal-to-noise ratio simple synthesis and sensitivity upon immobilization make BPLPs (Grimley et al., 2013). suited to diagnose CF by detecting abnormal levels of Cl− in However, both Cl-sensor and SuperClomeleon suffer from pH biological fluids, such as sweat, which is currently the gold standard sensitivity due to the T203Y mutation, which is essential for tight for diagnosis of CF. Additionally, Cl−-selective electrodes and Cl− binding to YFP. In fact, at low Cl−, variations in YFP optodes have been engineered for fiber optical measurement of Cl− fluorescence are as high as 15% between pH 6.8 and 7.2, with (Barker et al., 1998; Brasuel et al., 2003; Pospíšilová et al., 2015). changes in the Kd being the main source of error (Rhoden et al., Finally, a naturally occurring YFP that displays turn-on 2007). This leads to significant errors when applying these sensors sensitivity to Cl− seems promising. Unlike genetically encoded in neurons, which exhibit sizable pH shifts (Raimondo et al., 2012). sensors based on GFP from Aequorea victoria (avGFP), YFP from One approach to circumvent this limitation has been to the jellyfish Phialidium sp. (phiYFP) is self-ratiometric (Tutol et al., − simultaneously measure pH and correct for changes in Kd. 2019a). Cl binding alters the pKa of the chromophore Y66 such ClopHensor achieved this by fusing a DsRed monomer to EGFP- that the phenolic form is favored. However, the Cl− dissociation T203Y through a flexible linker (Arosio et al., 2010). EGFP-T203Y constant is out of the physiological range even at acidic pH, making is a self-ratiometric pH sensor with a pKa of 6.8, such that the pH can it impractical for deployment at physiological pH. Similarly, the be derived from the ratio of excitation between that at 488 nm and at tetrameric YFP from Brachiostoma lanceolatum (lanYFP) has a 458 nm. Furthermore, the T203Y mutation introduces the Cl−- Cl−-binding pocket (Tutol et al., 2019b). Monomerization of binding site in EGFP, and the Cl−-induced fluorescence change can lanYFP maintains the Cl−-binding pocket to form mNeonGreen, a be normalized to the DsRed intensity. Thus, Cl− concentrations can turn-on sensor for Cl−. However, sensitivity to Cl− depends on be derived from the ratio of the emission intensity of EGFP to that of K143 being decarboxylated, such that the sensor cannot function DsRed (excitation ratio 458 nm:560 nm), and corrected using the Kd above pH 5.5. Despite their current limitations, these scaffolds can value at the observed pH. However, DsRed aggregation has be optimized to minimize their pH sensitivity and tune Cl− hindered its applicability, and has been replaced with tandem- affinities to be compatible with physiological levels. All of the − Tomato as in ClopHensorN (Raimondo et al., 2013). DsRed has aforementioned Cl reporters are described in Table S2. Journal of Cell Science

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AB N O O N O2N NO2 N N tBu N N N N N N OH N N O O NH HN R R

C N O Cl− NH HN

N O O

R R NH HN tBu

O NH2 H2N O OH O R D N N O O R HO O OR OH O HO O N O n 8 8 O O S O OR O

Fig. 4. Structures of new classes of Cl− reporters. Bis-NBD (A), the squaraine rotaxane shuttle (B), and the aggregation-induced 1+ (C) function as switch-on reporters, whereas citrate-based biodegradable photoluminescent polymers (BPLPs) (D) are quenching-based sensors. Fluorescent portions of the sensors are highlighted in the color of their fluorescent emission.

What biologists want from their Cl− reporters (Chakraborty et al., 2017; Weinert et al., 2010). In addition, pH- Because of the reporter-specific intricacies that make them correctable genetically encoded reporters such as ClopHensor can be unsuitable in certain biological contexts, we engaged scientists applied, but their complex analysis makes using the small-molecule interested in different aspects of Cl− biology in a discussion on conjugates more feasible. particular pain-points related to current Cl− sensors, and identified Another set of considerations is how well the fluorescence what would be the desirable characteristics for upcoming Cl− characteristics suit the context. For example, reporters that are reporters. bright and not prone to photobleaching are needed for dynamic One major issue discussed by all of the researchers involved in this imaging. In these scenarios, genetically encoded sensors are better. dialogue is the level of quantification they need from their reporter. In However, even among small-molecule reporters, those conjugated general, genetically encoded reporters are ideal when simple data through macromolecules are more capable of dynamic imaging. acquisition and detectable readouts are more important than Often, conjugates such as MEQ-TMR-dextran show acceptable quantitative information. For example, Cl-sensor has been useful to photostability and quantum yields to obtain quantitative dynamic compare intracellular Cl− between neurons from wild-type and measurements. For example, it has been used to show defective Cl− mutant mice with altered KCC2 function, and to reveal the predicted accumulation in lysosomes lacking ClC-7 and in ClC-7unc and ClC-7td overall increase in intracellular Cl− (Ludwig et al., 2017). On the lysosomes (Weinert et al., 2010, 2014). In addition, the spectral other hand, the halide-sensitive YFP variants were critical for the regime occupied by the reporter is important. Most Cl− reporters use identification of therapeutics that target CFTR, TMEM16A and the GFP channel, therefore precluding the use of the GFP channel for SLC26A3 (De La Fuente et al., 2008; Galietta et al., 2001a,c; Haggie simultaneous sensing or protein labeling. Since ratiometric Cl− et al., 2018; Ma et al., 2002; Namkung et al., 2011). Additionally, sensors use one or two additional excitation wavelengths, this these variants were perfectly suited to molecularly identify limitation is exacerbated. Finally, the sensing regime is a legitimate TMEM16A, TMEM206 and LRRC8A as Cl− channels (Caputo concern. Most genetically encoded reporters have a low affinity et al., 2008; Qiu et al., 2014; Yang et al., 2019a). However, because of for Cl−, making them unusable in physiological contexts, but suitable the complicated pH sensitivity of these reporters, it is practically for screens using a higher affinity anion, such as iodide, as a proxy. For impossible to use them to detect small changes in Cl− or to obtain physiological applications, it is instead better to use a small-molecule quantitative information. This is especially an issue because Cl− sensor, such as Clensor, which can accurately report on Cl− across the channels, most notably CFTR and GABAAR, are permeable to entire physiological regime. − HCO3 , which affects pH fluctuations. Despite these complications, One final factor is the capacity of the reporter to be delivered and halide-sensitive YFP variants have also provided a valuable retained inside the desired compartment. In additional to measuring qualitative understanding of KCC2 activity and dysregulation Cl− fluxes across the cell membrane, researchers would also like to (Boffi et al., 2018; Ludwig et al., 2017; Wimmer et al., 2015). obtain this information for the membranes of intracellular organelles However, to obtain quantitative information, ratiometric reporters that and proteoliposomes. The growing number of Cl− channels and are insensitive to pH are needed. For example, Clensor and MEQ- transporters found in various organelles requires new Cl− reporters TMR-dextran have been used to show that high lumenal Cl−,caused specifically targeting different intracellular membranes. On the by the activity of ClC-7, is necessary for lysosomal degradation other hand, proteoliposomes offer an in vitro environment to study a Journal of Cell Science

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Plasma membrane Cytosol unique set of characteristics tailored to the particular application • Clensor • SPQ (Fig. 5). For example, probing an intracellular channel presents distinct • ChloropHore* • Lucigenin sensing considerations from a plasma membrane channel, and high- • ClopHsensor* • MQ conjugates throughput screens necessitate different reporters than detailed • YFP H148Q • Clomeleon quantitative characterizations. Rather than compiling a list of • Cl-Sensor characteristics constituting an ideal sensor, we have highlighted • ClopHensor features that make each sensor suited to a given scenario. As discussed here, the capacity to glean molecular or quantitative information on chloride transport in diverse biological contexts clearly depends on the appropriate selection of chloride reporters. By taking advantage of the unique advantages of available reporters and improving reporter designs, we can better address chloride dysregulation in diverse disease conditions. Brighter small-molecule sensors that are not prone to photobleaching will transform our ability to visualize Cl− flux and thereby target it in diseases such as epilepsy and stroke. Genetically Neutral compartments Acidic compartments (endocytic encoded sensors with simpler corrections for pH dependence will (ER, nucleus, mitochondria) and secretory organelles) allow us to screen for channels in acidic compartments and obtain • YFP-H148Q* • BAC-TMR dextran • Clomeleon* • Clensor quantitative information in user-friendly stable cell lines. Finally, • Cl-Sensor* • ChloropHore reporters localized to currently unexplored organelles will reveal • ClopHensor* • ClopHensor* molecular information on channels in the mitochondria, ER, and Golgi that have disease relevance but unexplained functions.

Fig. 5. Suitable chloride reporters based on the requirements of Acknowledgements particular subcellular locations. Indicated here are suitable reporters for the We thank Verenice Noyola for assistance in drawing schemes for figures. M.Z. plasma membrane, cytosol, acidic compartments and neutral compartments. thanks the NIH Chemistry-Biology Interface (CBI) Predoctoral Training Program for support. K.C. thanks the Schmidt Science Fellows Program, in partnership with the An asterisk indicates that a targeting signal or tag would need to be appended Rhodes Trust, for support. to the indicated sensor for use in that compartment. Competing interests Cl− transporter in isolation, and have been used to show the Cl− The authors declare no competing or financial interests. channel activity of many proteins, such as TMEM16A and the CLC Funding family (Park and MacKinnon, 2018; Ran et al., 1992; Terashima Our work in this area is supported by The Wellcome Trust DBT India Alliance. et al., 2013). In the case of whole-cell Cl− measurements, the delivery and abundance of the reporters is a major concern. Supplementary information Although quinoline-based small-molecule reporters show sufficient Supplementary information available online at cell permeability, acridinium-based dyes exhibit less permeability http://jcs.biologists.org/lookup/doi/10.1242/jcs.240390.supplemental (Fig. 3A,B). For organelles, the reporter must have the capacity to be References targeted, using a signal peptide or chemical tag, and function in the Alper, S. L. and Sharma, A. K. (2013). The SLC26 gene family of anion transporters unique lumenal environment. Small-molecule conjugates, such as and channels. Mol. Aspects Med. 34, 494-515. doi:10.1016/j.mam.2012.07.009 Clensor and BAC-TMR-dextran, are good choices in these Amatori, S., Ambrosi, G., Fanelli, M., Formica, M., Fusi, V., Giorgi, L., Macedi, E., scenarios, as they show robust localization along the endocytic Micheloni, M., Paoli, P., Pontellini, R. et al. (2012). Multi-use NBD-based tetra- amino macrocycle: fluorescent probe for metals and anions and live cell marker. pathway and are functional in acidic conditions. Finally, in the case Chem. Eur. J. 18, 4274-4284. doi:10.1002/chem.201103135 of in vitro measurements within proteoliposomes, the most Amatori, S., Ambrosi, G., Borgogelli, E., Fanelli, M., Formica, M., Fusi, V., important issue is retention within the liposome. Because the Giorgi, L., Macedi, E., Micheloni, M., Paoli, P. et al. (2014). Modulating the efflux of small molecules from liposomes confounds dynamic sensor response to halide using NBD-based azamacrocycles. Inorg. Chem. 53, 4560-4569. doi:10.1021/ic5001649 measurements, especially in cases where the channel of interest has Argenzio, E. and Moolenaar, W. H. (2016). Emerging biological roles of Cl- scramblase activity, such as TMEM16F, alternative non- intracellular channel proteins. J. Cell Sci. 129, 4165-4174. doi:10.1242/jcs. fluorescence-based methods are often applied. 189795 No single reporter will satisfy these needs in every biological Arosio, D. and Ratto, G. M. (2014). Twenty years of fluorescence imaging of intracellular chloride. Front. Cell Neurosci. 8, 258. doi:10.3389/fncel.2014.00258 context, which is why the field is always open to fine-tuning current Arosio, D., Ricci, F., Marchetti, L., Gualdani, R., Albertazzi, L. and Beltram, F. reporters and developing entirely new scaffolds. Hopefully, future (2010). Simultaneous intracellular chloride and pH measurements using a GFP- reporters fill the current gaps in our current sensing capabilities; we based sensor. Nat. Methods 7, 516-518. doi:10.1038/nmeth.1471 need brighter small molecules that are not prone to photobleaching, Ashton, T. D., Jolliffe, K. A. and Pfeffer, F. M. (2015). Luminescent probes for the bioimaging of small anionic species in vitro and in vivo. Chem. Soc. Rev. 44, genetically encoded reporters that have a simpler way to correct for 4547-4595. doi:10.1039/C4CS00372A pH dependence, and sensors that are functional within organelles Auzanneau, C., Thoreau, V., Kitzis, A. and Becq, F. (2003). A Novel voltage- distinct from the endocytic pathway. dependent chloride current activated by extracellular acidic pH in cultured rat Sertoli cells. J. Biol. Chem. 278, 19230-19236. doi:10.1074/jbc.M301096200 Ayoub, C., Wasylyk, C., Li, Y., Thomas, E., Marisa, L., Robé, A., Roux, M., Conclusions and future perspectives Abecassis, J., de Reynies,̀ A. and Wasylyk, B. (2010). ANO1 amplification and As discussed above, fluorescent chloride reporters are not a ‘one size expression in HNSCC with a high propensity for future distant metastasis and its fits all’ solution. Genetically encoded reporters have distinct functions in HNSCC cell lines. Br. J. Cancer 103, 715-726. doi:10.1038/sj.bjc. advantages and disadvantages as compared with small-molecule 6605823 Barasch, J., Kiss, B., Prince, A., Saiman, L., Gruenert, D. and al-Awqati, Q. probes, and novel classes offer new sensing capabilities. Probing each (1991). Defective acidification of intracellular organelles in cystic fibrosis. Nature physiological role of chloride transporters requires a reporter with a 352, 70-73. doi:10.1038/352070a0 Journal of Cell Science

9 REVIEW Journal of Cell Science (2020) 133, jcs240390. doi:10.1242/jcs.240390

Barker, S. L. R., Thorsrud, B. A. and Kopelman, R. (1998). Nitrite- and chloride- Devuyst, O., Christie, P. T., Courtoy, P. J., Beauwens, R. and Thakker, R. V. selective fluorescent nano-optodes and in vitro application to rat conceptuses. (1999). Intra-renal and subcellular distribution of the human chloride channel, Anal. Chem. 70, 100-104. doi:10.1021/ac970912s CLC-5, reveals a pathophysiological basis for Dent’s disease. Hum. Mol. Genet. Barro-Soria, R., Aldehni, F., Almaça, J., Witzgall, R., Schreiber, R. and 8, 247-257. doi:10.1093/hmg/8.2.247 Kunzelmann, K. (2010). ER-localized bestrophin 1 activates Ca2+-dependent Di, A., Brown, M. E., Deriy, L. V., Li, C., Szeto, F. L., Chen, Y., Huang, P., Tong, J., ion channels TMEM16A and SK4 possibly by acting as a counterion channel. Naren, A. P., Bindokas, V. et al. (2006). CFTR regulates phagosome acidification Pflugers Arch. 459, 485-497. doi:10.1007/s00424-009-0745-0 in macrophages and alters bactericidal activity. Nat. Cell Biol. 8, 933-944. doi:10. Ben-Ari, Y., Khalilov, I., Kahle, K. T. and Cherubini, E. (2012). The GABA 1038/ncb1456 excitatory/inhibitory shift in brain maturation and neurological disorders. Domingo-Fernández, R., Coll, R. C., Kearney, J., Breit, S. and O’Neill, L. A. J. Neuroscientist 18, 467-486. doi:10.1177/1073858412438697 (2017). The intracellular chloride channel proteins CLIC1 and CLIC4 induce IL-1β Biwersi, J. and Verkman, A. S. (1991). Cell-permeable fluorescent indicator for transcription and activate the NLRP3 inflammasome. J. Biol. Chem. 292, cytosolic chloride. Biochemistry 30, 7879-7883. doi:10.1021/bi00246a001 12077-12087. doi:10.1074/jbc.M117.797126 Biwersi, J. and Verkman, A. S. (1994). Functional CFTR in endosomal Doyon, N., Vinay, L., Prescott, S. A. and De Koninck, Y. (2016). Chloride compartment of CFTR-expressing fibroblasts and T84 cells. Am. J. Physiol. regulation: a dynamic equilibrium crucial for synaptic inhibition. Neuron 89, 266, C149-C156. doi:10.1152/ajpcell.1994.266.1.C149 1157-1172. doi:10.1016/j.neuron.2016.02.030 Biwersi, J., Tulk, B. and Verkman, A. S. (1994). Long-wavelength chloride- Du, K., Sharma, M. and Lukacs, G. L. (2005). The ΔF508 cystic fibrosis mutation sensitive fluorescent indicators. Anal. Biochem. 219, 139-143. doi:10.1006/abio. impairs domain-domain interactions and arrests post-translational folding of 1994.1242 CFTR. Nat. Struct. Mol. Biol. 12, 17-25. doi:10.1038/nsmb882 Blaesse, P., Airaksinen, M. S., Rivera, C. and Kaila, K. (2009). Cation-chloride Dunn, K. W., Park, J., Semrad, C. E., Gelman, D. L., Shevell, T. and McGraw, T. E. cotransporters and neuronal function. Neuron 61, 820-838. doi:10.1016/j.neuron. (1994). Regulation of endocytic trafficking and acidification are independent of the 2009.03.003 cystic fibrosis transmembrane regulator. J. Biol. Chem. 269, 5336-5345. Boffi, J. C., Knabbe, J., Kaiser, M. and Kuner, T. (2018). KCC2-dependent steady- Duran, C., Thompson, C. H., Xiao, Q. and Hartzell, H. C. (2010). Chloride state intracellular chloride concentration and pH in cortical layer 2/3 neurons of channels: often enigmatic, rarely predictable. Annu. Rev. Physiol. 72, 95-121. anesthetized and awake mice. Front. Cell Neurosci. 12, 7. doi:10.3389/fncel. doi:10.1146/annurev-physiol-021909-135811 2018.00007 Fernández-Salas, E., Suh, K. S., Speransky, V. V., Bowers, W. L., Levy, J. M., Brasuel, M. G., Miller, T. J., Kopelman, R. and Philbert, M. A. (2003). Liquid Adams, T., Pathak, K. R., Edwards, L. E., Hayes, D. D., Cheng, C. et al. (2002). polymer nano-PEBBLEs for Cl- analysis and biological applications. Analyst 128, mtCLIC/CLIC4, an organellular chloride channel protein, is increased by DNA 1262-1267. doi:10.1039/b305254k damage and participates in the apoptotic response to p53. Mol. Cell. Biol. 22, Bregestovski, P. and Arosio, D. (2012). Green fluorescent protein-based chloride 3610-3620. doi:10.1128/MCB.22.11.3610-3620.2002 ion sensors for in vivo imaging. In Fluorescent Proteins II (ed. G. Jung), pp. Frizzell, R. A. and Hanrahan, J. W. (2012). Physiology of epithelial chloride and 99-124. Berlin, Heidelberg: Springer Berlin Heidelberg. fluid secretion. Cold Spring Harb. Perspect. Med. 2, a009563. doi:10.1101/ Capurro, V., Gianotti, A., Caci, E., Ravazzolo, R., Galietta, L. J. V. and Zegarra- cshperspect.a009563 Moran, O. (2015). Functional analysis of acid-activated Cl− channels: properties Galietta, L. J. V., Springsteel, M. F., Eda, M., Niedzinski, E. J., By, K., Haddadin, and mechanisms of regulation. Biochim. Biophys. Acta 1848, 105-114. doi:10. M. J., Kurth, M. J., Nantz, M. H. and Verkman, A. S. (2001a). Novel CFTR 1016/j.bbamem.2014.10.008 chloride channel activators identified by screening of combinatorial libraries based Caputo, A., Caci, E., Ferrera, L., Pedemonte, N., Barsanti, C., Sondo, E., Pfeffer, on flavone and benzoquinolizinium lead compounds. J. Biol. Chem. 276, U., Ravazzolo, R., Zegarra-Moran, O. and Galietta, L. J. V. (2008). TMEM16A, a 19723-19728. doi:10.1074/jbc.M101892200 membrane protein associated with calcium-dependent chloride channel activity. Galietta, L. J. V., Haggie, P. M. and Verkman, A. S. (2001b). Green fluorescent Science 322, 590-594. doi:10.1126/science.1163518 protein-based halide indicators with improved chloride and iodide affinities. FEBS Chakraborty, K., Veetil, A. T., Jaffrey, S. R. and Krishnan, Y. (2016). Nucleic acid- Lett. 499, 220-224. doi:10.1016/S0014-5793(01)02561-3 based nanodevices in biological imaging. Annu. Rev. Biochem. 85, 349-373. Galietta, L. V. J., Jayaraman, S. and Verkman, A. S. (2001c). Cell-based assay for doi:10.1146/annurev-biochem-060815-014244 high-throughput quantitative screening of CFTR chloride transport agonists. Chakraborty, K., Leung, K. H. and Krishnan, Y. (2017). High lumenal chloride in Am. J. Physiol. Cell Physiol. 281, C1734-C1742. doi:10.1152/ajpcell.2001.281.5. the lysosome is critical for lysosome function. Elife 6, e28862. doi:10.7554/eLife. C1734 28862 Gassensmith, J. J., Matthys, S., Lee, J.-J., Wojcik, A., Kamat, P. V. and Smith, Collins, C. G., Peck, E. M., Kramer, P. J. and Smith, B. D. (2013). Squaraine B. D. (2010). Squaraine rotaxane as a reversible optical chloride sensor. Chem. rotaxane shuttle as a ratiometric deep-red optical chloride sensor. Chem. Sci. 4, Eur. J. 16, 2916-2921. doi:10.1002/chem.200902547 2557. doi:10.1039/c3sc50535a Geddes, C. D., Apperson, K., Karolin, J. and Birch, D. J. S. (2001). Chloride- Coull, J. A. M., Boudreau, D., Bachand, K., Prescott, S. A., Nault, F., Sık,́ A., De sensitive fluorescent indicators. Anal. Biochem. 293, 60-66. doi:10.1006/abio. Koninck, P. and De Koninck, Y. (2003). Trans-synaptic shift in anion gradient in 2001.5103 spinal lamina I neurons as a mechanism of neuropathic pain. Nature 424, Graves, A. R., Curran, P. K., Smith, C. L. and Mindell, J. A. (2008). The Cl-/H+ 938-942. doi:10.1038/nature01868 antiporter ClC-7 is the primary chloride permeation pathway in lysosomes. Nature Cuthbert, A. W. (2011). New horizons in the treatment of cystic fibrosis. 453, 788-792. doi:10.1038/nature06907 Br. J. Pharmacol. 163, 173-183. doi:10.1111/j.1476-5381.2010.01137.x Grimley, J. S., Li, L., Wang, W., Wen, L., Beese, L. S., Hellinga, H. W. and Dan, K., Veetil, A. T., Chakraborty, K. and Krishnan, Y. (2019). DNA nanodevices Augustine, G. J. (2013). Visualization of synaptic inhibition with an optogenetic map enzymatic activity in organelles. Nat. Nanotechnol. 14, 252-259. doi:10. sensor developed by cell-free protein engineering automation. J. Neurosci. 33, 1038/s41565-019-0365-6 16297-16309. doi:10.1523/JNEUROSCI.4616-11.2013 Dargaei, Z., Bang, J. Y., Mahadevan, V., Khademullah, C. S., Bedard, S., Parfitt, Grubb, B. R. and Boucher, R. C. (1999). Pathophysiology of gene-targeted mouse G. M., Kim, J. C. and Woodin, M. A. (2018). Restoring GABAergic inhibition models for cystic fibrosis. Physiol. Rev. 79, S193-S214. doi:10.1152/physrev. rescues memory deficits in a Huntington’s disease mouse model. Proc. Natl. 1999.79.1.S193 Acad. Sci. USA 115, E1618-E1626. doi:10.1073/pnas.1716871115 Guilbault, C., Saeed, Z., Downey, G. P. and Radzioch, D. (2007). Cystic fibrosis Deidda, G., Parrini, M., Naskar, S., Bozarth, I. F., Contestabile, A. and mouse models. Am. J. Respir. Cell Mol. Biol. 36, 1-7. doi:10.1165/rcmb.2006- Cancedda, L. (2015). Reversing excitatory GABAAR signaling restores 0184TR synaptic plasticity and memory in a mouse model of Down syndrome. Nat. Med. Günther, W., Lüchow, A., Cluzeaud, F., Vandewalle, A. and Jentsch, T. J. (1998). 21, 318-326. doi:10.1038/nm.3827 ClC-5, the chloride channel mutated in Dent’s disease, colocalizes with the proton De La Fuente, R., Namkung, W., Mills, A. and Verkman, A. S. (2008). Small- pump in endocytotically active kidney cells. Proc. Natl. Acad. Sci. USA 95, molecule screen identifies inhibitors of a human intestinal calcium-activated 8075-8080. doi:10.1073/pnas.95.14.8075 chloride channel. Mol. Pharmacol. 73, 758-768. doi:10.1124/mol.107.043208 Gururaja Rao, S., Ponnalagu, D., Patel, N. J. and Singh, H. (2018). Three Dell’Antone, P. (1979). Evidence for an ATP-driven “proton pump” in rat liver decades of chloride intracellular channel proteins: from organelle to organ lysosomes by basic dyes uptake. Biochem. Biophys. Res. Commun. 86, 180-189. physiology. Curr. Protoc. Pharmacol. 80, 11.21.1-11.21.17. doi:10.1002/cpph.36 doi:10.1016/0006-291X(79)90398-X Haggie, P. M. and Verkman, A. S. (2007). Cystic fibrosis transmembrane Deriy, L. V., Gomez, E. A., Jacobson, D. A., Wang, X., Hopson, J. A., Liu, X. Y., conductance regulator-independent phagosomal acidification in macrophages. Zhang, G., Bindokas, V. P., Philipson, L. H. and Nelson, D. J. (2009a). The J. Biol. Chem. 282, 31422-31428. doi:10.1074/jbc.M705296200 granular chloride channel ClC-3 is permissive for insulin secretion. Cell Metab. 10, Haggie, P. M. and Verkman, A. S. (2009a). Unimpaired lysosomal acidification in 316-323. doi:10.1016/j.cmet.2009.08.012 respiratory epithelial cells in cystic fibrosis. J. Biol. Chem. 284, 7681-7686. doi:10. Deriy, L. V., Gomez, E. A., Zhang, G., Beacham, D. W., Hopson, J. A., Gallan, 1074/jbc.M809161200 A. J., Shevchenko, P. D., Bindokas, V. P. and Nelson, D. J. (2009b). Disease- Haggie, P. M. and Verkman, A. S. (2009b). Defective organellar acidification as a causing mutations in the cystic fibrosis transmembrane conductance regulator cause of cystic fibrosis lung disease: reexamination of a recurring hypothesis. determine the functional responses of alveolar macrophages. J. Biol. Chem. 284, Am. J. Physiol. Lung Cell Mol. Physiol. 296, L859-L867. doi:10.1152/ajplung.

35926-35938. doi:10.1074/jbc.M109.057372 00018.2009 Journal of Cell Science

10 REVIEW Journal of Cell Science (2020) 133, jcs240390. doi:10.1242/jcs.240390

Haggie, P. M., Cil, O., Lee, S., Tan, J.-A., Rivera, A. A., Phuan, P.-W. and Krishnan, Y. and Simmel, F. C. (2011). Nucleic acid based molecular devices. Verkman, A. S. (2018). SLC26A3 inhibitor identified in small molecule screen Angew. Chem. Int. Ed. Engl. 50, 3124-3156. doi:10.1002/anie.200907223 blocks colonic fluid absorption and reduces constipation. JCI Insight 3, 121370. Kruger, W., Gilbert, D., Hawthorne, R., Hryciw, D. H., Frings, S., Poronnik, P. doi:10.1172/jci.insight.121370 and Lynch, J. W. (2005). A yellow fluorescent protein-based assay for high- Hara-Chikuma, M., Yang, B., Sonawane, N. D., Sasaki, S., Uchida, S. and throughput screening of glycine and GABAA receptor chloride channels. Verkman, A. S. (2005a). ClC-3 chloride channels facilitate endosomal Neurosci. Lett. 380, 340-345. doi:10.1016/j.neulet.2005.01.065 acidification and chloride accumulation. J. Biol. Chem. 280, 1241-1247. doi:10. Kuner, T. and Augustine, G. J. (2000). A genetically encoded ratiometric indicator 1074/jbc.M407030200 for chloride: capturing chloride transients in cultured hippocampal neurons. Hara-Chikuma, M., Wang, Y., Guggino, S. E., Guggino, W. B. and Verkman, A. S. Neuron 27, 447-459. doi:10.1016/S0896-6273(00)00056-8 (2005b). Impaired acidification in early endosomes of ClC-5 deficient proximal Kunzelmann, K., Milenkovic, V. M., Spitzner, M., Soria, R. B. and Schreiber, R. tubule. Biochem. Biophys. Res. Commun. 329, 941-946. doi:10.1016/j.bbrc.2005. (2007). Calcium-dependent chloride conductance in epithelia: is there a 02.060 contribution by Bestrophin? Pflugers Arch. 454, 879-889. doi:10.1007/s00424- Harikumar, P. and Reeves, J. P. (1983). The lysosomal proton pump is 007-0245-z electrogenic. J. Biol. Chem. 258, 10403-10410. Lambert, S. and Oberwinkler, J. (2005). Characterization of a proton-activated, Heimlich, G. and Cidlowski, J. A. (2006). Selective role of intracellular chloride outwardly rectifying anion channel. J. Physiol. (Lond.) 567, 191-213. doi:10.1113/ in the regulation of the intrinsic but not extrinsic pathway of apoptosis in Jurkat T- jphysiol.2005.089888 cells. J. Biol. Chem. 281, 2232-2241. doi:10.1074/jbc.M507367200 Lange, P. F., Wartosch, L., Jentsch, T. J. and Fuhrmann, J. C. (2006). ClC-7 Hirota, S., Trimble, N., Pertens, E. and Janssen, L. J. (2006). Intracellular Cl- requires Ostm1 as a beta-subunit to support bone resorption and lysosomal fluxes play a novel role in Ca2+ handling in airway smooth muscle. Am. J. Physiol. function. Nature 440, 220-223. doi:10.1038/nature04535 Lung Cell Mol. Physiol. 290, L1146-L1153. doi:10.1152/ajplung.00393.2005 Leung, K. H., Chakraborty, K., Saminathan, A. and Krishnan, Y. (2019). A DNA Huberfeld, G., Wittner, L., Clemenceau, S., Baulac, M., Kaila, K., Miles, R. and nanomachine chemically resolves lysosomes in live cells. Nat. Nanotechnol. 14, Rivera, C. (2007). Perturbed chloride homeostasis and GABAergic signaling in 176-183. doi:10.1038/s41565-018-0318-5 human temporal lobe epilepsy. J. Neurosci. 27, 9866-9873. doi:10.1523/ Li, P., Xie, T., Fan, N., Li, K. and Tang, B. (2012). Ratiometric fluorescence imaging JNEUROSCI.2761-07.2007 for distinguishing chloride concentration between normal and ischemic ventricular Inglefield, J. R. and Schwartz-Bloom, R. D. (1997). Confocal imaging of myocytes. Chem. Commun. 48, 2077-2079. doi:10.1039/C1CC15258K intracellular chloride in living brain slices: measurement of GABAA receptor Li, P., Zhang, S., Fan, N., Xiao, H., Zhang, W., Zhang, W., Wang, H. and Tang, B. activity. J. Neurosci. Methods 75, 127-135. doi:10.1016/S0165-0270(97)00054-X (2014). Quantitative fluorescence ratio imaging of intralysosomal chloride ions Jamali, S., Klier, M., Ames, S., Barros, L. F., McKenna, R., Deitmer, J. W. and with single excitation/dual maximum emission. Chem. Eur. J. 20, 11760-11767. Becker, H. M. (2015). Hypoxia-induced carbonic anhydrase IX facilitates lactate doi:10.1002/chem.201402999 flux in human breast cancer cells by non-catalytic function. Sci. Rep. 5, 13605. Lloyd, S. E., Pearce, S. H., Fisher, S. E., Steinmeyer, K., Schwappach, B., doi:10.1038/srep13605 Scheinman, S. J., Harding, B., Bolino, A., Devoto, M., Goodyer, P. et al. Jani, M. S., Veetil, A. T. and Krishnan, Y. (2019). Precision immunomodulation with (1996). A common molecular basis for three inherited kidney stone diseases. Nature 379, 445-449. doi:10.1038/379445a0 synthetic nucleic acid technologies. Nat. Rev. Mater 4, 451-458. doi:10.1038/ Ludwig, A., Rivera, C. and Uvarov, P. (2017). A noninvasive optical approach for s41578-019-0105-4 assessing chloride extrusion activity of the K-Cl cotransporter KCC2 in neuronal Jayaraman, S., Biwersi, J. and Verkman, A. S. (1999). Synthesis and cells. BMC Neurosci. 18, 23. doi:10.1186/s12868-017-0336-5 characterization of dual-wavelength Cl–sensitive fluorescent indicators for ratio Lukacs, G. L., Chang, X. B., Kartner, N., Rotstein, O. D., Riordan, J. R. and imaging. Am. J. Physiol. 276, C747-C757. doi:10.1152/ajpcell.1999.276.3.C747 Grinstein, S. (1992). The cystic fibrosis transmembrane regulator is present and Jayaraman, S., Haggie, P., Wachter, R. M., Remington, S. J. and Verkman, A. S. functional in endosomes. Role as a determinant of endosomal pH. J. Biol. Chem. (2000). Mechanism and cellular applications of a green fluorescent protein-based 267, 14568-14572. halide sensor. J. Biol. Chem. 275, 6047-6050. doi:10.1074/jbc.275.9.6047 Ma, T., Vetrivel, L., Yang, H., Pedemonte, N., Zegarra-Moran, O., Galietta, L. J. V. Jia, Y., Jucius, T. J., Cook, S. A. and Ackerman, S. L. (2015). Loss of Clcc1 results and Verkman, A. S. (2002). High-affinity activators of cystic fibrosis in ER stress, misfolded protein accumulation, and neurodegeneration. transmembrane conductance regulator (CFTR) chloride conductance identified J. Neurosci. 35, 3001-3009. doi:10.1523/JNEUROSCI.3678-14.2015 by high-throughput screening. J. Biol. Chem. 277, 37235-37241. doi:10.1074/jbc. Judah, J. D. and Thomas, G. M. H. (2006). Two distinct chloride ion requirements in M205932200 the constitutive protein secretory pathway. Eur. J. Cell Biol. 85, 825-836. doi:10. Ma, C., Zhang, F., Wang, Y., Zhu, X., Liu, X., Zhao, C. and Zhang, H. (2018). 1016/j.ejcb.2006.03.005 Synthesis and application of ratio fluorescence probe for chloride. Anal. Bioanal. Kaila, K. (1994). Ionic basis of GABAA receptor channel function in the nervous Chem. 410, 6507-6516. doi:10.1007/s00216-018-1250-0 system. Prog. Neurobiol. 42, 489-537. doi:10.1016/0301-0082(94)90049-3 Maeda, Y., Ide, T., Koike, M., Uchiyama, Y. and Kinoshita, T. (2008). GPHR is a Kaila, K., Price, T. J., Payne, J. A., Puskarjov, M. and Voipio, J. (2014). Cation- novel anion channel critical for acidification and functions of the Golgi apparatus. chloride cotransporters in neuronal development, plasticity and disease. Nat. Rev. Nat. Cell Biol. 10, 1135-1145. doi:10.1038/ncb1773 Neurosci. 15, 637-654. doi:10.1038/nrn3819 Mahadevan, V. and Woodin, M. A. (2016). Regulation of neuronal chloride Kaneko, H., Putzier, I., Frings, S., Kaupp, U. B. and Gensch, T. (2004). Chloride homeostasis by neuromodulators. J. Physiol. (Lond.) 594, 2593-2605. doi:10. accumulation in mammalian olfactory sensory neurons. J. Neurosci. 24, 1113/JP271593 7931-7938. doi:10.1523/JNEUROSCI.2115-04.2004 Mahadevan, V., Khademullah, C. S., Dargaei, Z., Chevrier, J., Uvarov, P., Kwan, Kang, C., Xie, L., Gunasekar, S. K., Mishra, A., Zhang, Y., Pai, S., Gao, Y., J., Bagshaw, R. D., Pawson, T., Emili, A., De Koninck, Y. et al. (2017). Native Kumar, A., Norris, A. W., Stephens, S. B. et al. (2018). SWELL1 is a glucose KCC2 interactome reveals PACSIN1 as a critical regulator of synaptic inhibition. β sensor regulating -cell excitability and systemic glycaemia. Nat. Commun. 9, 367. Elife 6, e28270. doi:10.7554/eLife.28270.032 doi:10.1038/s41467-017-02664-0 Markova, O., Mukhtarov, M., Real, E., Jacob, Y. and Bregestovski, P. (2008). Kasper, D., Planells-Cases, R., Fuhrmann, J. C., Scheel, O., Zeitz, O., Ruether, Genetically encoded chloride indicator with improved sensitivity. J. Neurosci. ̈ K., Schmitt, A., Poet, M., Steinfeld, R., Schweizer, M. et al. (2005). Loss of the Methods 170, 67-76. doi:10.1016/j.jneumeth.2007.12.016 chloride channel ClC-7 leads to lysosomal storage disease and Medina, I., Friedel, P., Rivera, C., Kahle, K. T., Kourdougli, N., Uvarov, P. and neurodegeneration. EMBO J. 24, 1079-1091. doi:10.1038/sj.emboj.7600576 Pellegrino, C. (2014). Current view on the functional regulation of the neuronal Kim, J. P., Xie, Z., Creer, M., Liu, Z. and Yang, J. (2017). Citrate-based fluorescent K(+)-Cl(−) cotransporter KCC2. Front. Cell Neurosci. 8, 27. doi:10.3389/fncel. materials for low-cost chloride sensing in the diagnosis of Cystic Fibrosis. Chem. 2014.00027 Sci. 8, 550-558. doi:10.1039/C6SC02962K Mindell, J. A. (2012). Lysosomal acidification mechanisms. Annu. Rev. Physiol. 74, Kimelberg, H. K., Goderie, S. K., Higman, S., Pang, S. and Waniewski, R. A. 69-86. doi:10.1146/annurev-physiol-012110-142317 (1990). Swelling-induced release of glutamate, aspartate, and taurine from Modi, M. G. S., Goswami, D., Gupta, G. D., Mayor, S. and Krishnan, Y. (2009). A astrocyte cultures. J. Neurosci. 10, 1583-1591. doi:10.1523/JNEUROSCI.10-05- DNA nanomachine that maps spatial and temporal pH changes inside living cells. 01583.1990 Nat. Nanotechnol. 4, 325-330. doi:10.1038/nnano.2009.83 Klein, P. M., Lu, A. C., Harper, M. E., McKown, H. M., Morgan, J. D. and Modi, S., Nizak, C., Surana, S., Halder, S. and Krishnan, Y. (2013). Two DNA Beenhakker, M. P. (2018). Tenuous inhibitory gabaergic signaling in the reticular nanomachines map pH changes along intersecting endocytic pathways inside the thalamus. J. Neurosci. 38, 1232-1248. doi:10.1523/JNEUROSCI.1345-17.2017 same cell. Nat. Nanotechnol. 8, 459-467. doi:10.1038/nnano.2013.92 Kornak, U., Kasper, D., Bösl, M. R., Kaiser, E., Schweizer, M., Schulz, A., Modi, S., Halder, S., Nizak, C. and Krishnan, Y. (2014). Recombinant antibody Friedrich, W., Delling, G. and Jentsch, T. J. (2001). Loss of the ClC-7 chloride mediated delivery of organelle-specific DNA pH sensors along endocytic channel leads to osteopetrosis in mice and man. Cell 104, 205-215. doi:10.1016/ pathways. Nanoscale 6, 1144-1152. doi:10.1039/C3NR03769J S0092-8674(01)00206-9 Mohammad-Panah, R., Harrison, R., Dhani, S., Ackerley, C., Huan, L.-J., Wang, Kovalchuk, Y. and Garaschuk, O. (2012). Two-photon chloride imaging using Y. and Bear, C. E. (2003). The chloride channel ClC-4 contributes to endosomal MQAE in vitro and in vivo. Cold Spring Harb. Protoc. 2012, 778-785. doi:10.1101/ acidification and trafficking. J. Biol. Chem. 278, 29267-29277. doi:10.1074/jbc.

pdb.prot070037 M304357200 Journal of Cell Science

11 REVIEW Journal of Cell Science (2020) 133, jcs240390. doi:10.1242/jcs.240390

Moore, Y. E., Kelley, M. R., Brandon, N. J., Deeb, T. Z. and Moss, S. J. (2017). an essential component of volume-regulated anion channel. Cell 157, 447-458. Seizing control of KCC2: A new therapeutic target for epilepsy. Trends Neurosci. doi:10.1016/j.cell.2014.03.024 40, 555-571. doi:10.1016/j.tins.2017.06.008 Quinton, P. M. (1983). Chloride impermeability in cystic fibrosis. Nature 301, Namkung, W., Yao, Z., Finkbeiner, W. E. and Verkman, A. S. (2011). Small- 421-422. doi:10.1038/301421a0 molecule activators of TMEM16A, a calcium-activated chloride channel, stimulate Raimondo, J. V., Irkle, A., Wefelmeyer, W., Newey, S. E. and Akerman, C. J. epithelial chloride secretion and intestinal contraction. FASEB J. 25, 4048-4062. (2012). Genetically encoded proton sensors reveal activity-dependent pH doi:10.1096/fj.11-191627 changes in neurons. Front. Mol. Neurosci. 5, 68. doi:10.3389/fnmol.2012.00068 Narayanaswamy, N., Chakraborty, K., Saminathan, A., Zeichner, E., Leung, Raimondo, J. V., Joyce, B., Kay, L., Schlagheck, T., Newey, S. E., Srinivas, S. K. H., Devany, J. and Krishnan, Y. (2019). A pH-correctable, DNA-based and Akerman, C. J. (2013). A genetically-encoded chloride and pH sensor for fluorescent reporter for organellar calcium. Nat. Methods 16, 95-102. doi:10.1038/ dissociating ion dynamics in the nervous system. Front. Cell Neurosci. 7, 202. s41592-018-0232-7 doi:10.3389/fncel.2013.00202 Neussert, R., Müller, C., Milenkovic, V. M. and Strauß, O. (2010). The presence of Ran, S., Fuller, C. M., Arrate, M. P., Latorre, R. and Benos, D. J. (1992). Functional bestrophin-1 modulates the Ca2+ recruitment from Ca2+ stores in the ER. reconstitution of a chloride channel protein from bovine trachea. J. Biol. Chem. Pflugers Arch. 460, 163-175. doi:10.1007/s00424-010-0840-2 267, 20630-20637. Nobles, M., Higgins, C. F. and Sardini, A. (2004). Extracellular acidification elicits a Rhoden, K. J., Cianchetta, S., Stivani, V., Portulano, C., Galietta, L. J. V. and chloride current that shares characteristics with ICl(swell). Am. J. Physiol. Cell Romeo, G. (2007). Cell-based imaging of sodium iodide symporter activity with Physiol. 287, C1426-C1435. doi:10.1152/ajpcell.00549.2002 the yellow fluorescent protein variant YFP-H148Q/I152L. Am. J. Physiol. Cell Nordeen, M. H., Jones, S. M., Howell, K. E. and Caldwell, J. H. (2000). GOLAC: an Physiol. 292, C814-C823. doi:10.1152/ajpcell.00291.2006 endogenous anion channel of the Golgi complex. Biophys. J. 78, 2918-2928. Riordan, J. R., Rommens, J. M., Kerem, B., Alon, N., Rozmahel, R., Grzelczak, doi:10.1016/S0006-3495(00)76832-9 Z., Zielenski, J., Lok, S., Plavsic, N. and Chou, J. L. (1989). Identification of the Novarino, G., Weinert, S., Rickheit, G. and Jentsch, T. J. (2010). Endosomal cystic fibrosis gene: cloning and characterization of complementary DNA. Science chloride-proton exchange rather than chloride conductance is crucial for renal 245, 1066-1073. doi:10.1126/science.2475911 endocytosis. Science 328, 1398-1401. doi:10.1126/science.1188070 Rivera, C., Voipio, J., Payne, J. A., Ruusuvuori, E., Lahtinen, H., Lamsa, K., − Ohkuma, S., Moriyama, Y. and Takano, T. (1982). Identification and Pirvola, U., Saarma, M. and Kaila, K. (1999). The K+/Cl co-transporter KCC2 characterization of a proton pump on lysosomes by fluorescein-isothiocyanate- renders GABA hyperpolarizing during neuronal maturation. Nature 397, 251-255. dextran fluorescence. Proc. Natl. Acad. Sci. USA 79, 2758-2762. doi:10.1073/ doi:10.1038/16697 pnas.79.9.2758 Rivinoja, A., Kokkonen, N., Kellokumpu, I. and Kellokumpu, S. (2006). Elevated O’Rourke, B. (2007). Mitochondrial ion channels. Annu. Rev. Physiol. 69, 19-49. Golgi pH in breast and colorectal cancer cells correlates with the expression of doi:10.1146/annurev.physiol.69.031905.163804 oncofetal carbohydrate T-antigen. J. Cell Physiol. 208, 167-174. doi:10.1002/jcp. Osei-Owusu, J., Yang, J., Vitery, M. D. C. and Qiu, Z. (2018). Molecular biology 20653 and physiology of volume-regulated anion channel (VRAC). Curr. Top. Membr. 81, Rommens, J. M., Iannuzzi, M. C., Kerem, B., Drumm, M. L., Melmer, G., Dean, 177-203. doi:10.1016/bs.ctm.2018.07.005 M., Rozmahel, R., Cole, J. L., Kennedy, D. and Hidaka, N. (1989). Identification Paredes, J. M., Idilli, A. I., Mariotti, L., Losi, G., Arslanbaeva, L. R., Sato, S. S., of the cystic fibrosis gene: chromosome walking and jumping. Science 245, 1059-1065. doi:10.1126/science.2772657 Artoni, P., Szczurkowska, J., Cancedda, L., Ratto, G. M. et al. (2016). Saha, S., Prakash, V., Halder, S., Chakraborty, K. and Krishnan, Y. (2015). A pH- Synchronous bioimaging of intracellular ph and chloride based on LSS independent DNA nanodevice for quantifying chloride transport in organelles of fluorescent protein. ACS Chem. Biol. 11, 1652-1660. doi:10.1021/acschembio. living cells. Nat. Nanotechnol. 10, 645-651. doi:10.1038/nnano.2015.130 6b00103 Saint-Criq, V. and Gray, M. A. (2017). Role of CFTR in epithelial physiology. Cell Park, E. and MacKinnon, R. (2018). Structure of the CLC-1 chloride channel from Mol. Life Sci. 74, 93-115. doi:10.1007/s00018-016-2391-y Homo sapiens. Elife 7, e36629. doi:10.7554/eLife.36629.031 Sato-Numata, K., Numata, T., Okada, T. and Okada, Y. (2013). Acid-sensitive Paroutis, P., Touret, N. and Grinstein, S. (2004). The pH of the secretory pathway: outwardly rectifying (ASOR) anion channels in human epithelial cells are highly measurement, determinants, and regulation. Physiology (Bethesda) 19, 207-215. sensitive to temperature and independent of ClC-3. Pflugers Arch. 465, doi:10.1152/physiol.00005.2004 1535-1543. doi:10.1007/s00424-013-1296-y Payne, J. A., Rivera, C., Voipio, J. and Kaila, K. (2003). Cation-chloride co- Schroeder, B. C., Cheng, T., Jan, Y. N. and Jan, L. Y. (2008). Expression cloning of transporters in neuronal communication, development and trauma. Trends TMEM16A as a calcium-activated chloride channel subunit. Cell 134, 1019-1029. Neurosci. 26, 199-206. doi:10.1016/S0166-2236(03)00068-7 doi:10.1016/j.cell.2008.09.003 Pilas, B. and Durack, G. (1997). A flow cytometric method for measurement of Seksek, O., Biwersi, J. and Verkman, A. S. (1996). Evidence against defective intracellular chloride concentration in lymphocytes using the halide-specific probe trans-Golgi acidification in cystic fibrosis. J. Biol. Chem. 271, 15542-15548. 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ). Cytometry 28, 316-322. doi:10. doi:10.1074/jbc.271.26.15542 1002/(SICI)1097-0320(19970801)28:4<316::AID-CYTO7>3.0.CO;2-9 Shah, V. S., Meyerholz, D. K., Tang, X. X., Reznikov, L., Abou Alaiwa, M., Ernst, ̈ ̈ Piwon, N., Gunther, W., Schwake, M., Bosl, M. R. and Jentsch, T. J. (2000). ClC-5 S. E., Karp, P. H., Wohlford-Lenane, C. L., Heilmann, K. P., Leidinger, M. R. ’ Cl- -channel disruption impairs endocytosis in a mouse model for Dent s disease. et al. (2016). Airway acidification initiates host defense abnormalities in cystic Nature 408, 369-373. doi:10.1038/35042597 fibrosis mice. Science 351, 503-507. doi:10.1126/science.aad5589 Planells-Cases, R., Lutter, D., Guyader, C., Gerhards, N. M., Ullrich, F., Elger, Shukla, A., Malik, M., Cataisson, C., Ho, Y., Friesen, T., Suh, K. S. and Yuspa, D. A., Kucukosmanoglu, A., Xu, G., Voss, F. K., Reincke, S. M. et al. (2015). S. H. (2009). TGF-β signalling is regulated by Schnurri-2-dependent nuclear Subunit composition of VRAC channels determines substrate specificity and translocation of CLIC4 and consequent stabilization of phospho-Smad2 and 3. cellular resistance to Pt-based anti-cancer drugs. EMBO J. 34, 2993-3008. doi:10. Nat. Cell Biol. 11, 777-784. doi:10.1038/ncb1885 15252/embj.201592409 Sonawane, N. D., Thiagarajah, J. R. and Verkman, A. S. (2002). Chloride Platt, C. D., Chou, J., Houlihan, P., Badran, Y. R., Kumar, L., Bainter, W., Poliani, concentration in endosomes measured using a ratioable fluorescent Cl- indicator: P. L., Perez, C. J., Dent, S. Y. R., Clapham, D. E. et al. (2017). Leucine-rich evidence for chloride accumulation during acidification. J. Biol. Chem. 277, repeat containing 8A (LRRC8A)-dependent volume-regulated anion channel 5506-5513. doi:10.1074/jbc.M110818200 activity is dispensable for T-cell development and function. J. Allergy Clin. Staley, K. J., Soldo, B. L. and Proctor, W. R. (1995). Ionic mechanisms of neuronal Immunol. 140, 1651-1659.e1. doi:10.1016/j.jaci.2016.12.974 excitation by inhibitory GABAA receptors. Science 269, 977-981. doi:10.1126/ Pollock, N. S., Kargacin, M. E. and Kargacin, G. J. (1998). Chloride channel science.7638623 blockers inhibit Ca2+ uptake by the smooth muscle sarcoplasmic reticulum. Steinberg, B. E., Huynh, K. K., Brodovitch, A., Jabs, S., Stauber, T., Jentsch, Biophys. J. 75, 1759-1766. doi:10.1016/S0006-3495(98)77617-9 T. J. and Grinstein, S. (2010). A cation counterflux supports lysosomal Ponnalagu, D. and Singh, H. (2017). Anion channels of mitochondria. Handb. Exp. acidification. J. Cell Biol. 189, 1171-1186. doi:10.1083/jcb.200911083 Pharmacol. 240, 71-101. doi:10.1007/164_2016_39 Stewart, A. K., Yamamoto, A., Nakakuki, M., Kondo, T., Alper, S. L. and Ponnalagu, D., Gururaja Rao, S., Farber, J., Xin, W., Hussain, A. T., Shah, K., Ishiguro, H. (2009). Functional coupling of apical Cl-/HCO3- exchange with Tanda, S., Berryman, M., Edwards, J. C. and Singh, H. (2016). Molecular CFTR in stimulated HCO3- secretion by guinea pig interlobular pancreatic duct. identity of cardiac mitochondrial chloride intracellular channel proteins. Am. J. Physiol. Gastrointest. Liver Physiol. 296, G1307-G1317. doi:10.1152/ajpgi. Mitochondrion 27, 6-14. doi:10.1016/j.mito.2016.01.001 90697.2008 Pospıś̌ilová, M., Kuncová, G. and Trögl, J. (2015). Fiber-optic chemical sensors Suh, K. S., Mutoh, M., Nagashima, K., Fernandez-Salas, E., Edwards, L. E., and fiber-optic bio-sensors. Sensors (Basel) 15, 25208-25259. doi:10.3390/ Hayes, D. D., Crutchley, J. M., Marin, K. G., Dumont, R. A., Levy, J. M. et al. s151025208 (2004). The organellular chloride channel protein CLIC4/mtCLIC translocates to Prakash, V., Saha, S., Chakraborty, K. and Krishnan, Y. (2016). Rational design the nucleus in response to cellular stress and accelerates apoptosis. J. Biol. of a quantitative, pH-insensitive, nucleic acid based fluorescent chloride reporter. Chem. 279, 4632-4641. doi:10.1074/jbc.M311632200 Chem. Sci. 7, 1946-1953. doi:10.1039/C5SC04002G Sulis Sato, S., Artoni, P., Landi, S., Cozzolino, O., Parra, R., Pracucci, E., Qiu, Z., Dubin, A. E., Mathur, J., Tu, B., Reddy, K., Miraglia, L. J., Reinhardt, J., Trovato, F., Szczurkowska, J., Luin, S., Arosio, D. et al. (2017). Simultaneous

Orth, A. P. and Patapoutian, A. (2014). SWELL1, a plasma membrane protein, is two-photon imaging of intracellular chloride concentration and pH in mouse Journal of Cell Science

12 REVIEW Journal of Cell Science (2020) 133, jcs240390. doi:10.1242/jcs.240390

pyramidal neurons in vivo. Proc. Natl. Acad. Sci. USA 114, E8770-E8779. doi:10. Wachter, R. M., Yarbrough, D., Kallio, K. and Remington, S. J. (2000). 1073/pnas.1702861114 Crystallographic and energetic analysis of binding of selected anions to the Sun, H., Tsunenari, T., Yau, K.-W. and Nathans, J. (2002). The vitelliform macular yellow variants of green fluorescent protein. J. Mol. Biol. 301, 157-171. doi:10. dystrophy protein defines a new family of chloride channels. Proc. Natl. Acad. Sci. 1006/jmbi.2000.3905 USA 99, 4008-4013. doi:10.1073/pnas.052692999 Wang, H.-Y., Shimizu, T., Numata, T. and Okada, Y. (2007). Role of acid-sensitive Szabadics, J., Varga, C., Molnár, G., Oláh, S., Barzó, P. and Tamás, G. (2006). outwardly rectifying anion channels in acidosis-induced cell death in human Excitatory effect of GABAergic axo-axonic cells in cortical microcircuits. Science epithelial cells. Pflugers Arch. 454, 223-233. doi:10.1007/s00424-006-0193-z 311, 233-235. doi:10.1126/science.1121325 Watt, M. M., Engle, J. M., Fairley, K. C., Robitshek, T. E., Haley, M. M. and Tang, X., Drotar, J., Li, K., Clairmont, C. D., Brumm, A. S., Sullins, A. J., Wu, H., Johnson, D. W. (2015). “Off-on” aggregation-based fluorescent sensor for the Liu, X. S., Wang, J., Gray, N. S. et al. (2019). Pharmacological enhancement of detection of chloride in water. Org. Biomol. Chem. 13, 4266-4270. doi:10.1039/ KCC2 gene expression exerts therapeutic effects on human Rett syndrome C4OB02409E neurons and Mecp2 mutant mice. Sci. Transl. Med. 11, eaau0164. doi:10.1126/ Weinert, S., Jabs, S., Supanchart, C., Schweizer, M., Gimber, N., Richter, M., scitranslmed.aau0164 Rademann, J., Stauber, T., Kornak, U. and Jentsch, T. J. (2010). Lysosomal Terashima, H., Picollo, A. and Accardi, A. (2013). Purified TMEM16A is sufficient pathology and osteopetrosis upon loss of H+-driven lysosomal Cl- accumulation. to form Ca2+-activated Cl- channels. Proc. Natl. Acad. Sci. USA 110, Science 328, 1401-1403. doi:10.1126/science.1188072 19354-19359. doi:10.1073/pnas.1312014110 Weinert, S., Jabs, S., Hohensee, S., Chan, W. L., Kornak, U. and Jentsch, T. J. Thekkan, S., Jani, M. S., Cui, C., Dan, K., Zhou, G., Becker, L. and Krishnan, Y. (2014). Transport activity and presence of ClC-7/Ostm1 complex account for (2018). A DNA-based fluorescent reporter maps HOCl production in the maturing different cellular functions. EMBO Rep. 15, 784-791. doi:10.15252/embr. phagosome. Nat. Chem. Biol. 15, 1165-1172. doi:10.1038/s41589-018-0176-3 201438553 Thompson, R. J., Nordeen, M. H., Howell, K. E. and Caldwell, J. H. (2002). A Weisz, O. A. (2003). Organelle acidification and disease. Traffic 4, 57-64. doi:10. large-conductance anion channel of the Golgi complex. Biophys. J. 83, 278-289. 1034/j.1600-0854.2003.40201.x doi:10.1016/S0006-3495(02)75168-0 Wemmie, J. A., Taugher, R. J. and Kreple, C. J. (2013). Acid-sensing ion channels Tomaskova, Z. and Ondrias, K. (2010). Mitochondrial chloride channels–What are in pain and disease. Nat. Rev. Neurosci. 14, 461-471. doi:10.1038/nrn3529 they for? FEBS Lett. 584, 2085-2092. doi:10.1016/j.febslet.2010.01.035 West, R. B., Corless, C. L., Chen, X., Rubin, B. P., Subramanian, S., Tutol, J. N., Peng, W. and Dodani, S. C. (2019a). Discovery and characterization of Montgomery, K., Zhu, S., Ball, C. A., Nielsen, T. O., Patel, R. et al. (2004). a naturally occurring, turn-on yellow fluorescent protein sensor for chloride. The novel marker, DOG1, is expressed ubiquitously in gastrointestinal stromal Biochemistry 58, 31-35. doi:10.1021/acs.biochem.8b00928 tumors irrespective of KIT or PDGFRA mutation status. Am. J. Pathol. 165, Tutol, J., Kam, H. and Dodani, S. (2019b). Identification and spectroscopic 107-113. doi:10.1016/S0002-9440(10)63279-8 characterization of mNeonGreen as a pH-dependent, turn-on fluorescent protein Wimmer, R. D., Schmitt, L. I., Davidson, T. J., Nakajima, M., Deisseroth, K. and sensor for chloride. Chembiochem 20, 1727-1727. doi:10.1002/cbic.201900408 Halassa, M. M. (2015). Thalamic control of sensory selection in divided attention. Tyzio, R., Nardou, R., Ferrari, D. C., Tsintsadze, T., Shahrokhi, A., Eftekhari, S., Khalilov, I., Tsintsadze, V., Brouchoud, C., Chazal, G. et al. (2014). Oxytocin- Nature 526, 705-709. doi:10.1038/nature15398 mediated GABA inhibition during delivery attenuates autism pathogenesis in Yang, Y. D., Cho, H., Koo, J. Y., Tak, M. H., Cho, Y., Shim, W.-S., Park, S. P., Lee, rodent offspring. Science 343, 675-679. doi:10.1126/science.1247190 J., Lee, B., Kim, B.-M. et al. (2008). TMEM16A confers receptor-activated Ullman, J. C., Yang, J., Sullivan, M., Bendor, J., Levy, J., Pham, E., Silm, K., calcium-dependent chloride conductance. Nature 455, 1210-1215. doi:10.1038/ Seifikar, H., Sohal, V. S., Nicoll, R. A. et al. (2018). A mouse model of autism nature07313 implicates endosome pH in the regulation of presynaptic calcium entry. Nat. Yang, J., Chen, J., del Carmen Vitery, M., Osei-Owusu, J., Chu, J., Yu, H., Sun, Commun. 9, 330. doi:10.1038/s41467-017-02716-5 S. and Qiu, Z. (2019a). PAC, an evolutionarily conserved membrane protein, is a Ullrich, F., Blin, S., Lazarow, K., Daubitz, T., von Kries, J. P. and Jentsch, T. J. proton-activated chloride channel. Science 364, 395-399. doi:10.1126/science. (2019). Identification of TMEM206 proteins as a pore of PAORAC/ASOR acid- aav9739 sensitive chloride channels. eLife 8, e49187. doi:10.7554/eLife.49187 Yang, J., Vitery, M. D. C., Chen, J., Osei-Owusu, J., Chu, J. and Qiu, Z. (2019b). Verkman, A. S. (1990). Development and biological applications of chloride- Glutamate-releasing SWELL1 channel in astrocytes modulates synaptic sensitive fluorescent indicators. Am. J. Physiol. 259, C375-C388. doi:10.1152/ transmission and promotes brain damage in stroke. Neuron 102, 813-827.e6. ajpcell.1990.259.3.C375 doi:10.1016/j.neuron.2019.03.029 Voss, F. K., Ullrich, F., Münch, J., Lazarow, K., Lutter, D., Mah, N., Andrade- Yingjun, G. and Xun, Q. (2013). Acid-sensing ion channels under hypoxia. Navarro, M. A., von Kries, J. P., Stauber, T. and Jentsch, T. J. (2014). Channels 7, 231-237. doi:10.4161/chan.25223 Identification of LRRC8 heteromers as an essential component of the volume- Zhang, Y., Stanton, J. B., Wu, J., Yu, K., Hartzell, H. C., Peachey, N. S., regulated anion channel VRAC. Science 344, 634-638. doi:10.1126/science. Marmorstein, L. Y. and Marmorstein, A. D. (2010). Suppression of Ca2+ 1252826 signaling in a mouse model of Best disease. Hum. Mol. Genet. 19, 1108-1118. Wachter, R. M. and Remington, S. J. (1999). Sensitivity of the yellow variant of doi:10.1093/hmg/ddp583 green fluorescent protein to halides and nitrate. Curr. Biol. 9, R628-R629. doi:10. Zhou, Y. and Danbolt, N. C. (2014). Glutamate as a neurotransmitter in the healthy 1016/S0960-9822(99)80408-4 brain. J. Neural Transm. 121, 799-817. doi:10.1007/s00702-014-1180-8 Journal of Cell Science