© 2020. Published by The Company of Biologists Ltd | Journal of Cell Science (2020) 133, jcs240390. doi:10.1242/jcs.240390 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 1 REVIEW Journal of Cell Science (2020) 133, jcs240390. doi:10.1242/jcs.240390 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