Genetically encoded optical sensors for monitoring of intracellular chloride and chloride-selective channels activity

Abstract

This review briefly discusses the main approaches for monitoring chloride (Cl−), the most abundant physiological anion. Noninvasive monitoring of intracellular Cl− ([Cl−]i) is a challenging task owing to two main difficulties: (i) the low transmembrane ratio for Cl−, approximately 10:1; and (ii) the small driving force for Cl−, as the Cl− reversal potential (ECl) is usually close to the resting potential of the cells. Thus, for reliable monitoring of intracellular Cl−, one has to use highly sensitive probes. From several methods for intracellular Cl− analysis, genetically encoded chloride indicators represent the most promising tools. Recent achievements in the development of genetically encoded chloride probes are based on the fact that yellow fluorescent protein (YFP) exhibits Cl−-sensitivity. YFP-based probes have been successfully used for quantitative analysis of Cl− transport in different cells and for high-throughput screening of modulators of Cl−-selective channels. Development of a ratiometric genetically encoded probe, Clomeleon, has provided a tool for noninvasive estimation of intracellular Cl− concentrations. While the sensitivity of this protein to Cl− is low (EC50 about 160 mM), it has been successfully used for monitoring intracellular Cl− in different cell types. Recently a CFP–YFP-based probe with a relatively high sensitivity to Cl− (EC50 about 30 mM) has been developed. This construct, termed Cl-Sensor, allows ratiometric monitoring using the fluorescence excitation ratio. Of particular interest are genetically encoded probes for monitoring of ion channel distribution and activity. A new molecular probe has been constructed by introducing into the cytoplasmic domain of the Cl−-selective glycine receptor (GlyR) channel the CFP–YFP-based Cl-Sensor. This construct, termed BioSensor-GlyR, has been successfully expressed in cell lines. The new genetically encoded chloride probes offer means of screening pharmacological agents, analysis of Cl− homeostasis and functions of Cl−-selective channels under different physiological and pathological conditions.

Activity‐dependent intracellular chloride accumulation and diffusion controls GABAA receptor‐mediated synaptic transmission

“…Therefore, quantitative knowledge of local Cl 2 concentration dynamics is necessary to understand not only membrane potential changes but also biochemical phenomena, as Cl 2 plays a ''second messenger'' role, modulating biochemical processes close to the cell surface (Hull and von Gersdorff, 2004). Spatial and temporal discrimination of flourescence methods for measuring Cl 2 concentration is not yet sufficient to estimate microdomain Cl 2 concentration changes, particularly in hardly accessible cellular compartments like dendrites (Kuner and Augustine, 2000;Marandi et al, 2002;Isomura et al, 2003;Berglund et al, 2008;Bregestovski et al, 2009). Thus, computational modeling that we present here is a valuable complementary method for the assessment of small scale Cl 2 dynamics.…”

Optogenetic Sensors for Monitoring Intracellular Chloride

“…A ratiometric indicator, meaning a fluorophore that changes its excitation or emission spectra, can circumvent this problem (Grynkiewicz et al 1985;Bright et al 1989;Kuner and Augustine 2000;Miyawaki et al 2005;Bregestovski et al 2009). …”

An Inhibitor of Na+/H+Exchanger (NHE), Ethyl-Isopropyl Amiloride (EIPA), Diminishes Proliferation of MKN28 Human Gastric Cancer Cells by Decreasing the Cytosolic Cl-Concentration via DIDS-Sensitive Pathways

“…The MQAE is insensitive to physiological changes in pH [27] MKN28 cells were cultured in a glass bottom dish with the culture medium containing 5 mM MQAE with EIPA or DMSO for 45 min at 37°C in a CO 2 incubator. MQAEloaded cells were washed with the culture medium containing EIPA or DMSO, and incubated for 20 min at 37°C.…”