Monitoring of chloride and activity of glycine receptor channels using genetically encoded fluorescent sensors

Abstract

Genetically encoded probes have become powerful tools for non-invasive monitoring of ions, distributions of proteins and the migration and formation of cellular components. We describe the functional expression of two molecular probes for non-invasive fluorescent monitoring of intracellular Cl ([Cl] i ) and the functioning of glycine receptor (GlyR) channels. The first probe is a recently developed cyan fluorescent protein-yellow fluorescent protein-based construct, termed Cl-Sensor, with relatively high sensitivity to Cl (K app w30 mM). In this study, we describe its expression in retina cells using in vivo electroporation and analyse changes in [Cl] i at depolarization and during the first three weeks of post-natal development. An application of 40 mM K C causes an elevation in [Cl] i of approximately 40 mM. In photoreceptors from retina slices of a 6-day-old rat (P6 rat), the mean [Cl] i is approximately 50 mM, and for P16 and P21 rats it is approximately 30-35 mM. The second construct, termed BioSensor-GlyR, is a GlyR channel with Cl-Sensor incorporated into the cytoplasmic domain. This is the first molecular probe for spectroscopic monitoring of the functioning of receptor-operated channels. These types of probes offer a means of screening pharmacological agents and monitoring Cl under different physiological and pathological conditions and permit spectroscopic monitoring of the activity of GlyRs expressed in heterologous systems and neurons.

Genetically encoded Cl-Sensor as a tool for monitoring of Cl-dependent processes in small neuronal compartments

“…4A). This closely coincided with our previous observations Mukhtarov et al, 2008) and was similar for all three vectors used: 28.8 ± 0.5 mM (n = 162) for Clontech-Cl-Sensor, 31.1 ± 0.6 mM (n = 105) for GW1-Cl-Sensor and 27.0 ± 0.6 mM (n = 95) for pLLU2G-Cl-Sensor (Fig. 4A).…”

“…This construct, called Cl-Sensor, allows ratiometric non-invasive estimation of [Cl] i using the fluorescence excitation ratio. This genetically encoded indicator has been used for estimation of [Cl − ] i in several cell lines, in the soma of cultured hippocampal neurons and photoreceptors of retinal slices Mukhtarov et al, 2008).…”

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

“…Recently, a new genetic probe, termed BioSensor-GlyR, has been developed (Mukhtarov et al, 2008). This construct is a Cl − -selective GlyR channel with Cl-Sensor incorporated into the long cytoplasmic domain (Figure 5A).…”

Optogenetic reporters

“…Intracellular chloride dynamics have been imaged in cultured neurons and retinal slices using Cl-sensor (Waseem et al, 2010). Additionally, a fusion of Cl-Sensor with the glycine receptor GlyR has been used to monitor chloride conduction and ion channel activity (Mukhtarov et al, 2008). Finally, transgenic mice that express a chloride-sensitive EYFP under control of the K V 3.1 promoter have also been generated (Metzger et al, 2002).…”