title: 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 (Kapp approximately 30 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+ 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.
authors.affiliation: Institut de Neurobiologie de la Méditerranée (INMED), INSERM U901, Parc Scientifique de Luminy, 13273 Marseille Cedex 09, France.
authors.forename: Marat
authors.initials: M
authors.lastname: Mukhtarov
authors.forename: Olga
authors.initials: O
authors.lastname: Markova
authors.forename: Eleonore
authors.initials: E
authors.lastname: Real
authors.forename: Yves
authors.initials: Y
authors.lastname: Jacob
authors.forename: Svetlana
authors.initials: S
authors.lastname: Buldakova
authors.forename: Piotr
authors.initials: P
authors.lastname: Bregestovski
mesh_terms: D000818:Animals; D016466:CHO Cells; D002712:Chlorides / Q000494:pharmacology*; D002713:Chlorine / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D005456:Fluorescent Dyes / Q000494:pharmacology*; D005998:Glycine / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000737:chemistry; D051381:Rats; D018009:Receptors, Glycine / Q000378:metabolism*
chemical_list: D002712:Chlorides; C485182:Cyan Fluorescent Protein; D005456:Fluorescent Dyes; D018009:Receptors, Glycine; D049452:Green Fluorescent Proteins; D002713:Chlorine; D011188:Potassium; D005998:Glycine