Monitoring of chloride and activity of glycine receptor channels using genetically encoded fluorescent sensors.
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.
Mukhtarov
Marat
M
Institut de Neurobiologie de la Méditerranée (INMED), INSERM U901, Parc Scientifique de Luminy, 13273 Marseille Cedex 09, France.
Markova
Olga
O
Real
Eleonore
E
Jacob
Yves
Y
Buldakova
Svetlana
S
Bregestovski
Piotr
P
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*
D002712:Chlorides; C485182:Cyan Fluorescent Protein; D005456:Fluorescent Dyes; D018009:Receptors, Glycine; D049452:Green Fluorescent Proteins; D002713:Chlorine; D011188:Potassium; D005998:Glycine