title: A noninvasive optical approach for assessing chloride extrusion activity of the K-Cl cotransporter KCC2 in neuronal cells.
abstract: Cation-chloride cotransporters (CCCs) are indispensable for maintaining chloride homeostasis in multiple cell types, but K-Cl cotransporter KCC2 is the only CCC member with an exclusively neuronal expression in mammals. KCC2 is critical for rendering fast hyperpolarizing responses of ionotropic γ-aminobutyric acid and glycine receptors in adult neurons, for neuronal migration in the developing central nervous system, and for the formation and maintenance of small dendritic protrusions-dendritic spines. Deficit in KCC2 expression and/or activity is associated with epilepsy and neuropathic pain, and effective strategies are required to search for novel drugs augmenting KCC2 function.
  
  
  
  We revised current methods to develop a noninvasive optical approach for assessing KCC2 transport activity using a previously characterized genetically encoded chloride sensor. Our protocol directly assesses dynamics of KCC2-mediated chloride efflux and allows measuring genuine KCC2 activity with good spatial and temporal resolution. As a proof of concept, we used this approach to compare transport activities of the two known KCC2 splice isoforms, KCC2a and KCC2b, in mouse neuronal Neuro-2a cells.
  
  
  
  Our noninvasive optical protocol proved to be efficient for assessment of furosemide-sensitive chloride fluxes. Transport activities of the N-terminal splice isoforms KCC2a and KCC2b obtained by the novel approach matched to those reported previously using standard methods for measuring chloride fluxes.
authors.affiliation: Neuroscience Center, University of Helsinki, Helsinki, Finland.|École Normale Supérieure, Institut de Biologie de l'ENS (IBENS), INSERM U1024, CNRS 8197, Paris, France.
authors.forename: Anastasia
authors.initials: A
authors.lastname: Ludwig
authors.affiliation: Neuroscience Center, University of Helsinki, Helsinki, Finland.|INSERM U901, Institut de Neurobiologie de la Méditerranée (INMED), Marseille, France.|UMR S901, Aix-Marseille Université, Marseille, France.
authors.forename: Claudio
authors.initials: C
authors.lastname: Rivera
authors.affiliation: Department of Anatomy, Faculty of Medicine, University of Helsinki, Helsinki, Finland. pavel.uvarov@helsinki.fi.|Department of Biosciences and Neuroscience Center, University of Helsinki, Helsinki, Finland. pavel.uvarov@helsinki.fi.
authors.forename: Pavel
authors.identifier: 0000-0001-8439-6150
authors.initials: P
authors.lastname: Uvarov
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002712:Chlorides / Q000378:metabolism*; D005665:Furosemide / Q000008:administration & dosage; D051379:Mice; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D061848:Optical Imaging / Q000379:methods*; D020033:Protein Isoforms / Q000378:metabolism; D049994:Sodium Potassium Chloride Symporter Inhibitors / Q000008:administration & dosage; D027981:Symporters / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000096922:K Cl- Cotransporters
chemical_list: D002712:Chlorides; D005665:Furosemide; D020033:Protein Isoforms; D049994:Sodium Potassium Chloride Symporter Inhibitors; D027981:Symporters; D000096922:K Cl- Cotransporters
keywords: GABA; Genetically encoded chloride sensor; Inhibition; KCC2; Slc12a5 gene