title: A sensitive membrane-targeted biosensor for monitoring changes in intracellular chloride in neuronal processes.
abstract: BACKGROUND
  Regulation of chloride gradients is a major mechanism by which excitability is regulated in neurons. Disruption of these gradients is implicated in various diseases, including cystic fibrosis, neuropathic pain and epilepsy. Relatively few studies have addressed chloride regulation in neuronal processes because probes capable of detecting changes in small compartments over a physiological range are limited.
  
  
  RESULTS
  In this study, a palmitoylation sequence was added to a variant of the yellow fluorescent protein previously described as a sensitive chloride indicator (YFPQS) to target the protein to the plasma membrane (mbYFPQS) of cultured midbrain neurons. The reporter partitions to the cytoplasmic face of the cellular membranes, including the plasma membrane throughout the neurons and fluorescence is stable over 30-40 min of repeated excitation showing less than 10% decrease in mbYFPQS fluorescence compared to baseline. The mbYFPQS has similar chloride sensitivity (k(50) =  41 mM) but has a shifted pKa compared to the unpalmitoylated YFPQS variant (cytYFPQS) that remains in the cytoplasm when expressed in midbrain neurons. Changes in mbYFPQS fluorescence were induced by the GABA(A) agonist muscimol and were similar in the soma and processes of the midbrain neurons. Amphetamine also increased mbYFPQS fluorescence in a subpopulation of cultured midbrain neurons that was reversed by the selective dopamine transporter (DAT) inhibitor, GBR12909, indicating that mbYFPQS is sensitive enough to detect endogenous DAT activity in midbrain dopamine (DA) neurons.
  
  
  CONCLUSIONS
  The mbYFPQS biosensor is a sensitive tool to study modulation of intracellular chloride levels in neuronal processes and is particularly advantageous for simultaneous whole-cell patch clamp and live-cell imaging experiments.
authors.affiliation: Department of Neurobiology, Medical School, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Spencer D
authors.initials: SD
authors.lastname: Watts
authors.forename: Katherine L
authors.initials: KL
authors.lastname: Suchland
authors.forename: Susan G
authors.initials: SG
authors.lastname: Amara
authors.forename: Susan L
authors.initials: SL
authors.lastname: Ingram
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D015374:Biosensing Techniques / Q000379:methods; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins / Q000378:metabolism; D059290:Dopaminergic Neurons / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008164:Luminescent Proteins / Q000378:metabolism*; D008636:Mesencephalon / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D001426:Bacterial Proteins; D002712:Chlorides; D050483:Dopamine Plasma Membrane Transport Proteins; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria