title: A genetically-encoded YFP sensor with enhanced chloride sensitivity, photostability and reduced ph interference demonstrates augmented transmembrane chloride movement by gerbil prestin (SLC26a5).
abstract: BACKGROUND
  Chloride is the major anion in cells, with many diseases arising from disordered Cl- regulation. For the non-invasive investigation of Cl- flux, YFP-H148Q and its derivatives chameleon and Cl-Sensor previously were introduced as genetically encoded chloride indicators. Neither the Cl- sensitivity nor the pH-susceptibility of these modifications to YFP is optimal for precise measurements of Cl- under physiological conditions. Furthermore, the relatively poor photostability of YFP derivatives hinders their application for dynamic and quantitative Cl- measurements. Dynamic and accurate measurement of physiological concentrations of chloride would significantly affect our ability to study effects of chloride on cellular events.
  
  
  RESULTS
  In this study, we developed a series of YFP derivatives to remove pH interference, increase photostability and enhance chloride sensitivity. The final product, EYFP-F46L/Q69K/H148Q/I152L/V163S/S175G/S205V/A206K (monomeric Cl-YFP), has a chloride Kd of 14 mM and pKa of 5.9. The bleach time constant of 175 seconds is over 15-fold greater than wild-type EYFP. We have used the sensor fused to the transmembrane protein prestin (gerbil prestin, SLC26a5), and shown for the first time physiological (mM) chloride flux in HEK cells expressing this protein. This modified fluorescent protein will facilitate investigations of dynamics of chloride ions and their mediation of cell function.
  
  
  CONCLUSIONS
  Modifications to YFP (EYFP-F46L/Q69K/H148Q/I152L/V163S/S175G/S205V/A206K (monomeric Cl-YFP) results in a photostable fluorescent protein that allows measurement of physiological changes in chloride concentration while remaining minimally affected by changes in pH.
authors.affiliation: Dept. of Surgery (Otolaryngology), Yale University School of Medicine, New Haven, Connecticut, United States of America.
authors.forename: Sheng
authors.initials: S
authors.lastname: Zhong
authors.affiliation: Dept. of Surgery (Otolaryngology), Yale University School of Medicine, New Haven, Connecticut, United States of America; Dept. of Neurobiology, Yale University School of Medicine, New Haven, Connecticut, United States of America; Dept. of Neurology, Yale University School of Medicine, New Haven, Connecticut, United States of America.
authors.forename: Dhasakumar
authors.initials: D
authors.lastname: Navaratnam
authors.affiliation: Dept. of Surgery (Otolaryngology), Yale University School of Medicine, New Haven, Connecticut, United States of America; Dept. of Neurology, Yale University School of Medicine, New Haven, Connecticut, United States of America; Dept. of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut, United States of America.
authors.forename: Joseph
authors.initials: J
authors.lastname: Santos-Sacchi
mesh_terms: D000818:Animals; D027321:Anion Transport Proteins / Q000235:genetics / Q000378:metabolism; D000838:Anions / Q000737:chemistry; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D005849:Gerbillinae / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008027:Light; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D038761:Photobleaching; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D027321:Anion Transport Proteins; D000838:Anions; D001426:Bacterial Proteins; D002712:Chlorides; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria