A genetically-encoded YFP sensor with enhanced chloride sensitivity, photostability and reduced ph interference demonstrates augmented transmembrane chloride movement by gerbil prestin (SLC26a5)

PLoS One. 2014 Jun 5;9(6):e99095. doi: 10.1371/journal.pone.0099095. eCollection 2014.

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.

Methodology/principal findings: 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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Anion Transport Proteins / genetics
  • Anion Transport Proteins / metabolism
  • Anions / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Chlorides / metabolism*
  • Gerbillinae / metabolism*
  • Hydrogen-Ion Concentration
  • Light
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism*
  • Mutation
  • Photobleaching
  • Protein Stability
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / genetics

Substances

  • Anion Transport Proteins
  • Anions
  • Bacterial Proteins
  • Chlorides
  • Luminescent Proteins
  • Recombinant Fusion Proteins
  • yellow fluorescent protein, Bacteria