Abstract
BackgroundChloride 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 FindingsIn 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.ConclusionsModifications 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.
Highlights
Chloride is the major anion in cells, and plays various physiological roles
V68L is included in native EYFP, and we opted to not include F64L in our new Cl2 sensor
We found that the mutant Q69K when added to EYFPH148Q/I152L/V163S folds poorly at 37uC, which was previously reported in EYFP-V68L/Q69K [27], possibly resulting from the extra length of the Lysine side chain and disturbing the hydrogenbond network in the halide-binding cavity
Summary
Chloride is the major anion in cells, and plays various physiological roles. For example, chloride is a key determinant of intestinal fluid secretion and cell volume primarily through affecting osmotic gradients [1,2,3]. As a natural consequence of affecting these diverse phenomena many diseases result from disordered Cl2 regulation [5] These diverse effects of chloride underscore the need to accurately and dynamically measure physiological intracellular chloride concentration. The unique membrane protein prestin in outer hair cells (OHCs), SLC26a5, functions as an ultrafast molecular motor, converting electrical to mechanical energy This protein is thought to bring about cochlea amplification in mammals, which is responsible for the exquisite sensitivity of mammalian hearing. Intracellular chloride ions in the 0–10 mM range regulate the behavior of prestin, shifting its voltage responsiveness by 22 mV per mM of chloride (with gluconate as the counter anion) These in vitro findings have been supported by in vivo experiments demonstrating effects of chloride on cochlear amplification [6]. Dynamic and accurate measurement of physiological concentrations of chloride would significantly affect our ability to study effects of chloride on cellular events
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