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A ratiometric pH reporter for imaging protein-dye conjugates in living cells.

ABSTRACT: A molecule that transfers energy through bonds from a donor to an acceptor was prepared with a pH sensitive donor function (fluorescein). At pH values above 6.5, minimal energy transfer occurred, and the probe emitted green fluorescence (ca. 520 nm) when excited at the donor (488 nm). Below pH 6.0 however, energy transfer is efficient; hence excitation at the donor causes emission at the acceptor part (600 nm). This probe was used to image a conjugate of the probe with bovine serum albumin that was imported into endosomes or in the cytosol using the noncovalently bound carrier, Pep-1, at 37 and 4 degrees C, respectively. The more acidic environment of the endosomes was conspicuous from the red fluorescence of the probe.

SUBMITTER: Han J

PROVIDER: S-EPMC2720788 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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A ratiometric pH reporter for imaging protein-dye conjugates in living cells.

Han Junyan J Loudet Aurore A Barhoumi Rola R Burghardt Robert C RC Burgess Kevin K

Journal of the American Chemical Society 20090201 5

A molecule that transfers energy through bonds from a donor to an acceptor was prepared with a pH sensitive donor function (fluorescein). At pH values above 6.5, minimal energy transfer occurred, and the probe emitted green fluorescence (ca. 520 nm) when excited at the donor (488 nm). Below pH 6.0 however, energy transfer is efficient; hence excitation at the donor causes emission at the acceptor part (600 nm). This probe was used to image a conjugate of the probe with bovine serum albumin that ...[more]

PMID: 19146412

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Project description:Potassium is the most abundant intracellular metal in the body, playing vital roles in regulating intracellular fluid volume, nutrient transport, and cell-to-cell communication through nerve and muscle contraction. On the other hand, aberrant alterations in K+ homeostasis contribute to a diverse array of diseases spanning cardiovascular and neurological disorders to diabetes to kidney disease to cancer. There is an unmet need for studies of K+ physiology and pathology owing to the large differences in intracellular versus extracellular K+ concentrations ([K+]intra = 150 mM, [K+]extra = 3-5 mM). With a relative dearth of methods to reliably measure dynamic changes in intracellular K+ in biological specimens that meet the dual challenges of low affinity and high selectivity for K+, particularly over Na+, currently available fluorescent K+ sensors are largely optimized with high-affinity receptors that are more amenable for extracellular K+ detection. We report the design, synthesis, and biological evaluation of Ratiometric Potassium Sensor 1 (RPS-1), a dual-fluorophore sensor that enables ratiometric fluorescence imaging of intracellular potassium in living systems. RPS-1 links a potassium-responsive fluorescent sensor fragment (PS525) with a low-affinity, high-selectivity crown ether receptor for K+ to a potassium-insensitive reference fluorophore (Coumarin 343) as an internal calibration standard through ester bonds. Upon intracellular delivery, esterase-directed cleavage splits these two dyes into separate fragments to enable ratiometric detection of K+. RPS-1 responds to K+ in aqueous buffer with high selectivity over competing metal ions and is sensitive to potassium ions at steady-state intracellular levels and can respond to decreases or increases from that basal set point. Moreover, RPS-1 was applied for comparative screening of K+ pools across a panel of different cancer cell lines, revealing elevations in basal intracellular K+ in metastatic breast cancer cell lines vs. normal breast cells. This work provides a unique chemical tool for the study of intracellular potassium dynamics and a starting point for the design of other ratiometric fluorescent sensors based on two-fluorophore approaches that do not rely on FRET or related energy transfer designs.

Dataset Information

A ratiometric pH reporter for imaging protein-dye conjugates in living cells.

Publications

A ratiometric pH reporter for imaging protein-dye conjugates in living cells.

Similar Datasets

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