D4cpv-calsequestrin: a sensitive ratiometric biosensor accurately targeted to the calcium store of skeletal muscle

Current fluorescent monitors of free [Ca(2+)] in the sarcoplasmic reticulum (SR) of skeletal muscle cells are of limited quantitative value. They provide either a nonratio signal that is difficult to calibrate and is not specific or, in the case of Forster resonant energy transfer (FRET) biosensors,...

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Published in:The Journal of general physiology 2011-08, Vol.138 (2), p.211-229
Main Authors: Sztretye, Monika, Yi, Jianxun, Figueroa, Lourdes, Zhou, Jingsong, Royer, Leandro, Ríos, Eduardo
Format: Article
Language:English
Subjects:
Animals
Biosensing Techniques - methods
Biosensors
Calcium
Calcium (reticular)
Calcium - analysis
Calcium - metabolism
calsequestrin
Calsequestrin - chemistry
Calsequestrin - metabolism
Cell Membrane - metabolism
Cell Membrane Permeability - physiology
Cells
Cells, Cultured
fluorescence resonance energy transfer
Ligands
Mice
Muscle, Skeletal - chemistry
Muscle, Skeletal - metabolism
Musculoskeletal system
Protein biosynthesis
Rodents
Sarcoplasmic reticulum
Sarcoplasmic Reticulum - metabolism
Skeletal muscle
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Summary:Current fluorescent monitors of free [Ca(2+)] in the sarcoplasmic reticulum (SR) of skeletal muscle cells are of limited quantitative value. They provide either a nonratio signal that is difficult to calibrate and is not specific or, in the case of Forster resonant energy transfer (FRET) biosensors, a signal of small dynamic range, which may be degraded further by imperfect targeting and interference from endogenous ligands of calsequestrin. We describe a novel tool that uses the cameleon D4cpv, which has a greater dynamic range and lower susceptibility to endogenous ligands than earlier cameleons. D4cpv was targeted to the SR by fusion with the cDNA of calsequestrin 1 or a variant that binds less Ca(2+). "D4cpv-Casq1," expressed in adult mouse at concentrations up to 22 µmole/liter of muscle cell, displayed the accurate targeting of calsequestrin and stayed inside cells after permeabilization of surface and t system membranes, which confirmed its strict targeting. FRET ratio changes of D4cpv-Casq1 were calibrated inside cells, with an effective K(D) of 222 µM and a dynamic range [(R(max) - R(min))/R(min)] of 2.5, which are improvements over comparable sensors. Both the maximal ratio, R(max), and its resting value were slightly lower in areas of high expression, a variation that was inversely correlated to distance from the sites of protein synthesis. The average [Ca(2+)](SR) in 74 viable cells at rest was 416 µM. The distribution of individual ratio values was Gaussian, but that of the calculated [Ca(2+)](SR) was skewed, with a tail of very large values, up to 6 mM. Model calculations reproduce this skewness as the consequence of quantifiably small variations in biosensor performance. Local variability, a perceived weakness of biosensors, thus becomes quantifiable. It is demonstrably small in D4cpv. D4cpv-Casq1 therefore provides substantial improvements in sensitivity, specificity, and reproducibility over existing monitors of SR free Ca(2+) concentration.
ISSN:0022-1295
1540-7748
DOI:10.1085/jgp.201010591