title: D4cpv-calsequestrin: a sensitive ratiometric biosensor accurately targeted to the calcium store of skeletal muscle.
abstract: 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.
authors.affiliation: Section of Cellular Signaling, Department of Molecular Biophysics and Physiology, Rush University, Chicago, IL 60612, USA.
authors.forename: Monika
authors.initials: M
authors.lastname: Sztretye
authors.forename: Jianxun
authors.initials: J
authors.lastname: Yi
authors.forename: Lourdes
authors.initials: L
authors.lastname: Figueroa
authors.forename: Jingsong
authors.initials: J
authors.lastname: Zhou
authors.forename: Leandro
authors.initials: L
authors.lastname: Royer
authors.forename: Eduardo
authors.initials: E
authors.lastname: Ríos
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D002155:Calsequestrin / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000502:physiology; D002478:Cells, Cultured; D008024:Ligands; D051379:Mice; D018482:Muscle, Skeletal / Q000737:chemistry / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism
chemical_list: D002155:Calsequestrin; D008024:Ligands; D002118:Calcium