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. © 2011 Sztretye et al.