SR Ca2+ buffering power, BP, decreases during Ca2+-depleting depolarizations of mouse skeletal muscle. During Ca2+ release the stage of high BP is characterized by a “hump” in the release flux waveform. After the depolarization BP returns slowly to its initial value, as demonstrated by the absence of a hump in the flux induced by the second pulse of pairs separated by 600 ms. These time-dependent features were described as “buffer hysteresis” and shown to be contributed by calsequestrin in the Sztretye et al., companion poster. SR release flux and BP were measured in calsequestrin 1-null cells expressing the biosensor D4cpv-calsequestrin. Null cells had lower BP and generally lacked the hump in the flux. In some regions of these cells, however, [biosensor] reached very high values. The spatial heterogeneity of expression permitted comparison of flux and buffer properties evaluated with the same depolarizing pulse simultaneously in regions of widely different [biosensor]. The hump of Ca2+ release flux and other features of buffer hysteresis were restored in regions of calsequestrin-null cells where D4cpv-calsequestrin reached above 8 μmol/liter of cytosol. The restoration was partial or nil at [biosensor] below 3 μM. At 10 μM a functional calsequestrin moiety in the biosensor should provide 800 μM binding sites (or 400 μM Ca2+ at 50% occupancy), a significant contribution compared with the estimates of amount released (1240 μM in WT, 867 μM in the null). Therefore, in addition to a targeted biosensor D4cpv-calsequestrin is a fluorescently tagged Ca2+ buffer. Moreover, the restoration of hysteretic Ca2+ buffering features indicates that this biosensor contributes the full buffer functionality of calsequestrin. To our knowledge, this is the first example of a molecule with the functional properties of both a biosensor and a native protein.Funded by NIAMS/NIH.