Ratiometric FURA-2 imaging was used to measure cytosolic [Ca²⁺]_i in WT and Tmtc4-KO P3–P5 cochlear cultures. (A) Representative spontaneous intracellular Ca²⁺ wave in the inner sulcus of the organ of Corti. Regions of interest (7-μm square) in the inner and outer sulci were identified and FURA-2 excitation ratios (proportional to cytosolic Ca²⁺ concentration) measured. Ca²⁺ peaks were extracted and analyzed. (B) Aggregate ratio time course for Ca²⁺ peaks normalized to baseline and peak maximum show identical initial kinetics and faster return to baseline in WT cochleae treated with the SERCA2B activator CDN1163 (CDN) plus the SERCA2B inhibitor thapsigargin (TG) (n = 88 peaks) compared with TG (n = 65 peaks) alone, demonstrating the ability of known modulators of ER Ca²⁺ reuptake to affect Ca²⁺ peak decay time. (C–E) Analysis of 273 and 653 peaks, respectively, from 6 WT and 6 KO cochleae. (C) Peaks in Tmtc4-KO cochleae (n = 653) showed prolongation of return to baseline cytosolic Ca²⁺ level relative to WT controls (n = 273). (B and C) Black bars: points at which aggregate ratios were statistically significantly different. Peak frequency (D) (mean ± SEM) and height (E) (mean ± SEM) were greater in KO cochleae. (F) Decay time in WT cochleae was measured in dissecting solution (DS) and with drugs (CDN: 10 μM CDN1163; TG: 1 μM TG; and TG+CDN: 10 μM CDN1163 and 1 μM TG) as well as for Tmtc4-KO cochleae in DS. Data indicate difference in mean ± SE of decay time relative to that measured in WT cochleae in DS (number of peaks analyzed for each comparison in parentheses).*P < 0.01 for DS in WT cochleae. #P < 0.0001 versus TG in WT cochleae (unpaired, 2-tailed t test for all comparisons).