Figure 1. Ca²⁺ release from intracellular stores is required for spontaneous currents and crenation in inner supporting cells.

(A) Model of ATP-mediated depolarization of inner hair cells. ATP: adenosine triphosphate, PLC: phospholipase C, IP₃: inositol triphosphate, TMEM16A: transmembrane member 16A (Ca²⁺-activated Cl^– channel). Inhibitors of key steps in this pathway are indicated. (B) Whole-cell voltage-clamp recordings from inner supporting cells after pre-incubating with indicated inhibitors. (C) Quantification of ISC spontaneous current frequency in the presence of the indicated inhibitors. Data shown as mean ± SEM. n = 16 cells, 11 cochleae from six postnatal day (P) 6–8 rats (control), 18 cells, 10 cochleae from five rats (BAPTA-AM; 100 μM), 20 cells, 11 cochleae from six rats (Thapsigargin; 2 μM), 14 cells, 11 cochleae from seven rats, (U73122; 10 μM), and 16 cells, 10 cochleae from five rats (U73343; 10 μM). ****p<5e-5, one-way ANOVA. (D) Quantification of ISC spontaneous current amplitude in the presence of indicated inhibitors. Data shown as mean ± SEM. n values are reported in (C) (one-way ANOVA; ****p<5e-5, **p<0.005, ns: not significant). (E) Quantification of ISC spontaneous current charge transfer (integral) in the presence of indicated inhibitors. Data shown as mean ± SEM. n values are reported in (C) (one-way ANOVA; ****p<5e-5, **p<0.005, ns: not significant). (F) Quantification of ISC crenation (cell shrinkage) frequency in the presence of indicated inhibitors. Data shown as mean ± SEM. n = 19 videos, 11 cochleae from six rats (control), 15 videos, 8 cochleae from four rats (BAPTA-AM), 22 videos, 12 cochleae from six rats (Thapsigargin), 23 videos, 17 cochleae from 10 rats (U73122), and 20 videos, 10 cochleae from five rats (U73343) (one-way ANOVA; ****p<5e-5, **p<0.005, ns: not significant). See Figure 1—source data 1 for plotted values and statistics.

Figure 1—figure supplement 1. Inhibition of calcium-induced calcium release does not alter spontaneous currents or crenations.

(A) Spontaneous inward currents recorded from an ISC before and after application of ryanodine (10 μM). Recordings were performed near physiological temperature 32–34°C. (B) Quantification of event frequency, amplitude, and charge transfer (integral). Each window measured was 5 min in length. n = 11 ISCs, 11 cochleae from 8 P6-8 mice (two-tailed paired Student's t test with Bonferroni correction; ns, not significant). (C) Intrinsic optical imaging performed before and after application of ryanodine (10 μM). Detected crenations are outlined in colors based on time of occurrence as indicated by timeline below image. Imaging was performed near physiological temperature 32–34°C. (D) Quantification of crenation frequency and area before and after application of ryanodine. n = 13 cochleae from 8 P6-8 mice (two-tailed paired Student's t test with Bonferroni correction; ns, not significant). See Figure 1—figure supplement 1—source data 1 for plotted values and statistics.