Novel PMCA2 Mutation and Hearing Loss in Mouse Figure 3. Hair cell counts in Oblivion heterozygous and wildtype animals. Hair cell counts show that outer hair cell loss in heterozygotes (Obl/+) at P75 is present in the base (C) but not the middle (D) of the cochlea. By P121, outer hair cell loss is seen in the base (E) and the middle (F) of the cochlear duct. IHC loss is seen in the base of the cochlea only at later ages (E). This suggests that hair cell loss in Obl/+ mutants progresses in a base to apex direction, with OHC loss occurring first and IHC loss later. No OHC or IHC loss was seen in the base (A) or middle (B) regions of the cochlea at P20. Hair cell counts calculated as mean number of hair cells per 100 mm at P20 (+/+ n = 4–7, Obl/+ n = 7), P72–75 (+/+ n = 4, Obl/+ n = 5) and P121 (+/+ n = 3–4, Obl/+ n = 4–5). Error bars indicate 95% confidence interval, T-test, * P-value,0.05. doi:10.1371/journal.pgen.1000238.g003 transient to baseline were controlled primarily by the PMCA pump: the much larger amounts of the overexpressed PMCA2 pump overshadowed the endoplasmic reticulum Ca2+ pump (SERCA) (see [16]) and the contribution of plasma membrane Ca2+ influx channels opened by emptying of intracellular stores to the shaping of the Ca2+ trace was disregarded, as their effect would be the same in the wild type and Obl measurements. The overexpressed Obl pump did not further depress the limited ability of the wild type w/a pump to control the height of the Ca2+ peak (Figure 6). The mutation, however, severely affected the resting activity of the pump that drove the return of the Ca2+ trace to baseline after the peak. The half time of the declining phase was 64.1563.02 sec (n = 6) in control, 6.5560.72 sec (n = 9) in the wild type w/a variant and 45.5065.97 sec (n = 4), p,0.001, in Obl. Ca2+ Dynamics in the Hair Cell Stereocilia of Wild Type, Obl/+ , and Obl/Obl Mice To characterize Ca2+ dynamics in the stereocilia of hair cells, cultures of immature utricular maculae were obtained from wild type and mutant mice (see Materials and Methods). Ca2+- dependent changes in fluorescence evoked by the photorelease of intracellular caged Ca2+ (4 ns single UV pulse) were monitored with a temporal resolution of 6 ms using confocal laser scanning microscopy. Immunofluorescence labelling with isoform specific antibodies showed that PMCA2 was correctly located in the stereocilia of homozygous mutant organotypic cultures of utricular maculae (Figure 7A) and organ of Corti (Figure 8). Figure 7B shows a macular hair cell loaded with Fluo-4. Fluorescence changes in the stereocilia were monitored repeatedly by a line-scan positioned along the hair bundle and extending into the cell soma (dashed line). Time-dependent post UV pulse changes in fluorescence at different parts of the line scan are illustrated in Figure 7C. The time course of the stimulus-evoked changes in fluorescence (DF), normalized to basal (pre-stimulus) fluorescence (F0), are compared for wild type (wt, blue trace) heterozygous Oblivion mice (Obl/+, green trace) and homozygous Oblivion mice (Obl/Obl, black trace) in Figure 7D. The time courses matched well at the peak, although the [Ca2+]i transient decayed more slowly for the Obl/Obl mice. To highlight the differences, the traces are re-plotted in Figure 7E on an expanded time scale (solid lines), together with their respective confidence intervals (dash- dotted lines). A single exponential fit to the first 10 s of these transients yielded significantly longer decay time constants for the Obl/+ and Obl/Obl cultures: twt = 2.860.4 s for wt, tObl/+ = 4.261 s for Obl/+ mice (p = 0.02, if compared to wt using the ANOVA test) and tObl/Obl = 6.961.3 s for Obl/Obl mice (p,0.01). No significant divergence in the early phase (first 2 s) of the decay was found for Obl/+ mice relative to wt, consistent with the lack of evident phenotypic vestibular defects in these mice. At later times, the decay tended to diverge possibly due to the contribution of complex processes such as Ca2+-induced Ca2+ release. These contributions were not investigated further. Discussion Progressive, age-related hearing loss affects 60% of humans over the age of 70 [18]. The condition is a multifactorial disorder to which genetic variation, disease and environmental influences such as acoustic trauma are all contributing factors, making identifica- tion of the genes involved in humans difficult. The mouse is an ideal resource to study the genetics of progressive hearing loss due PLoS Genetics | www.plosgenetics.org 4 October 2008 | Volume 4 | Issue 10 | e1000238