Novel PMCA2 Mutation and Hearing Loss in Mouse Figure 2. Analysis of wildtype, Obl/+, and Obl/Obl organ of Corti by scanning electron microscopy. At 3–4 months, normal animals show three rows of outer hair cells (OHC) and one row of inner hair cells (IHC) in the apex (A), middle (B), and base (C) of the cochlea. Obl/+ have extensive OHC loss and some IHC loss in the base of the cochlea (F) and a few missing OHC in the middle of the cochlea (E). The apex appears normal (D). At 1 month of age, the phenotype in the Obl/Obl mice is extremely variable. In some regions of the base, middle and apex, the phenotype is similar to that seen in Obl/+. However, in other parts of the apex (G) and middle (H) regions of the cochlea, there are missing patches of OHCs. In some regions of the base, there is a complete degeneration of the organ of Corti, with no IHC, OHC, or supporting cells such as pillar cells present (I). Scale bar = 10 mm. doi:10.1371/journal.pgen.1000238.g002 Obl/Obl homozygotes were more severely affected than heterozy- gotes. However, there were many remaining hair cells with relatively normal appearance in the mutants, including a W- shaped arrangement of stereocilia, especially in the apical turn. Stereocilia fusion was seen in some, an early indicator of hair cell degeneration. At P20, no significant hair cell loss was detected in Obl/+ mutants compared to their littermate controls (Figure 3A and 3B), despite the fact that we saw significantly raised ABR thresholds in another cohort of P20 heterozygotes (Figure 1). Hair cell counts from the basal and middle turns at P75 showed no significant OHC degeneration in the middle turn and no significant IHC loss throughout the cochlea in Obl/+ heterozy- gotes (Figure 3C and 3D). By P121, there was significant OHC and IHC loss in basal and middle turns in Obl/+ (Figure 3E and 3F). This suggests that the hair cell loss seen in these mutants is a secondary consequence of the hair cell not functioning correctly, rather than being the primary cause of raised thresholds in Obl/+ mutants. In Obl/Obl mutants at P30 there was highly variable hair cell degeneration, both within and between animals. In some regions there was scattered hair cell loss with a pattern similar to that seen in heterozygotes (Figure 2G and 2H), while in some regions towards the base there was complete degeneration of the organ of Corti with a complete absence of specialised cells, including supporting cells such as pillar cells (Figure 2I). Mapping and Identification of the Oblivion Mutation Obl/+ mutants on a C3HeB/FeJ background were outcrossed to C57BL/6J and mutant F1 progeny were backcrossed to the original C3HeB/FeJ strain. Backcross litters were assessed for absence of a Preyer reflex and a genome-wide scan was performed on their DNA using 60 polymorphic microsatellite markers. We identified a region of linkage on chromosome 6 (Figure 4A) between markers D6Mit104 and D6Mit218, corresponding to a physical distance of 16 Mb. This region contained a good candidate gene: Atp2b2. Genomic DNA was used to sequence the 19 coding exons of the gene, including the splice sites. We identified a C/T heterozygous peak in Obl/+ mutants, suggesting a CRT transition (2630CRT) in exon 15 of Atp2b2, predicted to change a serine to a phenylalanine (S877F; Figure 4) in the mutant allele. This change was also confirmed by a restriction test assay that was used to genotype the colony. This assay was used to screen 19 inbred strains for the Obl mutation and none were found to have it, suggesting that it is not a common polymorphism. We found non-complementation of Obl with the deafwaddler mutant allele, Atp2b2dfw, confirming that the missense mutation we found was the pathogenic mutation (see Text S1). Functional Analysis of the Mutated PMCA2 Pump Overexpressed in Model Cells To investigate the effects of the serine to phenylalanine change on the functionality of the pump, mammalian expression plasmids for the S877F and the wild type variant of the PMCA2 pump were prepared and expressed in CHO cells. Appropriate controls (Western blotting and quantitative immunocytochemistry) estab- lished that the two pump variants were expressed at about the same levels, and were correctly delivered to the plasma membrane (Figure 5). CHO cells were transfected with the Ca2+ sensitive photoprotein aequorin (cytAEQ, [3]) and stimulated with ATP, an agonist of purinergic P2Y receptors that produces InsP3 generating a cytosolic Ca2+ transient. Under the experimental conditions, the height of the Ca2+ peak, and the kinetics of the return of the Ca2+ PLoS Genetics | www.plosgenetics.org 3 October 2008 | Volume 4 | Issue 10 | e1000238