Figure 4. Oblivion chromosomal location, mutation, and PMCA2 sequence alignment. (A) Obl mutation maps to a 16 Mb region on mouse Chromosome 6, between D6Mit104 and D6Mit218. Each box represents a marker typed, and each chromosomal arrangement detected is shown, with the number of animals possessing that chromosome given underneath. If a marker was not typed, then no box is present. (B) Sequence of Atp2b2 in Obl/+ mouse showing the CRT mutation and predicted sequence. (C) Sequence alignment of several PMCA family members showing Ser877 is highly conserved. doi:10.1371/journal.pgen.1000238.g004 to the possibility of controlling both genetic background and the environment. The present study has identified a new ENU-induced allele of Atp2b2, Atp2b2Obl in the Obl mouse mutant. This allele contains a C to T missense mutation in exon 15 of Atp2b2, causing a non- conservative amino acid substitution of serine by phenylalanine in transmembrane domain 6 of the PMCA2 pump [1,19]. The serine at position 877 is highly conserved in both human and mouse and also between other members of the PMCA family (Figures 4C and S2). The S877F mutation is of special interest on at least two accounts. One is the finding that substitutions in the transmem- brane residues of the PMCA pump frequently impair its correct plasma membrane targeting [20]. However, the mutant Obl pump was correctly delivered to the plasma membrane in both the model cells and in native stereocilia. In another Atp2b2 mutant allele, Wriggle mouse Sagami (wri), a missense mutation in transmem- brane domain 4 completely abolished the expression of PMCA2 protein in stereocilia of cochlear hair cells [21]. The second reason Novel PMCA2 Mutation and Hearing Loss in Mouse for interest stems from studies of the SERCA pump of endoplasmic reticulum, considered a model for all P-type pumps. Conserved residues within transmembrane domain 6 of SERCA have been shown to be components of the channel through which Ca2+ is translocated [22] and are also present in the PMCA pump. It is pertinent to quote at this point an earlier study on some point mutations in transmembrane domain 6 of another isoform of the pump, PMCA4 [23]. In that study, one of the mutants (S877A) appears to have higher activity than the wild type pump. The reason for the different effect of the S877A mutation of PMCA4 and the S877F mutation of PMCA2 that we report here is not clear. However, the PMCA4 study is difficult to compare with the present one, since it analyzed the pump in crude microsomes of overexpressing COS cells, and offered no information on the localization of the expressed pump in the plasma membrane. The Obl mutation is the seventh mutation in mouse Atp2b2 to be reported. In the deafwaddler (dfw) mouse a missense mutation in Atp2b2 leads to a partial loss of function [8] and PMCA2 retains 30% of its Ca2+ pumping activity [24]. A spontaneous T692K mutation in Atp2b2 led to clear ataxic behaviour, with normal mRNA levels, in a second unnamed mutant [25]. A further four mutations of mouse Atp2b2 have been described: deafwaddler 2J (dfw2J), deafwaddler 3J (dfw3J), wriggle mouse sagami (wri) and a targeted mutation. Analysis of mRNA transcripts and protein levels suggests that these latter four mutations are all alleles [13,21,26]. Homozygotes for these alleles show severe ataxia by 10 days of age as well as profound deafness. The profound deafness and ataxia detected in Obl homozygotes is similar to the phenotype of these other known Atp2b2 mutants [13,26,27]. Non- complementation between Obl and dfw confirmed that these two mouse mutants are likely to be allelic. The mutations identified in the Atp2b2 gene are shown in Figure S1. Both the Obl and the dfw mutations significantly reduce the non- activated Ca2+ exporting ability of the PMCA2 protein. Obser- vations on organotypic cultures showed that the defect of the pump observed in model cells also reduced its ability to remove the Ca2+ produced by UV photolysis in stereocilia. Thus, the Obl mutant has lost a significant portion of the non stimulated, longer- term Ca2+ exporting ability in respect to the w/a PMCA2 pump. In the present work we show that hearing loss in Obl/+ mice is detected at P20 and progresses in its severity with increasing age. Threshold shifts of up to 60-70 dB were found at frequencies corresponding to the basal and middle turns of the cochlea, where the majority of outer hair cell loss was detected. Analysis of Obl/+ mice at P20 showed no OHC or IHC loss despite the fact that they had a significant hearing impairment, confirming the previous suggestion that although hair cells are present they are not functioning normally [28]. IHC loss was detected in the base and middle of the cochlea in Obl/+ mutants at 4 months of age. The early degeneration of the OHCs seen in heterozygous Obl (and dfw) mice, leading to complete degeneration of the organ of Corti in the base of the cochlea, is similar to that seen in some human patients with age- related hearing loss [29,30]. It is not clear why hair cell dysfunction leads to hair cell degeneration in these mouse mutants, but prolonged abnormal calcium homeostasis may contribute to hair cell death. In dfw mice at approximately P60, distortion product otoacoustic emission studies show that PMCA2 is important for the correct functioning of outer hair cells, especially at high frequencies [31]. Mice expressing the Atp2b2dfw2J allele, [9] demonstrated a lowered endocochlear potential and reduced endolymphatic calcium concentration, and thus have a reduced capacity for mechanoelectrical transduction. Taken together, these features may explain why ABR thresholds in PLoS Genetics | www.plosgenetics.org 5 October 2008 | Volume 4 | Issue 10 | e1000238