Author Summary Progressive hearing loss is very common in the human population, but we know little about the causes. Environmental and genetic factors each may contribute. Knowledge of the genetic variants involved in hearing loss and understanding of the molecular and cellular mecha- nism of their action will aid the development of better treatments. One of the few genes known to be involved, in both mouse and humans, is Atp2b2, which encodes a calcium pump. We have discovered a new mutation in this gene leading to hearing loss in the mouse mutant oblivion. The mutation leads to a serine to phenylalanine substitution in a transmembrane domain. Mutations affecting such transmembrane domains are usually ex- pected to interfere with the normal process of inserting the protein in the membrane and transporting it to its final destination on the plasma membrane of the cell. Surprisingly, the pump is produced and is targeted to the plasma membrane, in both cultured cells expressing the mutant gene and sensory hair cells from the oblivion mutant inner ear. However, we show it has impaired calcium pumping ability, which can account for the progressive hearing loss as well as the progressive degeneration of the sensory hair cells that we observe in the mutants. The Oblivion (Obl) mutant was identified as a new mouse mutant with progressive hearing loss from a large scale ENU mutagenesis screen [17]. The aim of this screen is to provide new models for deafness, especially progressive deafness which is common in the human population, and to identify the genes and underlying pathology in these new mutants. Here we report that the progressive hearing loss in Obl is due to a missense mutation in the gene Atp2b2, encoding PMCA2. We describe the hearing impairment and hair cell pathology in the mutants, the dysfunction of Ca2+ export by the mutated PMCA2 pump cloned and overexpressed in model cells and in cultures of utricles from the mutant mice. Results Progressive Hearing Loss in Oblivion Mutants Obl/+ heterozygotes have a normal Preyer reflex at one month old, but by two months only 58% offspring from Obl/+6+/+ matings showed a Preyer reflex (Table S1), suggesting progressive hearing loss in Obl/+ mice. No vestibular defect, indicated by head-tossing or circling behaviour, was seen in these heterozy- gotes, although no detailed analysis of vestibular function was performed. To measure auditory thresholds, auditory brainstem responses (ABR), a reflection of cochlear and brainstem neural activity, were recorded in P20, P59–62 and P89–91 mice on their original C3HeB/FeJ genetic background (Figure 1). ABRs of wild-type mice showed an improvement in thresholds below 12 kHz from P20 to P59–62, perhaps indicative of maturation of the auditory system. From P20 to P89–91, wild type mice showed mild and progressive elevations of thresholds above 12 kHz. Obl/+ mice demonstrated a severe and age-related progressive hearing loss. Obl/+ mice showed significantly raised thresholds at all frequencies, compared to age-matched wild-type controls (t- test, p,0.05), of up to 60–70 dB or more. In contrast to the Preyer reflex tests, even at P20 Obl/+ showed large threshold elevations. At P59–62, the heterozygotes showed further threshold elevations which were most severe at higher frequencies, above 18 kHz. By Novel PMCA2 Mutation and Hearing Loss in Mouse Figure 1. Auditory brainstem response thresholds in Oblivion heterozygous and wildtype animals. Obl/+ mutants show signif- icantly raised auditory brainstem response (ABR) thresholds compared to wildtype mice at P20 (circles), P59–62 (squares), and P89–91 (triangles). Mean ABR thresholds with standard error bars are given for +/+ mice (filled symbols) and Obl/+ mice (open symbols). The dotted line without symbols indicates the maximum dB SPL output for the sound system at each frequency. The position along the length of the cochlear duct that best responds to each frequency (represented by % of total distance from the base) is indicated by the frequency place map at the top of the graph. Mean click ABR thresholds are plotted at an arbitrary point on the frequency axis; the position does not relate to the frequency content of the click stimulus. doi:10.1371/journal.pgen.1000238.g001 P89–91, high frequency losses were compounded by severe losses across the entire range measured. This indicated a progressive hearing loss in Obl/+ mice. The improvement of low frequency (3– 6 kHz) ABR thresholds between P20 and P59–62 may indicate maturation of the developing auditory system between these ages. Obl/Obl homozygous mutants show a very severe hearing and vestibular phenotype and are significantly smaller (10.5 g, SD 2.02) than age and sex matched Obl/+ littermates (17.3 g, SD 2.11; t-test, p,0.05, 31–34 days old). They (a) fail to develop a Preyer reflex; (b) fall from side to side whilst walking; (c) are unable to right themselves; and (d) curl towards their belly when lifted by their tail and do not display a reaching response when lowered towards a surface. Homozygotes also show hind limb stiffness and appear ataxic, which are not general features of vestibular dysfunction. Structure of the Inner Ear The gross morphology of the middle ear ossicles and inner ear appeared normal in Oblivion mutants, both heterozygotes and homozygotes. Scanning electron microscopy in Obl/+ mutants at 3–4 months of age showed degeneration of hair cells, with the basal turn more severely affected than the apex, and outer hair cells (OHCs) more affected than inner hair cells (IHCs), a pattern that is commonly reported in damaged cochleas (Figures 2 and 3). PLoS Genetics | www.plosgenetics.org 2 October 2008 | Volume 4 | Issue 10 | e1000238