Figure 5. Expression and immunolocalization of recombinant PMCA2 pumps in CHO cells. (A) Western blotting analysis. Upper panel, 48 h after transfection CHO cells were washed twice with cold PBS buffer and scraped in lysis buffer (Tris-HCl 10 mM, EDTA 1 mM, PMSF 2 mM and DTT 1 mM). After centrifugation at 10006 g for 5 min, the cells were resuspended in 80 ml of lysis buffer and subjected to three cycles of freeze and thaw. The proteins of the lysates were quantified using the Bradford Reagent (Sigma-Aldrich). 15 mg of proteins were loaded on 10% polyacrylamide gel and transferred to nitrocellulose membranes which were incubated with polyclonal PMCA2 antibody 2N and monoclonal b-actin antibody (Sigma-Aldrich). After incubation with HRP-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), the blots were developed with ECL reagents (Amersham Life Science). The band of ,130 kDa corresponds to PMCA2 and the band of ,42 kDa to b-actin. Lower panel, the relative expression of wt and Obl PMCA2 was determined by scanning the immunoblots digitally with Kodak 1D Image Analysis Software. The amount of Ca2+ pump protein was normalized for b-actin and the quantity of proteins was compared. (B) Immunolocalization of wt and Obl PMCA2 in transiently transfected CHO cells. The interaction with 2N Novel PMCA2 Mutation and Hearing Loss in Mouse antibody was revealed by the AlexaFluor488-conjugated secondary antibody. Upper panel, plasma membrane pattern of the overexpressed pumps (details in Materials and Methods). (a) wild type w/a pump. (b) Obl/Obl pump. Lower panel, fluorescence level in the plasma membrane quantified as described in Material and Methods. The SDs are indicated by the bars. doi:10.1371/journal.pgen.1000238.g005 Obl/+ mice are elevated above controls, but are still recordable. An interesting observation from the ABRs recorded in P59–62 and P89–91 Obl/+ mice is that click thresholds are more sensitive than the best tone threshold, by a factor of 15–22 dB. Clicks produce a more synchronised activation of a wider region of the basilar membrane compared to tone pips, and this may produce a summation of activity, reflected in lower click-evoked ABR thresholds. When Obl was placed on a mixed C3HeJ/FeB and C57BL/6J background, the progressive hearing loss in heterozygotes seemed more severe (data not shown). Onset and severity of progressive hearing loss seen in heterozygotes of other Atp2b2 mutant alleles have been shown to vary considerably depending on the genetic background on which the mutation arose [13,21,26,28,32]. This is due to the presence of modifier alleles, one of which has been identified as the G753A variant of Cdh23 at the ahl locus (also known as modifier of deafwaddler, mdfw) [32,33]. Interactions between heterozygous Atp2b2 mutations and Cdh23 mutations have been shown to worsen the progressive hearing loss seen in some human patients. Heterozygous mutations (ATP2B2V586M) increase the severity of the progressive hearing loss seen in human patients with mutations in CDH23 [34]. The ATP2B2V586M mutation reduced the level of PMCA2 protein produced by 50%, although on its own it was not sufficient to cause hearing impairment in humans [34]. More recently, hearing loss has been reported in a human patient with an ATP2B2G293S mutation and a CDH23T1999S mutation. The parents of the patient carried either the ATP2B2 or the CDH23 mutation, but had no hearing impairment [16]. The ATP2B2 gene has recently been implicated in the deafness in 3p- syndrome. The syndrome is characterised by developmental delay, growth retardation and craniofacial abnormalities (see [35]), which is sometimes, but not always, associated with a severe sensorineural hearing loss. In 3p- syndrome patients with a hearing loss, a deletion in the 3p25.3 locus was mapped to a region containing 18 genes including ATP2B2. It is likely that haploinsufficiency of ATP2B2 is responsible for the deafness associated with this syndrome [36]. As progressive hearing loss is so common in the human population and we know so little about its molecular basis, identifying other mutations in and modifiers of the Atp2b2 locus in mouse inbred strains may be of importance in identifying new loci involved in progressive and age-related hearing loss in humans. Materials and Methods Mice The founder mouse carrying the Oblivion mutation (gene symbol Obl, original identifier DEA14) arose from the ENU mutagenesis program at Munich [17]. Mutations were generated by injecting 3 doses of 80–90 mg/kg bodyweight of N-ethyl-N- nitrosourea (ENU) into C3HeB/FeJ males. F1 progeny were screened for a range of phenotypes, including deafness and balance disorders. The founder Obl/+ mutant was identified by the absence of a Preyer reflex at 3 months. The mutant colony was maintained on the C3HeB/FeJ background, and all ABR and PLoS Genetics | www.plosgenetics.org 6 October 2008 | Volume 4 | Issue 10 | e1000238