Fig. 3
A missense mutation in Atp2b2 results in semidominant progressive hearing loss. a Sequence traces from an unaffected and an affected mouse showing the variant Atp2b2Tkh (MEBJ colony), p.R969G. b Clustal alignment from mouse, human, chicken, anole lizard, frog and zebrafish showing that the affected amino acid is highly conserved (red box). c Mean ABR thresholds from wildtype (black inverted triangles), heterozygote (blue circles) and homozygote (red triangles) mice at P28-P31 (n = 10 wildtypes, 27 heterozygotes, 9 homozygotes), P55-P58 (n = 9 wildtypes, 24 heterozygotes, 6 homozygotes) and P71-P101 (n = 9 wildtypes, 24 heterozygotes, 6 homozygotes). Error bars are standard deviations. d Jag1 and Atp2b2 qPCR on RNA from the organ of Corti at P4 (n = 6 wildtypes, 6 heterozygotes and 6 homozygotes). There was no difference between the Jag1 levels of wildtypes, heterozygotes and homozygotes (p = 0.307, Welch’s one-way ANOVA). We found a marginally significant difference in Atp2b2 levels (p = 0.036, Welch’s one-way ANOVA), but this was not borne out by the post hoc Games-Howell multiple comparison test (p = 0.052 for wildtypes compared to heterozygotes, p = 0.379 for wildtypes compared to homozygotes, p = 0.553 for heterozygotes compared to homozygotes). The bars show the mean expression levels, and error bars are standard deviations. e PMCA2 antibody stains at P4 (n = 3 wildtypes, 3 heterozygotes, 3 homozygotes), showing hair cells from the region 43% of the distance along the organ of Corti from base to apex. Images are representative examples for each genotype, and no differences were observed between genotypes. Arrowheads indicate the hair cells, red for the inner hair cell and black for the outer hair cells. Scale bar = 20 µm. f Schematic of PMCA2 protein showing the 10 transmembrane helices and the 11 known missense mutations [24, 25, 29,30,31,32,33,34, 36]). g Model of PMCA2 protein with the amino acid affected by the Tkh allele in orange. The four other missense mutations located in or near the transmembrane helices are also visible, shown in green (Obv [31]), dark red (Deaf11 [25]), olive yellow (wri [33]) and purple (M1Btlr [29]). The wildtype amino acid structures are shown for all four residues. Data underlying plots in this figure are in Additional File 3