Spiden Sarah L, Bortolozzi Mario, Di Leva Francesca, de Angelis Martin Hrabé, Fuchs Helmut, Lim Dmitry, Ortolano Saida, Ingham Neil J, Brini Marisa, Carafoli Ernesto, Mammano Fabio, Steel Karen P
Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridge, UK.
PLoS Genet. 2008 Oct;4(10):e1000238. doi: 10.1371/journal.pgen.1000238. Epub 2008 Oct 31.
Progressive hearing loss is common in the human population, but we have few clues to the molecular basis. Mouse mutants with progressive hearing loss offer valuable insights, and ENU (N-ethyl-N-nitrosourea) mutagenesis is a useful way of generating models. We have characterised a new ENU-induced mouse mutant, Oblivion (allele symbol Obl), showing semi-dominant inheritance of hearing impairment. Obl/+ mutants showed increasing hearing impairment from post-natal day (P)20 to P90, and loss of auditory function was followed by a corresponding base to apex progression of hair cell degeneration. Obl/Obl mutants were small, showed severe vestibular dysfunction by 2 weeks of age, and were completely deaf from birth; sensory hair cells were completely degenerate in the basal turn of the cochlea, although hair cells appeared normal in the apex. We mapped the mutation to Chromosome 6. Mutation analysis of Atp2b2 showed a missense mutation (2630C-->T) in exon 15, causing a serine to phenylalanine substitution (S877F) in transmembrane domain 6 of the PMCA2 pump, the resident Ca(2+) pump of hair cell stereocilia. Transmembrane domain mutations in these pumps generally are believed to be incompatible with normal targeting of the protein to the plasma membrane. However, analyses of hair cells in cultured utricular maculae of Obl/Obl mice and of the mutant Obl pump in model cells showed that the protein was correctly targeted to the plasma membrane. Biochemical and biophysical characterisation showed that the pump had lost a significant portion of its non-stimulated Ca(2+) exporting ability. These findings can explain the progressive loss of auditory function, and indicate the limits in our ability to predict mechanism from sequence alone.
进行性听力损失在人群中很常见，但我们对其分子基础知之甚少。患有进行性听力损失的小鼠突变体提供了有价值的见解，而ENU（N-乙基-N-亚硝基脲）诱变是生成模型的一种有用方法。我们鉴定了一种新的ENU诱导的小鼠突变体，遗忘（等位基因符号Obl），其表现出听力障碍的半显性遗传。Obl/+突变体从出生后第（P）20天到P90天听力障碍逐渐加重，听觉功能丧失后，毛细胞变性相应地从基部向顶部发展。Obl/Obl突变体体型较小，2周龄时出现严重的前庭功能障碍，出生时即完全失聪；耳蜗基部转弯处的感觉毛细胞完全退化，尽管顶部的毛细胞看起来正常。我们将该突变定位到6号染色体。对Atp2b2的突变分析显示外显子15中有一个错义突变（2630C→T），导致PMCA2泵跨膜结构域6中的丝氨酸被苯丙氨酸取代（S877F），PMCA2泵是毛细胞静纤毛中的驻留Ca(2+)泵。一般认为这些泵的跨膜结构域突变与蛋白质正常靶向质膜不兼容。然而，对Obl/Obl小鼠培养的椭圆囊斑中的毛细胞和模型细胞中的突变Obl泵的分析表明，该蛋白质正确地靶向质膜。生化和生物物理特性表明，该泵已失去了其大部分非刺激状态下的Ca(2+)输出能力。这些发现可以解释听觉功能的进行性丧失，并表明我们仅从序列预测机制的能力存在局限性。
