The Novel Mouse Mutation Oblivion Inactivates the PMCA2 Pump and Causes Progressive Hearing Loss Sarah L. Spiden1,2., Mario Bortolozzi3., Francesca Di Leva4., Martin Hrabe´ de Angelis5, Helmut Fuchs5, Dmitry Lim3, Saida Ortolano 3 , Neil J. Ingham1, Marisa Brini4", Ernesto Carafoli3", Fabio Mammano3,6", Karen P. Steel1,2"* 1 Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridge, United Kingdom, 2 MRC Institute of Hearing Research, University Park, Nottingham, United Kingdom, 3 Venetian Institute of Molecular Medicine (VIMM), Padua, Italy, 4 Department of Biochemistry and Department of Experimental Veterinary Sciences, University of Padua, Padua, Italy, 5 Helmholtz Zentrum Mu¨ nchen, Institute of Experimental Genetics, Neuherberg, Germany, 6 Department of Physics, University of Padua, Padua, Italy Abstract 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 (2630CRT) in exon 15, causing a serine to phenylalanine substitution (S877F) in transmembrane domain 6 of the PMCA2 pump, the resident Ca2+ 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 Ca2+ 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. Citation: Spiden SL, Bortolozzi M, Di Leva F, de Angelis MH, Fuchs H, et al. (2008) The Novel Mouse Mutation Oblivion Inactivates the PMCA2 Pump and Causes Progressive Hearing Loss. PLoS Genet 4(10): e1000238. doi:10.1371/journal.pgen.1000238 Editor: Wayne N. Frankel, The Jackson Laboratory, United States of America Received February 28, 2008; Accepted September 25, 2008; Published October 31, 2008 Copyright: 2008 Spiden et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Medical Research Council, Wellcome Trust, Deafness Research UK, the EC (contracts CT97-2715, QLG2-CT-1999-00988, and EUROHEAR LSHG-CT-20054-512063), the Telethon Foundation of Italy (Project GGP04169), and the Italian Ministry of University and Research (PRIN 2005, FIRB 2001). Competing Interests: The authors have declared that no competing interests exist. * E-mail: kps@sanger.ac.uk . These authors contributed equally to this work. " These four authors are joint senior authors on this work. Introduction PMCA2 is one of four isoforms of the plasma membrane Ca2+ pumps of mammalian cells [1,2]. The expression of PMCA2 and PMCA3 is largely restricted to brain and muscle, whereas PMCA1 and 4 are ubiquitously expressed. PMCA2 and PMCA3 are more active in exporting Ca2+ than the ubiquitous isoforms [3], probably due to their higher affinity for the activator calmodulin. PMCA2, however, is peculiar in its very high activity even in the absence of calmodulin [4,5]. In the ear, PMCA2 is expressed at high levels in outer hair cell stereocilia and apical membranes and at moderate levels in inner hair cell stereocilia and in the spiral ganglion [6–10]. It actively extrudes Ca2+ that has entered the hair cell during mechanoelectrical transduction [11]. This maintains the low intracellular levels of Ca2+ and may create a relatively higher concentration of Ca2+ in the endolymph surrounding the stereocilia, contributing to the maintenance of the electrochemical gradient needed for transduction to occur [11]. Bulk concentration of Ca2+ in mammalian cochlear endolymph is estimated at ,20 mM [12]. PMCA2 is also important in maintaining sufficient extracellular Ca2+ in the vestibular system for the formation of the otoconia, the calcium carbonate crystals needed for sensing gravity and acceleration [13]. Transcripts for PMCA2 undergo alternative splicing at two sites. Site A is closer to the N-terminus and site C closer to the C- terminus. In the PMCA2 variant expressed in stereocilia [14,15], the splicing introduces three exons at site A, generating variant w, and two alternative exons at site C, generating variant a. The C- site insert leads to a truncated pump that contains only about half of the original calmodulin binding domain [4,5]. The doubly inserted w/a variant, seen in stereocilia, has an unusually limited ability to increase activity rapidly when challenged with a Ca2+ pulse, but has about the same high non-stimulated activity as the full-length z/b variant [16]. PLoS Genetics | www.plosgenetics.org 1 October 2008 | Volume 4 | Issue 10 | e1000238