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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 48, pp. 37693–37703, November 26, 2010 © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

The Novel PMCA2 Pump Mutation Tommy Impairs Cytosolic Calcium Clearance in Hair Cells and Links to Deafness in Mice*
Received for publication, July 30, 2010, and in revised form, September3, 2010 Published, JBC Papers in Press, September 8, 2010, DOI 10.1074/jbc.M110.170092

Mario Bortolozzi‡§1, Marisa Brini¶1, Nick Parkinson 1,2, Giulia Crispino§, Pietro Scimemi**, Romolo Daniele De Siati**, Francesca Di Leva¶3, Andrew Parker , Saida Ortolano§4, Edoardo Arslan**, Steve D. Brown 5, Ernesto Carafoli§5, and Fabio Mammano‡§5,6 From the ‡Department of Physics “G. Galilei,” Universityof Padua, Padua 35131, Italy, the §Venetian Institute of Molecular Medicine, Padua 35129, Italy, the ¶Department of Biological Chemistry, University of Padua, Padua 35121, Italy, the MRC Mammalian Genetics Unit, MRC Harwell, Oxfordshire OX11 0RD, United Kingdom, and the **Department of Medical and Surgical Specialities-Audiology and Phoniatric Service, University of Padua, Padua 35128, Italy
Themechanotransduction process in hair cells in the inner ear is associated with the influx of calcium from the endolymph. Calcium is exported back to the endolymph via the splice variant w/a of the PMCA2 of the stereocilia membrane. To further investigate the role of the pump, we have identified and characterized a novel ENU-induced mouse mutation, Tommy, in the PMCA2 gene. The mutation causes anon-conservative E629K change in the second intracellular loop of the pump that harbors the active site. Tommy mice show profound hearing impairment from P18, with significant differences in hearing thresholds between wild type and heterozygotes. Expression of mutant PMCA2 in CHO cells shows calcium extrusion impairment; specifically, the long term, non-stimulated calcium extrusion activity of thepump is inhibited. Calcium extrusion was investigated directly in neonatal organotypic cultures of the utricle sensory epithelium in Tommy mice. Confocal imaging combined with flash photolysis of caged calcium showed impairment of calcium export in both Tommy heterozygotes and homozygotes. Immunofluorescence studies of the organ of Corti in homozygous Tommy mice showed a progressive base to apexdegeneration of hair cells after P40. Our results on the Tommy mutation along with previously observed interactions between cadherin-23 and PMCA2 mutations in mouse and humans underline the importance of maintaining the appropriate calcium concentrations in the endolymph to control the rigidity of cadherin and ensure the function of interstereocilia links, including tip links, of the stereociliabundle.

Deflection of the stereocilia bundle that protrudes from hair cells into the endolymph causes the opening of mechanotrans-

* This work was supported by the European Commission under the Sixth
Research Frame Program of the European Union (FP6 Integrated Project EuroHear, Grant LSHGCT20054512063) (to F. M., E. C., and S. D. B.), by the Fondazione Cariparo (Progetti di Eccellenza 2006–2007), Telethon Italy Grant GGP09137, by the Italian Ministry of Research (PRIN 2007, Grant 2007BZ4RX3_003) (to F. M.), and by Telethon Foundation Grant GGP04169 and the Italian Ministry of University and Research (PRIN 2005) (to M. Brini). Author’s Choice—Final version full access. 1 These authors are joint first authors. 2 Present address: Systems Biology Laboratory, Abingdon, UK. 3 Present address:CIBIO, University of Trento, Trento 38123, Italy. 4 Present address: Neuropathology, Patological Anatomy Department, University Hospital of Vigo, Vigo 36203, Spain. 5 Both are senior authors. 6 To whom correspondence should be addressed: via G. Orus 2, 35129, Padova, Italy. E-mail: fabio.mammano@unipd.it.

duction (MET)7 channels and influx of K and Ca2 into the cells, ensuing in graded...