sulfinpyrazone and Pluronic F-127 (0.1% w/v). For de-esterifica- tion, cultures were transferred to an experimental chamber mounted on the stage of a confocal imaging setup (Biorad Radiance 2100) incorporating an upright microscope (Eclipse E600FN, Nikon, Tokyo, Japan) and superfused for 20 min with a medium composed of HBSS supplemented with 4.4 g/l glucose and 2 mM anhydrous CaCl2 (pH 7.4, Osm 330). Experiments were performed with a 1006 water-immersion objective (N.A. 1.00, LUMPlanFl, Olympus, Tokyo, Japan) using the same perfusion medium. Fluo-4 fluorescence was excited by the 488 nm line of an argon laser coupled by fiber optics to the confocal microscope. Fluorescence emission was selected around 528 nm using a narrow-band (50 nm) interference filter. Fluores- cence images of utricle hair cells in the organotypic cultures were acquired with a resolution of 5126512 pixel by scanning at 512 lines per second under control of the Biorad Laser Sharp software. To be retained for subsequent recording, imaged cells had hair bundles extending for their entire length in a single confocal plane (bundle planarity condition), a possibility afforded by having the culture attached to a curved surface. Confocal Line-Scan Recording and UV Photolysis of Caged Ca2+ in Utricular Hair Cells Dynamic fluorescence data were acquired in the ‘linescan’ mode to produce a scan series of fluorescence intensity values, F, measured in the photomultiplier tube PMT units from a value of 0 to a saturating value of 255. Laser intensity and PMT gain were adjusted to accommodate the dynamic range of changes in F. Typical background fluorescence values, measured from regions devoid of obvious cellular structures, were ,1 PMT units, while pre-stimulus (basal) levels, F0, averaged over the entire length of the hair bundle, were ,8 PMT units. In all experiments pinhole aperture was adjusted to the same value, yielding confocal section with 3 mm thickness. To assay the Ca2+ extrusion activity of the PMCAs, an area of ,3000 mm2, comprising a few hair cells in the cultured utricular macula, was exposed to UV radiation generated by an air- cooled 337 nm pulsed nitrogen laser (Model VSL-337ND-S, Spectra Physics, Mountain View, CA, USA) connected to the microscope through a 600 mm optical fiber. UV light was directed onto the sample by reflection off a 400 DCLP dichromatic beam splitter (Chroma) positioned at 45u just above the microscope objective lens. A single laser pulse (4 ns) delivering a maximum of 326 mJ of energy (at the laser output) was used to photorelease Ca2 from the caged state (NP-EGTA bound to Ca2+) in the brief time interval between the 2500th and the 2501st scan line. In a typical record lasting about 120 s, 20 000 consecutive lines were acquired. Off-Line Analysis of Fluorescence Transients All data were analyzed offline on a personal computer using the Matlab 7.0 (The MathWorks, Inc., Natick, MA) software environment. Data are presented as DF/F0 where DF = F2F0. In these expressions, raw pixel values are spatial averages along the hair bundle. Maximal percent fluorescence changes, (Fmax2F0)/F0, were about 280%. To estimate the slow time constant, t, of recovery to baseline, transients peaking at DFmax, were fitted by a single exponential function during the first 10 s from the UV pulse. Data are given as mean6standard error of the mean (S.E.M.). Novel PMCA2 Mutation and Hearing Loss in Mouse Supplementary materials. Figure S1 Mutations identified to date affecting PMCA2 protein in mice. Three mutations are missense mutations leading to amino acid substitutions (dfw, wri, Obl), two are small deletions that lead to frame shift mutations and premature truncation of the PMCA2 protein (dfw2J, dfw3J), and one is a targeted allele (Atp2b2tm1Ges) [8,13,21,26]. Adapted from [21]. Found at: doi:10.1371/journal.pgen.1000238.s001 (0.29 MB TIF) Figure S2 Amino acid alignments of PMCA isoforms. Align- ment of the amino acids residues in transmembrane domain 6 of PMCA isoforms in different species (A) and of PMCAs and other P-type ATPases (B). The similarity analysis was performed using the ClustalW program. GenBank accession numbers are listed: NP_001674 (Homo sapiens), Q9ROK7 (Mus musculus), NP_036640 (Rattus norvegicus), XP_509257 (Pan troglodytes), NP_777121 (Bos taurus), NP_999517 (Sus scrofa), Q00804 (Oryctolagus cuniculus), AAK11272 (Rana catesbeiana), AAH77905 (Xenopus laevis), AAR28532 (Procambarus clarkii), AAR13013 (Stylophora pistillata), AAK68551 (Caenorhabditis elegans), XP_653525 (Entamoeba histoly- tica), AAP46286 (Trypanosoma brucei), EAL62716 (Dictyostelium discoideum), NP_849716 (Arabidopsis thaliana), AAB81284 (Paramecium tetraurelia), NP_001001323 (PMCA1), NP_068768 (PMCA3), NP_001675 (PMCA4), NP_004311 (SERCA1), NP_733765 (SERCA2), NP_777615 (SERCA3), AAF35375 (SPCA1), NP_000693 (Na+/K+ ATPase), and AAH31609 (Na+/K+ ATPase). Found at: doi:10.1371/journal.pgen.1000238.s002 (1.73 MB TIF) Table S1 Progressive hearing loss in litters from Obl/+6+/+ matings. Progressive hearing loss detected in Obl/+ mutants starts at 1 month of age (6%), with most being deaf by 2 months of age (42%). 50% offspring from Obl/+6+/+ mating are expected to show phenotype. Found at: doi:10.1371/journal.pgen.1000238.s003 (0.04 MB DOC) Table S2 Microsatellite markers polymorphic between C3HeB/ FeJ and C57BL/6J. Found at: doi:10.1371/journal.pgen.1000238.s004 (0.17 MB DOC) Table S3 Sequencing primers for Atp2b2. Found at: doi:10.1371/journal.pgen.1000238.s005 (0.07 MB DOC) Text S1 Supplementary materials. Found at: doi:10.1371/journal.pgen.1000238.s006 (0.05 MB DOC) Acknowledgments We thank Dr. Corne Kros for providing dfw mice, Dr. Agnieszka Rzadzinska for expert advice on scanning electron microscopy, Ms. Sabrina Facciolo for skilled technical assistance, Dr. Emanuel Strehler for the donation of PMCA2 clones, Dr. Manuela Zaccolo for the pCDNA3.1/ zeo-mRFP, and Ms. Michelle Fleming for genotyping the mice used for ABR recordings. Author Contributions Conceived and designed the experiments: SLS MB FDL DL SO NJI MB EC FM KPS. Performed the experiments: SLS MB FDL DL SO NJI. Analyzed the data: SLS MHdA HF NJI MB EC FM KPS. Contributed reagents/materials/analysis tools: MHdA HF. Wrote the paper: SLS NJI MB EC FM KPS. References 1. Carafoli E (1992) The Ca2+ pump of the plasma membrane. J Biol Chem 267: 2115–2118. 2. Strehler EE, Zacharias DA (2001) Role of alternative splicing in generating isoform diversity among plasma membrane calcium pumps. Physiol Rev 81: 21–50. PLoS Genetics | www.plosgenetics.org 11 October 2008 | Volume 4 | Issue 10 | e1000238