by independent digestion with SalI-EcoRI and EcoRI-KpnI and inserted into XhoI-KpnI sites of pmRFP-c1 in a three-part ligation reaction resulting in pmRFP-PMCA2wa. The construct was controlled by sequencing. Site-directed mutagenesis was carried out to obtain the mutant cloned in the appropriate vector. pmRFP- PMCA2w/a was used as target and experiments were performed according to the manufacturer’s standard protocol (Stratagene, Cedar Creek, TX) The following primers were used: Obl 59 CATCATGGACACATTTGCTTTCCTGGCCCTGGCAACA- GAGC 39 (forward) and 59 GCTCTGTTGCCAGGGCCAG- GAAAGCAAATGTGTCCATGATG 39 (reverse) Immunolocalization of the Expressed Pumps and Membrane Fluorescence Computation CHO cells were grown in Ham’s F12 medium, supplemented with 10% fetal calf serum (FCS). Before transfection, they were seeded onto 13 mm glass coverslips and allowed to grow to 50% confluence. Transfection with 3 mg of plasmid DNA (or 1.5 :1.5 mg in the case of co-transfection) was carried out with a Ca- phosphate procedure [39]. Immunocytochemistry quantified the expressed pump proteins in the plasma membrane of transfected cells. CHO cells expressing the PMCA2 variants, were stained with polyclonal isoform-specific PMCA antibody 2N (Affinity Bioreagent, Inc., Golden, CO) or a monoclonal antibody recognizing all pump isoforms (5F10, Affinity Bioreagent, Inc., Golden, CO), at a 1:100 dilution in PBS. Staining was carried out with Alexa 488 labelled anti-rabbit or anti-mouse secondary antibodies (Molecular Probes, Invitrogen Corp., Carlsbad, CA) at a 1:50 dilution in PBS. Cells were imaged on a spinning disk confocal microscope (Ultraview; Perkin-Elmer) by using a X60 oil- immersion objective at a N.A. (PlanAPo; Nikon, Tokyo, Japan). Regions of interest were selected by applying an edge-finding (Sobel) digital filter, thus limiting the analysis to plasma membrane areas. The total fluorescence intensity in membrane-delimiting regions of interest was quantified with software developed in our laboratory. For each construct fluorescence was averaged over a total of 50 cells in 3 different slides. Ca2+ Measurements with Recombinant Aequorin Transfected cytAEQ were reconstituted by incubating CHO cells for 1–3 h with 5 mM coelenterazine in Dulbecco’s modified Eagle’s medium (D-MEM) supplemented with 1% FCS, at 37uC in a 5% CO2 atmosphere. Additions to the KRB medium (1 mM CaCl2, 100 mM ATP) were made as specified in the figure legends. The experiments and luminescence calibration into [Ca2+] values were carried out according to [40]. The experiments were terminated by lysing the cells with 100 mM digitonin in a hypotonic Ca2+-rich solution (10 mM CaCl2 in H2O) to discharge the remaining aequorin pool. Briefly, a 13-mm round coverslip with the transfected cells was placed in a perfused thermostated chamber in close proximity to a low-noise photomultiplier, with a built-in amplifier discriminator. The output of the discriminator was captured by a Thorn-EMI photon-counting board and stored in an IBM-compatible computer for further analyses. Lumines- cence was calibrated off-line into [Ca2+] values by using a computer algorithm based on the Ca2+ response curve of wt aequorin. Data are reported as mean6SD. Statistical differences were evaluated by Student’s 2-tailed t-test for unpaired samples. A p value,0.01 was considered statistically significant. Preparation of Organotypic Utricle Cultures To access utricular maculae of wild type or mutant mice between postnatal day 3 (P3) and P4, the otic capsule was opened Novel PMCA2 Mutation and Hearing Loss in Mouse medially and the endolymphatic compartment of the macula cut open. The otolithic membrane was removed after 15 min incubation in dissection saline to which 0.1 g/l bacterial subtilisin (type XXIV; Sigma-Aldrich, St. Louis, MO) had been added. Dissection saline was composed of Hank’s Balanced Salt Solution (HBSS; part number H6648, Sigma-Aldrich) with 10 mM HEPES, 10.000 U/l penicillin and 25 mg/l fungizone. HBSS contained (in g/l): 0.4 KCl, 0.06 KH2PO4 (anhydrous), 0.35 NaHCO3, 8.0 NaCl, 0.048 Na2HPO4 (anhydrous), 1 D-glucose. The epithelium was fixed by Cell-Tak (BD Biosciences, Bedford, MA), mixed with 90% NaHCO3, to the lateral side of a glass capillary (1.5 mm diameter, 5 mm length), which had been previously glued to a microscope slide by a small drop of Sylgard Silicon Elastomer (Dow Corning, Wiesbaden, Germany). Cultures were preserved for one day at 37uC in a complete medium of 95% D-MEM/Ham’s F-12 (1:1) (concentration 1X, liquid form, containing L-glutamine but no HEPES; Gibco, Invitrogen Corp., Carlsbad, CA) and 5% fetal bovine serum. Immunolocalization of PMCA2 in the Stereocilia of Hair Cells in Utricle Cultures Organotypic cultures dissected from P3 wild type and mutant mice pups were maintained over night at 37uC in D-MEM/Ham’s F-12 (1:1) medium with 5% fetal bovine serum. Tissue preparations were fixed in paraformaldehyde 4% for 20 min at room temperature, rinsed in washing solution (PBS containing BSA 2%) and permeabilised with washing solution containing Triton 0.1% for 1 h at room temperature. Incubation with primary PMCA antibody 2N was carried out overnight at 4uC using a 1:100 dilution in washing solution. FITC-conjugated rabbit anti-IgG antibody (Invitrogen) was used as secondary antibody for pump detection (1:200 dilution in washing solution, 2 h at room temperature). The preparation was mounted on a coverslip and imaged up side down on an inverted spinning disk confocal microscope (Ultraview; Perkin-Elmner) using a 606 oil-immersion objective at a 1.4 N.A. (PlanApo; Nikon, Tokyo, Japan). Whole Cochlea Immunohistochemistry Cochleae dissected from P5 mice were fixed in 4% parafor- maldehyde for 20 min at room temperature, rinsed in PBS containing 2% BSA (rinse solution) and permeabilized for 1 hour at room temperature with 0.1% Triton, dissolved in rinse solution. Tissues were stained with polyclonal isoform-specific PMCA antibody 2N (Affinity Bioreagent, Inc., Golden, CO) by incubation overnight at 4uC with specific polyclonal antibodies (2.5 mg/ml) (Invitrogen) diluted in rinse solution. A FITC conjugated rabbit anti–IgG antibody (5 mg/ml, Invitrogen) was used as secondary antibody, following incubation in rinse solution for 2 hours at room temperature. F–Actin was stained by incubation for 1 hour at room temperature with rhodamine phalloidin (7 mM, Invitro- gen, R415) diluted in rinse solution. Stained samples were sandwiched between two coverslips and imaged with a 606 oil– immersion objective (NA 1.4, Plan Apo, Nikon, Tokyo, Japan) attached to an inverted microscope (Eclipse 200, Nikon) equipped with a Nipkow disk confocal scanning head (Ultraview, Perkin Elmner, USA). Confocal fluorescence images were captured with a scientific grade cooled CCD camera (Orca, Hamamatsu Photon- ics, Hamamatzu City, Shizuoka, Japan). Confocal Imaging of Organotypic Utricle Cultures Cultures were loaded with 10 mM cell permeant Fluo-4 AM (Invitrogen) for 50 min at 37uC in D-MEM supplemented with 10 mM cell permeant NP-EGTA AM (Invitrogen), 25 mM PLoS Genetics | www.plosgenetics.org 10 October 2008 | Volume 4 | Issue 10 | e1000238