We use the term “isoform” to refer to enzymes that catalyze the same reaction (e.g., PMCAs) but are produced by different genes. We adopt the naming system for full-length PMCA molecules used by Chicka and Strehler (2003)
and furthermore label splice variants with the isoform number and letter designation. Accordingly, 2w refers to the “w” splice variant of PMCA2; PMCA2w/a refers to PMCA2 with variant 2w at splice-site A and variant 2a at splice-site C. In another example, PMCA2w refers to PMCA2 with 2w at splice-site A and either variant at splice-site C.
Generating antibodies against PMCA2 splice-site A variants
Polyclonal rabbit antibodies were raised against human PMCA2 peptides with C-terminal cysteine residues (PMCA2w: GDGLQLPAADGAAASNAADSC, amino acids 307–326; PMCA2z: DDKKAKQQDGAAAMC, amino acids 300–312). Peptides were synthesized in the Mayo Clinic Protein Core facility (Rochester, MN) and used to immunize two rabbits each (Cocalico Biologicals, Reamstown, PA). Antibodies were affinity purified using positive and negative selection against peptides corresponding to rat PMCA2w (GDGLQLPAADGAAPANAAGSC), PMCA2x (KKGKMQ-GGGC), and PMCA2z (DKKAKQGGGC) using methods described previously (Dumont et al., 2001
). Antibodies used here are listed in .
Constructing PMCA2a variant expression vectors
The coding region of PMCA2z/a was amplified with Phusion DNA polymerase (MJ Research, Waltham, MA) from pMT2-PMCA2z/a (gift from Dr. J. Penniston, Mayo Clinic, Rochester, MN), creating a 5′ Xba
I site and a 3′ Hin
d III site. PMCA2z/a was subcloned into the Xba
I and Hin
d III sites of pJPA5 (gift from Dr. J. Adelman, Oregon Health & Science University, Portland, OR), which contained an N-terminal C8 epitope tag [PRGPDRPEGIEE (Abacioglu et al., 1994
)] downstream from the cytomegalovirus promoter. To create PMCA2w/a, we used site-directed mutagenesis to create unique Pac
I and Nhe
I sites flanking splice-site A. Overlapping oligonucleotides were synthesized, annealed, and amplified to create the 2w splice sequence. After the products were introduced into the Pac
I and Nhe
I sites of PMCA2z/a, the introduced restriction sites were then reverted to the native sequence, creating PMCA2w/a.
Splice-site analysis by reverse transcription-PCR
Purified cochlear total RNA (RNeasy kit; Qiagen, Valencia, CA) from two adult rats was primed with random hexamers and reversed transcribed (ThermoScript RT-PCR System; Invitrogen, Carlsbad, CA) to synthesize first-strand cDNA. PMCA2 splice region A sequences were amplified by nested PCR. Primers for the initial PCR were (5′ to 3′) GGACGGATGGTGGTGACTG (R2A + 1) and AGATGGCTGTGGCGTTACC (R2A − 1); primers for the second PCR were GCTGTGGGTGTCAACTCTC (R2A + 2) and ACCACCAGCACCGTCACAC (R2A − 2). The amplification protocol comprised 4 min at 94°C, 30 cycles of 30 s at 94°C, 30 s at 55°C, 45 s at 72°C, and a final 10 min at 72°C. The DNA template for the second round of PCR consisted of 2.5 μl of first-round product. PCR products were directly cloned into pCR-TOPO vector (Invitrogen) and sequenced to confirm the splice-form identity.
Proteins were separated by SDS-PAGE and transferred to blotting membranes essentially as described previously (Dumont et al., 2001
). Under these conditions, PMCA2 migrated at ~170 kDa; treatment of samples with 50 mg/ml urea increased its migration to 140 kDa, which corresponds to the molecular mass of the protein (data not shown).
Hair bundles were purified from postnatal day 7 (P7) to P10 Wistar rat utricles by using a modified twist-off technique (Gillespie and Hudspeth, 1991
). Utricles were dissected in minimum essential medium (MEM; Invitrogen) supplemented with 25 mm
HEPES at pH 7.5, adhered to coverslips with Cell-Tak (BD Biosciences, Bedford, MA), and embedded in 4% low-melting-point agarose at 37°C. After bundles were isolated mechanically (Gillespie and Hudspeth, 1991
), they were superfused with MEM during excision. We estimated bundle recovery visually, in which one-ear equivalent corresponds to 100% of the bundles from one utricle (Gillespie and Hudspeth, 1991
). Detergent-soluble proteins were extracted from bundles and maculae separately as described previously (Dumont et al., 2001
), with slight modifications. The tissues were incubated in extraction buffer at 4°C for 30 min, gently flicking the tube every 10 min. Detergent-soluble proteins were isolated by centrifuging samples for 30 min at 12,000×g
. Extracts were incubated on ice for 4 h with 4 μ
g of affinity-purified anti-PMCA2 antibody. Immune complexes were precipitated by adding 15 μ
l of Protein-G Plus agarose (Santa Cruz Biotechnology, Santa Cruz, CA), incubating for 2 h at 4°C with gentle agitation, and centrifuging to sediment the antibody–agarose complex. After three 250 μ
l washes with extraction buffer, precipitated proteins were eluted from the agarose by incubating at 65°C for 20 min in an SDS-PAGE sample buffer that included 2% SDS and 100 mm
DTT. Sequential immunoprecipitations were performed with R2z, R2w, and NR2. Proteins from the final supernatant were precipitated with 6 vol of acetone at −20°C and solubilized with SDS-PAGE sample buffer. Immunodetection of PMCA (Dumont et al., 2001
) used pan-PMCA monoclonal antibody 5F10 (Affinity Bioreagents, Golden, CO) diluted to 1:5000, followed by a 1:20,000 dilution of peroxidase-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA).
For immunoprecipitation from COS-7 cells, cells were grown to ~90% confluency in 6-well plates (Fisher Scientific, Pittsburgh, PA) using DMEM (Invitrogen) supplemented with 10% fetal bovine serum, 10 U/ml penicillin, and 10 μ
g/ml streptomycin. Cells were transfected with 1.5 μ
g of pJPA5-PMCA2a variant DNA using 6 μ
l of Fugene 6 (Roche Applied Science, Indianapolis, IN). After 48 h, the cells were washed twice in cold PBS and lysed as described for tissue. Detergent-soluble proteins were isolated by centrifuging samples for 30 min at 12,000 × g
. Extracts were incubated overnight at 4°C with 2 μ
g of affinity-purified R2w, R2z, mouse monoclonal anti-C8 (gift from Dr. T. Strassmaier, Oregon Health & Science University, Portland, OR), or F2a, an antibody specific for the 2a splice form of PMCA2 (Dumont et al., 2001
). Immune complexes were precipitated, eluted, and detected as described above.
COS-7 cells were grown to ~90% confluency on a coverslip in 24-well plates. Cells were transfected with 0.5 μg of pJPA5-PMCA2a variant DNA using the TransIT-COS Transfection kit (Mirus, Madison, WI). After 48 h, the cells were washed with PBS and fixed in 3% formaldehyde in PBS for 20 min. After another wash in PBS, the cells were permeabilized for 15 min with 0.2% saponin in blocking solution, which contained 3% normal donkey serum and 10 mg/ml bovine albumin serum (BSA) in PBS, and treated for 40 min with blocking solution without detergent. Cells were incubated for 2 h with primary antibody diluted in blocking solution (10 μg/ml R2w or 2.5 μg/ml NR2), washed in PBS, and incubated with 7.5 μg/ml Cy3-conjugated donkey anti-rabbit IgG (Invitrogen, Eugene, OR) in blocking solution for 2 h. Cells were washed in PBS, and the coverslips were mounted with Vectashield (Vector Laboratories, Burlingame, CA) and viewed with a Plan Apo 60× (numerical aperture, 1.40) oil lens on a Nikon Eclipse E800 upright microscope equipped with a Photometrics CoolSnap CCD camera and MetaMorph Imaging system (version 6.1r4; Molecular Devices, Sunnyvale, CA).
Auditory and vestibular organs from P21–P23 rats were dissected in MEM supplemented with 25 mm HEPES, pH 7.5. Tissues were fixed, permeabilized, and blocked as described for COS cells. Tissues were incubated overnight with primary antibody (1:500 5F10 ascites and 10 μg/ml R2w) diluted in blocking solution, washed in PBS, and incubated with secondary antibodies (7.5 μg/ml Cy3-conjugated donkey anti-rabbit IgG and Cy5-conjugated donkey anti-mouse IgG; Invitrogen) and 0.25 μm FITC-phalloidin (Sigma, St. Louis, MO) in the blocking solution for 2 h. In some experiments, the R2w antibody was incubated with 50 μg/ml antigenic peptide for 1 h before application to tissue. Tissues were washed in PBS, mounted with Vectashield, and viewed with a Plan Apo 60× (numerical aperture, 1.40) oil lens on a Nikon TE 300 inverted microscope with a Bio-Rad (Hercules, CA) 1024 confocal imaging system. Acquired images were processed with ImageJ (version 1.32j) and Photoshop (version 7.0; Adobe Systems, San Jose, CA).
Culturing and transfecting sensory epithelia
Inner-ear sensory epithelia were harvested from P2–P3 rat pups in DMEM/F-12 (Invitrogen). The cochlea was dissected away from the modiolus, Reissner's membrane was opened, and the tectorial membrane was removed. The organ of Corti was cut into two to three pieces. The saccule, utricle, and ampullae were also harvested, and the overlying otoconia and membranes were removed. The sensory epithelia were cultured in 24-well plates on coverslips coated with 100–200 μg/ml rat tail type I collagen (Sigma) and bathed in DMEM/F-12 supplemented with 10% FBS and 15 μg/ml penicillin; cultures were maintained at 37°C and 5% CO2. Before transfection, tissues were cultured for 24 h to allow fibroblast outgrowth; this step allows firm attachment of the epithelia to the collagen substrate.
A Helios gene gun (Bio-Rad) was used to transfect inner-ear cultures (Schneider et al., 2002
; Belyantseva et al., 2003
; Rzadzinska et al., 2004
). Gene-gun delivery of plasmids is the only established method that allows successful introduction of foreign genes into hair cells without using viral vectors (Holt, 2002
) or making transgenic mice (Gao et al., 2004
). Bullets were prepared by precipitating plasmid DNA onto 1-μ
m-diameter gold microcarriers at a ratio of 2 μ
g of DNA per 1 mg of gold particles; when transfecting two plasmids, each was precipitated at 1 μ
g/mg gold. The inner wall of Tefzel tubing was coated with the DNA-coated gold microcarriers and cut into individual bullets containing ~1 μ
g of DNA. Cartridges were loaded into the bullet chamber of the gene gun, and a blast of helium gas was used to strip the inner wall of the tubing and bombard the culture with the gold microcarriers. For each culture, a Bio-Rad diffusion screen was placed ~2–4 mm from coverslip-bound cultures; the gene-gun barrel (tip, 3–4 cm distant from the bullet exit) was placed directly against the diffusion screen, and one cartridge of DNA microcarriers was shot using a pressure of 140 psi. The gold microcarriers usually contained two plasmids, a fusion of green fluorescent protein and β
-actin (GFP-actin; Clontech, Palo Alto, CA), and one of the C8-tagged PMCA2a expression constructs. In some experiments, a single plasmid encoding GFP-PMCA2w/a was used for transfection.
Cultures were fixed 24 h after transfection using 4% formaldehyde in PBS for 25 min, permeabilized in 0.2% saponin for 15 min, and incubated for 1 h in blocking solution (3% donkey or goat serum and 10 mg/ml BSA). Tissues were incubated overnight in 2.5 μg/ml mouse monoclonal anti-C8 in blocking solution, washed in PBS, and incubated with a secondary antibody (5 μg/ml of either Cy3- or Rhodamine Red-X-conjugated donkey anti-mouse IgG) and 0.25 μm Alexa 633 or 660 phalloidin (Invitrogen). Tissues were again washed in PBS, mounted in Vectashield, and viewed by confocal microscopy as described for immunofluorescence.
Images were analyzed using ImageJ version 1.34. For quantitation of bundles or basolateral membrane, a region of interest was selected from an individual confocal section, and the mean pixel intensity was determined with the Measure tool. A similar region from an untransfected cell was measured to determine background pixel intensity. A hair-bundle region of interest encompassed the entire bundle visible in a single confocal section. For the basolateral membrane, we chose a section between the cuticular plate and nucleus; a narrow region of interest along the brightest section of the membrane was chosen. Because the density of membrane present in a single confocal section will be different in bundles and basolateral membranes, ratios are most valuable for comparison between transfection conditions, rather than for directly determining the relative PMCA concentration in the two compartments.