Animal and tissue preparation
Sprague-Dawley rats and C57BL6 mice ranging in age from embryonic day 16 (E16) to young adult (6 weeks) were either bred in-house or obtained from Harlan Labs (Indianapolis, IN). Appearance of the vaginal plug was representative of gestation day 1 (E1). For embryonic and postnatal animals, the crown-to-rump lengths were measured and used to verify the developmental stage of each animal. All animals were euthanized with near-lethal intraperitoneal injections of sodium pentobarbital (Nembutal, 100 mg/kg) or by hypothermia (only animals less than 1 week). The day of birth (E21 for rats; E19 for mice) represented postnatal day 0 (P0). All experimental procedures were approved by the Animal Studies Committee and conducted according to the guidelines for Animal Research at Washington University school of Medicine.
Whole organ of Corti and hair cells RT-PCR
Following anesthesia and temporal bone removal, the organ of Corti was microdissected and immediately placed into RNA extraction solution (Qiagen, La Jolla, CA). Total RNA was isolated and resuspended according to manufacturer’s instructions. Different amounts of RNA were then used as template for RT-PCR reaction. Reverse transcription reactions using random hexamers and the Retroscript kit (Ambion, Austin, Texas) were performed at 42°C for 60 minutes. Primers were designed to amplify a unique 378-bp fragment of intracellular loop of nAChR α9 subunit sequence (GenBank Accession #NM_022930), a 280-bp fragment of intracellular loop of nAChR α10 subunit sequence (GenBank Accession #NM_022639), a 303-bp fragment of rapsyn sequence (GenBank Accession #NM009023), a 422-bp fragment of RIC-3 sequence (GenBank Accession #NM_178780), and a 220-bp or 450-bp fragment of GAPDH (Clontech). The GAPDH (G3P) primers were used as internal controls. Forward and reverse primers of each fragment were as follows: nAChR α9 (forward: 5′attcacttctgtggagc3′ and reverse: 5′ccactcgctgcccttggag3′); nAChR α10 (forward: 5′ctgcactactgtggccc3′ and reverse: 5′cttccaatcttcgtggcgg3′); rapsyn (forward: 5′gcccacaacaacgatgac3′ and reverse: 5′gacagacgagacgaaacg3′); RIC-3 (forward: 5′atggaggactgggaaggtaaaatg3′ and reverse: 5′aagagaagcacagtatgtccaa3′); MuSK (forward: 5′tgccgaaggaggaaagaatg3′ and reverse: 5′gataccacaccaggagaccc3′). Purified populations of rat OHCs were also obtained using a previously described technique (Glowatzki and Fuchs, 2000
) from the apical turns of 2-week-old rats. Typically, 200 hair cells were harvested at one time, and this population was used for subsequent molecular experiments. Different primers were also designed to amplify unique 680-bp and 222-bp fragments of rapsyn sequence. All the PCR reactions were performed at 94 °C for 5 min followed by 40 cycles of 94 °C for 30 s, 60 °C for 1 min, 72 °C for 2 min, one cycle of 72 °C for 10 min, and a 4 °C hold. Amplified PCR products were run on 1% agarose gel. Candidate bands were cut out and sequenced to confirm identity.
Cloning of full-length human α9 and α10 acetylcholine receptor subunits (complete coding sequences of nAChR α9, 1440 bp; nAChR α10, 1353 bp) were isolated by standard PCR techniques. The PCR products were then subcloned directly into the eukaryotic expression vector pcDNA4/HisMax-TOPO according to manufacturer’s instructions (Invitrogen). Full-length of rat α9 and α10 receptor subunit complete coding regions were also isolated by standard PCR and subcloned into mammalian expression vectors pEGFP-C1 (Clontech) and pCS2-Venus (a generous gift from Dr. Atsushi Miyawaki, Riken Brain Science Institute, Japan) respectively. Following subcloning, their identity and proper reading frame was confirmed. Full-length mouse rapsyn was subcloned using the eukaryotic expression vector pCI (Promega, Madison, WI, USA) kindly provided by Dr. Joe Henry Steinbach, Washington University School of Medicine, St. Louis, USA. Full-length human RIC-3 was subcloned into pcDNA3 expression vector (Invitrogen) kindly provided by Dr. Millet Treinin, Hebrew University-Hadassah Medical School, Jerusalem, Israel.
Yeast two-hybrid mating of nAChR (amino acid 322–448) α9 or α10 subunit vs. rapsyn
We used the Matchmaker Gal4 Two Hybrid System 3 (BD Biosciences, Palo Alto CA). The intracellular loops of the human nAChR subunits α9 (ICD, amino acid 322–448) and α10 (ICD, amino acid 323–416) were amplified using standard PCR technique and sub-cloned into the Clontech yeast-two-hybrid vector pGBKT7. A portion of either rat cochlear or muscle rapsyn (coiled-coil domain, CCD; amino acid 298–332) protein was amplified and subcloned into the Clontech yeast-two-hybrid vector pGADT7. Construct inserts were sequenced to be sure the fusion proteins were in-frame and that no mutations were introduced. The yeast line AH109 (Saccharomyces cerevisiae) was then co-transfected with pGADT7-rapsyn and either pGBKT7-α9 or pGBKT7-α10 using the LiAc technique according to manufacturer’s directions. Transformed yeast were then plated on the following media: SD/-Trp (selecting for the pGBD vector), CD/-Leu (selecting for the pGAD vector), SD/-Trp-Leu (selecting for co-transformants), and SD/-His-Leu-Trp (selecting for a weak two-hybrid interaction) and SD/-Ade-His-Leu-Trp (selecting for a strong two-hybrid interaction). After 5 days, growth on each plate was scored as 4+ (>1000 colonies/plate), 3+ (100–1000 colonies/plate), 2+ (11–100 colonies/plate), 1+ (1–10 colonies/plate), or 0 (no growth).
Matchmaker in vitro immunoprecipitation
In vitro immunoprecipitation (IP) was used to independently confirm putative protein–protein interactions. After detecting protein–protein interactions through the in vivo yeast two-hybrid screen, the vectors were used directly in the in vitro transcription–translation reaction that was performed by using the rabbit reticolocyte lysate system (TNT kit; Promega, Madison, WI) according to the manufacturer’s instructions. The reaction was incubated for 90 min at 30°C in the presence of S35 methionine. Proteins were transcribed and translated in vitro from positive control vectors pGBKT7-murine p53 fused with c-Myc epitope and pGADT7-SV40 large T-antigen fused with HA epitope. These vectors were also used as positive controls because murine p53 and SV40 large T-antigen associate during an in vivo co-IP and interact in vitro during a yeast two-hybrid screen (Clontech kit). Product proteins (S35-α9 + cMyc tag and S35-rapsyn + AHtag) were mixed, split into two tubes, and incubated for 1 h at room temperature. The anti-AH antibody was then added to one of the tubes and the anti-cMyc antibody to the other tube. The complex was isolated after a 1 h incubation at room temperature by binding to protein A beads. The beads were washed following the manufacturer’s instructions and resuspended in Lammli buffer, heated for 5 min at 80°C, and electrophoresed in 15% SDS-PAGE gel. Kodak (Rochester, NY) x-ray films were exposed on the dry gels and developed, or the dry gel was visualized by autoradiography. The translated products were also incubated with c-Myc or HA antibodies and protein A and analyzed by Western blot (see below).
Culture and transfection of mammalian cells
HEK293T and QT6 cells were obtained from American Type Culture Collection (ATCC). The HEK293T cells were maintained at 37°C in DMEM supplemented with 10% fetal calf serum and penicillin/streptomycin mix at 1:1000. QT6 cells were grown at 37° C in Medium-199 containing Earle’s salts supplemented with 5% fetal calf serum and 10% Tryptose phosphate broth with penicillin/streptomycin. CL4 (LLC-PK1) cells kindly provided by Dr. James Bartles (Feinberg School of Medicine, Northwestern University, Chicago, IL, USA) were grown at 37°C in MEM alpha medium 1X with L-glutamine and without ribonucleotides and deoxyribonucleotides (Gibco) containing 10% fetal bovine serum supplemented with penicillin/streptomycin. For immunofluorescence staining, cells were plated on uncoated glass coverslips (Fisher Scientific, Pittsburgh, PA) placed in 35 mm tissue culture dishes and allowed to reach relatively high density (70–90% confluency). For Western blotting and immunoprecipitation experiments (below), 3×105 cells were plated on a 6-cm dish. After 24 hours the cells were transfected with α9, α10 along with rapsyn or RIC-3 DNA constructs (~ 2 μg) in equal molar ratios. All transfections were performed with lipofectamine 2000 (Invitrogen) and transfection efficiency, estimated to be 60–85% monitored by expression of a green fluorescent protein vector (pEGFP-C1, Clontech). Cells were examined for protein expression 24–48 hour after transfection.
HEK293T and CL4 cells were harvested by rinsing the 60-mm dish twice with ice-cold Tris-buffered saline (TBS), and lysed in lysis buffer containing (50 mM Tris pH 7.6, 150 mM NaCl, 2mM Na3VO4, 1% Triton X-100 and protease inhibitor cocktail). Cells were scraped, sonicated, and lysates were transferred to Eppendorf tubes and rotated at 4°C for 45 minutes. Laemmli SDS sample buffer was added to proteins and equal amounts of cell lysates were separated on 12% SDS-PAGE. Proteins were transferred to nitrocellulose membranes (BioRad), and blocked for 1 hour with 5% milk in TBS containing 0.05% Tween-20 (TBS-T). Membranes were incubated with His antibody (Clone 4D11, mouse monoclonal IgG, Upstate, Lake Placid, NY) at a 1:1000 dilution for the detection of nAChR α9 and the immunoblots were stripped and reprobed with the actin antibody (Sigma) to ensure equal protein loading, rapsyn antibody (MA1-746, Affinity Bioreagents, Golden, CO) at a 1:1000 dilution, and sheep polyclonal anti-RIC-3a antibody (a generous gift from Dr. Christopher Connolly, University of Dundee, Scotland) at a 1:500 dilution. The blots were washed three times with TBS-T followed by incubation for 1 hour with a 1:10,000 dilution of goat anti-mouse HRP conjugate (BioRad) or rabbit anti-sheep HRP conjugate (Pierce). Membranes were subsequently washed three times (10 min each wash) with TBS-T and developed with the Super signal ECL reagent (Pierce).
Total lysates from HEK293T cells were prepared as mentioned previously. To obtain mouse cochlear lysates, cochleae were homogenized in ice-cold lysis buffer containing (50 mM Tris pH 7.6, 150 mM NaCl, 10 mM NaF, 25 mM β–glycerophosphate, 20 mM sodium pyrophosphate, 2 mM EDTA, 10% glycerol, 2 mM Na3VO4, 1% Triton X-100 and protease inhibitor cocktail). Lysates were rotated at 40C for 1 hour and centrifuged at 14,000 rpm at 4°C for 30 minutes to remove the insoluble fraction. A portion of the total lysate was saved for protein immunoblotting.
To imunoprecipitate the nAChR α9 subunit, 10 μl of anti-α9 subunit (E-17, Santa Cruz Biotechnology) antibody were added to the lysates and incubated overnight at 4°C with gentle rocking. Afterwards, 50 μl of protein A-Sepharose beads (Amersham Biosciences) was added, and the solution incubated overnight to capture the immunoprecipitates. Protein A beads were collected by centrifugation at 14,000 rpm for 5 minutes, washed three times with lysis buffer, and bound proteins eluted with 45 μl of 2X Laemmli SDS sample buffer. Proteins were then analyzed by Western blotting using antibodies against rapsyn, or RIC-3a as described above.
HEK293T and CL4 cells transiently expressing full-length of human nAChR α9/α10 + mouse rapsyn, or full-length of human nAChR α9/α10 + mouse rapsyn + human RIC-3 were plated on uncoated glass coverslips (Fisher Scientific) placed in 35 mm tissue culture dishes and allowed to reach high density. Cells expressing a green fluorescent protein EGFP were used as controls. Thirty-six hours post-transfection, cells were incubated with Alexa-Fluor 488-labeled-α-bungarotoxin (α-Bgtx) or with Alexa-Fluor 594-labeled-α-Bgtx for EGFP controls at 1:100 dilution for 1 hours at room temperature. After incubation the α-Bgtx was removed, cells were fixed with 2% paraformaldehyde, washed with TBS, and incubated with blocking buffer (TBS with 5% BSA or donkey serum). Cells were then incubated overnight with primary antibodies: α9 goat polyclonal antibody (E-17, Santa Cruz Biotechnology) at 1:100 dilution, rapsyn mouse monoclonal antibody (MA1-746, Affinity Bioreagents, Golden, CO) at 1:500 dilution, RIC-3b sheep polyclonal (a generous gift from Dr. Christopher Connolly, University of Dundee, Scotland) at 1:200 and anti-GFP mouse monoclonal antibody (mab3580, Chemicon) at 1:250 in blocking buffer containing (TBS, 2 % BSA or donkey serum). Alexa flour conjugated secondary antibodies made in donkey were used at 1:500 dilution as appropriate and incubated for 2 hours at room temperature.
To label nAChRs in the inner ear with α-Bgtx, sensory organs were isolated and immediately immersed in oxygenated Hank’s buffer and incubated with fluorescently tagged α-Bgtx following either mechanical dissociation or sectioning. For immunocytochemistry, inner ear sections or dissociated organs were fixed with 2% paraformaldehyde and incubated with primary antibodies against vesicular acetylcholine transporter (VAChT) and calbindin (hair cell marker, 1:2000 dilution, Swant). Most preparations were also pre- or post-labeled with fluorescent phalloidin to label the hair-cell stereocilia. Muscle tissue isolated from rat diaphragm was processed as above and used as controls.
For immunocytochemical experiments, we used a minimum of three (3) animals at each age time point. For each animal, one ear was typically used for sensory organ whole mount preparations and one ear used for Vibratome sectioning. In multiple labeling experiments, antisera were applied to serial tissue section sets that included one section for multiple labeling and single labeling control sections. Primary antisera were against rapsyn (anti-mouse, 1:500, MA1-746, Affinity Bioreagents), α9 subunit (anti-goat, 1:100, E-17, Santa Cruz Biotechnology), RIC-3b (anti-sheep, 1:200). Primary antibodies were made in 2% normal chick serum containing 0.1% Triton X-100 and applied to cochlear tissues overnight at 4°C. In certain instances, hair cell bodies were visualized by incubating cochlear tissues with antibodies against rat calbindin (1:2000 dilution, Swant), α-parvalbumin or myosin VI antibodies. Alexa flour conjugated secondary antibodies made in chick were used at 1:500 dilution as appropriate and incubated for 2 hours at room temperature to visualize immunostaining using confocal microscopy. The specificity of primary antisera was confirmed in experiments using secondary antisera in the absence of primary antibody (data not shown).
Measurement of intracellular calcium
CL4 (LLC-PK1) cells expressing full-length of human α-9/10 nAChR subunits along with mouse rapsyn and human RIC-3 were plated on uncoated glass cover slips as described previously. Cells expressing a green fluorescent protein EGFP were used as controls. Intracellular free-calcium (Ca2+
) levels were measured using a confocal laser-scanning microscope (Zeiss-BioRad, Radiance 2000 MP), interfaced to a Nikon TE300 inverted microscope, and the fluorescent Ca2+
indicators Fluo-4-AM or X-Rhod-1-AM dissolved in DMSO (Molecular Probe, Eugene, OR) as described previously (Haynes et al., 2004
; Light et al., 2003
) with some modifications. Cells were loaded for 1 hour at room temperature with either 10 μM Fluo-4-AM or X-Rhod-1-AM in HBSS assay buffer containing 1.4 mM CaCl2
(Sigma-Aldrich, St. Louis, MO). The use of these dyes permit the accurate measurement of Ca2+
signals in cells transfected with fluorescent proteins. Equal volumes of the non-ionic detergent pluronic F-127 to that of Fluo-4-AM or X-Rhod-1-AM were used to assist in the dispersion of the non-polar dye in aqueous media and improve loading. To investigate whether activation of these receptor subunits was sufficient to evoke transient Ca2+
levels, 100 μM of acetylcholine was used. Fluo-4-AM and X-Rhod-1-AM were excited at 488 or 585 nm, respectively and the fluorescence intensity of individual cells was measured within one field of view (n ≥ 20 cells). Intracellular Ca2+
levels were measured at room temperature, 24–36 hours after transfection, from cells identified under fluorescence microscopy as YFP positive. Identical imaging parameters were maintained for all conditions. All experiments were done in triplicate. Images were captured every 1–2 seconds for about 400 second interval. Maximal fluorescence (maximal cytosolic calcium flux) was obtained at the end of each observation by adding 5–100 μM ionomycin made in DMSO, followed by 10–30 mM EGTA to release Ca2+
from Fluo-4-AM or X-Rhod-1-AM to obtain minimal fluorescence (minimal cytosolic calcium flux).
Using a laser scanning confocal microscope (Zeiss, Bio-Rad, Hercules, CA, USA), cells or inner ear sensory organs were sequentially scanned at high (500–1000×) magnification, exciting the green (488 nm), red (543 nm) and far-red (637 nm) channels. Fluorescent emissions were separated with appropriate blocking and emission filters, scanned at slow (50 lines/s) scan speeds for high resolution, and independently detected with 8-bit accuracy by photomultiplier tubes using, if necessary, accumulation was performed to increase signal and reduce noise. Three-dimensional images of serially reconstructed image stacks from the confocal microscope were rendered using Volocity (v4.xx; Improvision, Lexington, MA, USA). Wholemount images in the X–Y plane were digitally rotated and viewed in the X–Z plane. Z-projections of image stacks were also performed. Single images were exported to Canvas (vX, ACD Systems, Canada) and image quality (brightness/contrast or histogram levels) was adjusted to maximize signal and minimize background.
Quantitative colocalization analysis was performed on two fluorescence channels with Volocity software using 3D image stacks obtained from the confocal microscope. Image stacks were captured sequentially to minimize any crosstalk between channels. Threshold values were determined for each channel to minimize signal background. After generating a colocalization map, the colocalization voxels are displayed (masked) in white color and the highlighted colocalization voxels merged to a summed (projected) image. The colocalization coefficients (Mx and My) were calculated for each of the two channels according to the method of Manders et al. (1993)
All data are expressed as means ± SEM. A paired student t-test was used to test for differences between transfectants as appropriate, with P< 0.05 considered significant. We also used a one-way analysis of variance (ANOVA) to test for differences among transfection groups, followed by Tukeys’ post hoc test for comparisons between groups.