Computational protocol: Hair-Cell Mechanotransduction Persists in TRP Channel Knockout Mice

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Protocol publication

[…] Expression data for all 33 mouse TRP channels were drawn from Scheffer et al., 2015, as posted on the SHIELD database (shield.hms.harvard.edu). Read counts were analyzed based on the 20207 RefSeq genes in the DNAnexus set. Pkd1l1 is not in that set, so reads for Pkd1l1 were identified by specifically querying the raw data set. [...] Organ of Corti epithelia were dissected at P3-P5 in L-15 medium (Life Technologies), placed in glass bottom Petri dishes (WPI Inc.) and cultured for 2–3 days in DMEM/F12 medium (Life Technologies) supplemented with 5% FBS and 10 mg/l ampicillin at 37°C (10% CO2) as previously described []. Experiments were performed at room temperature in L-15 containing the following inorganic salts (in mM): NaCl (137), KCl (5.4), CaCl2 (1.26), MgCl2 (1.0), Na2HPO4 (1.0), KH2PO4 (0.44), MgSO4 (0.81). Hair cells were observed with an inverted microscope (TE 2000, Nikon) using a 100X 1.3 NA oil-immersion objective lens and DIC optics. Pipettes for whole-cell patch-clamp recordings were filled with intracellular solution containing (in mM): CsCl (140), MgCl2 (2.5), Na2ATP (2.5), EGTA (1.0), HEPES (5.0). The pipette resistance was typically 4–6 MΩ when measured in the bath. Patch clamp recordings were performed with an AxoPatch 200B amplifier (Molecular Devices) controlled by pClamp 9 software package. Hair cells were held at –60 mV between the short periods of transduction recordings, when the holding potential was temporarily hyperpolarized to –90 mV. All recorded hair cells were located 35–45% away from the apex of the organ of Corti explant.Hair bundles were deflected using a stiff glass probe, fire-polished to fit the shape of the stereocilia bundle (~5–7 μm). The probe was mounted on a piezo actuator (PA 8/14 SG, Piezosystem Jena), equipped with a strain gauge sensor to provide a direct reading of the probe’s axial displacement. The piezo was driven by a custom-made amplifier, providing a rapid step deflection within ~40 μs (10–90% risetime). The angle between the axis of the probe movement and the bottom surface of a dish was kept constant at ~30 degrees. […]

Pipeline specifications

Software tools DNAnexus, pCLAMP
Applications Miscellaneous, Patch-clamp
Organisms Mus musculus