Ultrastructural Identification and Colocalization of Interacting Proteins in the Murine Cochlea by Post-Embedding Immunogold Transmission Electron Microscopy.

Verification of the presence and location of a protein within tissue can be accomplished by western blotting and immunohistochemistry, using either paraffin or frozen sections. Affinity purification by reciprocal coimmunoprecipitations using the tissue of interest can demonstrate the existence of an interacting pair of proteins. Ultimately, the ability to visualize the interaction at the cellular level is desired. Precise location(s) of interacting proteins in situ can be accomplished by ultrastructural localization with high-quality primary antibodies and small-particle-size Au-conjugated secondary antibodies. Visualization can be obtained with a transmission electron microscope fitted with a high-resolution camera permitting magnifications that exceed 2 × 10(5), and, to date, resolution capability of 20+ Mpixels, thus enabling localization of the target protein to within nanometers of the actual location. Here, we report the method by which immunolocalization at the level of the electron microscope is accomplished using the post-embedding technique, i.e., performing antibody labeling of proteins on ultrathin sections of tissue embedded in acrylic resin.

The use of 2-D gels to identify novel protein-protein interactions in the cochlea.

Functional proteomics comprises a wide range of technologies for the identification of novel protein-protein interactions and biological markers. Studies of protein-protein interactions have gained from the development of techniques and technologies such as immunoprecipitation, preparative two-dimensional (2-D) gel electrophoresis for peptide mass fingerprinting (PMF), using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). These applications enabled the discovery of putative protein partners without a priori knowledge of which one(s) might be relevant. Here, we report the methods by which membrane proteins are isolated from cochlear tissues and prepared for identification by mass spectrometry techniques.

In vivo verification of protein interactions in the inner ear by coimmunoprecipitation.

Genomics has provided us with vast amounts of data and thus, the challenge to identify and characterize gene products. Proteomics analysis, using methods such as yeast two-hybrid screenings, isoelectric focusing, and mass spectroscopy, generate potentially useful information. To determine functional relationships between and among proteins, however, the initial data for putative protein interactions must first be validated. One technique, which is considered the gold standard, is coimmunoprecipitation.

Identification and localization of an arachidonic acid-sensitive potassium channel in the cochlea.

Receptor cells of the auditory and vestibular end organs of vertebrates acquire various types of potassium channels during development. Their expression and kinetics can differ along the tonotopic axis as well as in different cell types of the sensory epithelium. These variations can play a crucial role in modulating sensory transduction and cochlear tuning. Whole-cell tight-seal recordings of isolated hair cells revealed the presence of an arachidonic acid-sensitive A-type channel in the short (outer) hair cells of the chicken cochlea. This polyunsaturated fatty acid blocked the A-current, thereby increasing the amplitude and duration of the voltage response in these cells. We identified the gene encoding this channel as belonging to a member of the Shal subfamily, Kv4.2. Expression of the recombinant channel shows half-activation and inactivation potentials shifted to more positive values relative to native channels, suggesting that the native channel is coexpressed with an accessory subunit. RT-PCR revealed that transcription begins early in development, whereas in situ hybridization showed mRNA expression limited to the intermediate and short hair cells located in specific regions of the adult cochlea. Additional localization, using immunofluorescent staining, revealed clustering in apical-lateral regions of the receptor cell as well as in the cochlear ganglion. These experiments provide evidence that in addition to membrane proteins modulating excitation in these receptor cells, fatty acids contribute to the coding of auditory stimuli via these channels.