Comprehensive protein analysis of the transport system in a connective tissue of the inner ear

The cochlea in the inner ear is filled with a unique extracellular solution that exhibits 150 mM [K+] and a highly positive potential of +80 mV. This called 'endolymph' also shows stable pH of 7.5 and osmolarity of 320 mOsm. These properties are essential for hearing and likely to be maintained by transport of not only ions but also bioactive substances including hormones and amino acids across cochlear lateral wall. The lateral wall is made up of two different components, an epithelial-like tissue, stria vascularis, and a connective tissue, spiral ligament. In our earlier work, we comprehensively analyzed membrane proteins in the stria with mass spectrometry (MS) and described a profile of the transporters and ion channels (Uetsuka et al., Eur J Neurosci 42:1984-2002, 2015). Because molecular architecture of the transport system in the ligament remains uncertain, in the present study we examined this tissue in rats with liquid chromatography (LC)-MS/MS. Spiral ligament is tightly attached to its neighbour, stria vascularis. In the first experimental process we perfused a biotin-containing solution to the surface of the ligament to selectively label the membrane proteins. Thereafter we extracted the proteins and purified the biotinylated fractions with affinity beads. LC-MS analysis of the samples identified 820 molecules. Of these, 474 proteins (58% of total) belong to membrane proteins, which contain five ion channels and 19 transporters. One of the former and four of the latter have not yet been detected in the ligament. The protein library described here may be useful to elucidate not only overall network of the transport system in the lateral wall but also pathological processes of hearing disorders.