Proceedings of The Physiological Society
Durham University (2010) Proc Physiol Soc 21, C13 and PC13
The distribution of polycystic kidney disease channels in the organ of Corti
S. Mahendrasingam2, R. Fettiplace3, C. M. Hackney1
1. Biomedical Science, University of Sheffield, Sheffield, Yorkshire, United Kingdom. 2. Institute for Science and Technology in Medicine, Keele University, Stoke-on-Trent, Staffordshire, United Kingdom. 3. Dept of Physiology, University of Wisconsin-Madison, Wisconsin, Wisconsin, United States.
Polycystic kidney disease (PKD) channels, also known as TRPP channels, mediate the detection of fluid flow in cilia of renal epithelial cells and have characteristics in common with the mechanotransduction channels in inner ear hair cells. They are non-selective cation channels sensitive to extracellular calcium ion levels that have a large unitary conductance similar to the hair cell mechanotransduction channel (Fettiplace 2009). It is possible that one of the channel forming isoforms, PKD2, PKD2L and PKD2L1, have components in common with the hair cell mechanotransduction channel. Here we investigated the distribution of post-embedding immunogold labelling for the PKD2 component in the organ of Corti with respect to the sensory hair cells. Rats were anaesthetised with an overdose of sodium pentobarbitone (0.8 ml/kg) according to individual weights. After complete loss of the pedal reflex (checked by toe pinch), they were decapitated and the cochleae excised. The cochleae and utricles of four rats that could hear (P16 and P26) were perfused with 4% paraformaldehyde and 0.1% glutaraldehyde in sodium phosphate buffer (pH 7.4), immersion fixed in the same fixative and cochlear segments were dissected out in the same buffer. These were dehydrated through an alcohol series and embedded in LR White resin at a 50oC and labelled with an antibody to the PKD channels (Bethyl Labs, USA). Immunoblots of organ of Corti were performed with the antibody on tissue extracts from the cochleae of two rats that had undergone polyacrylamide gel electrophoresis and transferred to nitrocellulose sheets. Immunoblotting showed a main band at 110 kDa, a molecular weight corresponding to the PKD2 protein. In the organ of Corti, labelling of the microtubules was also observed. These organelles transport proteins to sites where they are used by the cell so such transport recognition is a plausible hypothesis for this labelling. Labelling of stereociliary membranes was apparent in both inner and outer hair cells with some evidence of labelling towards the stereociliary tips. The membrane of the kinocilium in utricular hair bundles showed a similar density of labelling to their stereociliary membranes, so the stronger labelling of this organelle with immunofluoresence and confocal laser microscopy that has been reported by others may be accounted for by microtubular labelling. We conclude that labelling for PKD2 is present in the stereocilia and the detection of PKD2 in the inner ear warrants more physiological investigation because there would be therapeutic benefits of determining further the characteristics of stereociliary ion channels.
Where applicable, experiments conform with Society ethical requirements