Proceedings of The Physiological Society

Durham University (2010) Proc Physiol Soc 21, C14 and PC14

Oral Communications

The distribution of plasma membrane calcium ATPase 2 in stereociliary bundles of mammalian cochlear hair cells.

J. A. Tickle1, S. Mahendrasingam1, C. M. Hackney2, R. Fettiplace3, D. N. Furness1

1. Institute for Science and Technology in Medicine, Keele University, Stoke-on-Trent, Staffordshire, United Kingdom. 2. Biomedical Science, University of Sheffield, Sheffield, Derbyshire, United Kingdom. 3. Physiology, University of Wisconsin-Madison, Madison, Wisconsin, United States.

  • Ultrathin section of an immunogold labelled OHC bundle. Note the gold particles (black dots) tend to lie over the membrane of the stereocilia, and are present on the tallest (tall) and intermediate (int) rows. The short row is not represented in this image.

The predomnant plasma membrane calcium ATPase (PMCA) within the stereociliary bundle of outer hair cells (OHC) of the mammalian cochlea is thought to be PMCA2s (Dumont et al, 2001). PMCA2a may be needed to regulate calcium in the bundle during mechanoelectrical transduction (MET), where calcium enters through the hair-cell MET channels. In rat OHC bundles there are three rows of stereocilia, short, intermediate and tall. Recently, it has been shown that MET channels are located only in the short and intermediate rows of the three (Beurg et al., 2009) probably associated with the lower end of the tip link that emanates from the stereociliary tips. We hypothesised, therefore, that PMCA2a would be at higher concentration in the short and intermediate rows. To test this hypothesis we performed post-embedding immunogold labelling on hair bundles from three regions of the rat cochlea. Two 26 day-old Sprague-Dawley rats were killed and the cochleae were removed and perfused with 4% p-formaldehyde and 0.1% glutaraldehyde in sodium phosphate buffer for 2 h, dissected, dehydrated and embedded in LR-White resin. Ultrathin sections of apical, middle and basal regions were cut and labelled with an antibody to PMCA2a and b isoforms (Abcam ab3529). The PMCA2b isoform is not thought to be expressed at high levels in the OHC. The primary antibodies were revealed by 10 or 15 nm gold-conjugated secondary antibodies. PMCA2 labelling was concentrated along the stereociliary membrane (Fig. 1) in all three rows. By counting gold particles per unit membrane length, similar PMCA2 labelling densities were found in all three rows, with a slightly, but not significantly, lower density in the tallest row (ANOVA; F = 2.019 (2,323) p>0.05). The PMCA2 labelling was present along the entire length of each stereocilium in each row, but most concentrated on the the shaft compared with the extreme tip or basal (ankle) region. The distributions were similar in all cochlear regions. These results indicate that PMCA2 is found in all three rows of stereocilia with no evidence of an overall change in density corresponding to the presence or absence of the MET channels. This implies that PMCA2 is required not only to regulate calcium entering via the MET channel but also that which diffuses into the stereocilia from other cellular regions. There will also be a contribution to rapid calcium buffering by parvalbumin beta and calbindin D28K which are present in the stereocilia, although at lower concentration than in the cell body (Hackney et al 2005).

Where applicable, experiments conform with Society ethical requirements