CALCIUM ATPASE DEFECTS AND CONGENITAL HEARING LOSS: IN VITRO AND IN VIVO STUDIES

The stereocilia of the hair cells, the sensory receptors of auditory and vestibular epithelia, contain a rare, C-terminally truncated variant of isoform 2 of the plasma membrane Calcium pump (PMCA 2). The pump ejects Ca that has entered into the cell during the conversion of mechanical stimuli into electrical signals. Genetic deafness resulting from mutations that inactivate the pump has been described in two mice strains, and has been produced by ablating the PMCA 2 gene in mice. Interestingly, a mutation in the domain of the PMCA 2 pump which induces deafness in mice has been found in a human case of genetic deafness. The project has two main correlated aims. The first is to understand the reason for the preference of stereocilia for PMCA 2: logically, this pump isoform should have properties that make it ideally suited to satisfy the special Ca regulation demands of hair cells. The truncated PMCA 2 will be overexpressed in model cells, and its ability to control their Ca will be compared to that of the other PMCA isoforms using Ca probes (aequorin). A search for protein partners, possibly involved in the isoform-specific regulation of the PMCAs, will also be performed using a yeast two-hybrid system. The second aim is to restore function in hair cells from mice in which the PMCA 2 gene has been ablated. The missing PMCA 2, but also other variants, will be reinstated in organotypic cultures of the auditory and vestibular sensory epithelia. The DNAs will be introduced into hair cells with adenoviral vectors, and function will be evaluated from the Ca dependence of mechano-transduction using the patch clamp technique combined with the direct stimulation of the hair bundles, and with the monitoring of cell Ca following stimuli that elevate it in the stereocilia. The project is expected to advance the understanding of the role of Ca in mechano-transduction, and to provide conceptual/experimental means for the future genetic therapy of hereditary deafness.