Ionized calcium (Ca2+) acts as an intracellular signal which is essential to hearing. To manage this signal, Ca2+ channels and pumps are necessary. Recent studies have shown that the plasma membrane Ca2+ pump (PMCA), which is highly concentrated in the tips of the stereocilia, is the sole Ca2+ extrusion system in hair cells. Knockout mice lacking isoform 2 of the PMCA are deaf and show severe balance deficits. Mice of strains named "deafwaddler" and "wriggle," which have similar deficits, have recently been found to be homozygous for a mutation in the same PMCA2 gene. Based on these findings, the proposal will explore the functional properties and specific interacting proteins of the major PMCA isoform(s) of the inner ear. The specific aims are (1) to determine which PMCA ioforms and alternative splice variants are expressed in rodent cochlea and vestibular hair cells; (2) to determine the functional properties of the major "deafwaddler" and "wriggle" PMCA mutants and of additional single amino acid mutants; and (3) to identify and characterize the proteins that interact with the major PMCA ioform of the hair cells, which may be responsible for its unique localization to the stereocilia tips. These experiments will establish a new area of investigation into the role of the plasma membrane Ca2+ pump as the controller of local Ca2+ regulation in the inner ear and should lead to a better appreciation of its contribution to the process of hearing.