Plasma membrane Ca2+-ATPase isoform 2a is the PMCA of hair bundles

Mechanoelectrical transduction channels of hair cells allow for the entry o f appreciable amounts of Ca2+, which regulates adaptation and triggers the mechanical activity of hair bundles. Most Ca2+ that enters transduction cha nnels is extruded by the plasma membrane Ca2+-ATPase (PMCA), a Ca2+ pump th at is highly concentrated in hair bundles and may be essential for normal h air cell function. Because PMCA isozymes and splice forms are regulated dif ferentially and have distinct biochemical properties, we determined the ide ntity of hair bundle PMCA in frog and rat hair cells. By screening a bullfr og saccular cDNA library, we identified abundant PMCA1b and PMCA2a clones a s well as rare PMCA2b and PMCA2c clones. Using immunocytochemistry and immu noprecipitation experiments, we showed in bullfrog sacculus that PMCA1b is the major isozyme of hair cell and supporting cell basolateral membranes an d that PMCA2a is the only PMCA present in hair bundles. This complete segre gation of PMCA1 and PMCA2 isozymes holds for rat auditory and vestibular ha ir cells; PMCA2a is the only PMCA isoform in hair bundles of outer hair cel ls and vestibular hair cells and is the predominant PMCA of hair bundles of inner hair cells. Our data suggest that hair cells control plasma membrane Ca2+-pumping activity by targeting specific PMCA isozymes to distinct subc ellular locations. Because PMCA2a is the only Ca2+ pump present at apprecia ble levels in hair bundles, the biochemical properties of this pump must ac count fully for the physiological features of transmembrane Ca2+ pumping in bundles.