Abstract
The phospholipid- and Ca2+-binding protein annexin A5 (ANXA5) is the most abundant membrane-associated protein of ~P23 mouse vestibular hair bundles, the inner ear’s sensory organelle. Using quantitative mass spectrometry, we estimated that ANXA5 accounts for ~15,000 copies per stereocilium, or ~2% of the total protein there. Although seven other annexin genes are expressed in mouse utricles, mass spectrometry showed that none were present at levels near ANXA5 in bundles and none were upregulated in stereocilia of Anxa5−/− mice. Annexins have been proposed to mediate Ca2+-dependent repair of membrane lesions, which could be part of the repair mechanism in hair cells after noise damage. Nevertheless, mature Anxa5−/− mice not only have normal hearing and balance function, but following noise exposure, they are identical to wild-type mice in their temporary or permanent changes in hearing sensitivity. We suggest that despite the unusually high levels of ANXA5 in bundles, it does not play a role in the bundle’s key function, mechanotransduction, at least until after two months of age in the cochlea and six months of age in the vestibular system. These results reinforce the lack of correlation between abundance of a protein in a specific compartment or cellular structure and its functional significance.
Highlights
Understanding how the sensory cells of the inner ear, hair cells, act to encode auditory or vestibular stimuli[1,2] requires deep understanding of the biochemical and physiological functions of the hundreds of proteins that make up the sensory hair bundle[3]
While annexin A5 (ANXA5) may participate in some way in hair-cell function, we saw no defects using standard vestibular and auditory tests; as these tests assess the fundamental roles of the inner ear, these negative results suggest that any function of ANXA5 in hair cells must be restricted to highly specialized conditions
While quantitation using MS1 or MS2 intensities is consistent from protein to protein only on average, our quantitative results were strengthened by the similarity of estimated ANXA5 abundance in mouse and chick utricle hair bundles
Summary
Understanding how the sensory cells of the inner ear, hair cells, act to encode auditory or vestibular stimuli[1,2] requires deep understanding of the biochemical and physiological functions of the hundreds of proteins that make up the sensory hair bundle[3]. Bundles from the chick utricle, a vestibular organ that detects linear acceleration, have ~11,000 molecules of ANXA5 (annexin A5) per stereocilium and almost no other annexins[3]. While this level makes ANXA5 the most abundant membrane-associated protein in chick bundles, its functional significance there is not known. We found that hair bundles of mouse utricles are rich in ANXA5, which was the most abundant membrane-associated protein there and whose concentration was considerably greater than that of other annexins in bundles. Because of the lack of other annexin isoforms that might compensate for the loss of ANXA5, bundles might be a good place to discern the function of this protein. ANXA5 may be present in stereocilia because of favorable environmental conditions, not because there is a need for annexins for function of this structure
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