**3.2 Otopetrin 1 may mobilize Ca2+ for CaCO3 formation**

Otopetrin (Otop1), a protein with multiple transmembrane domains, is essential for the formation of otoconia/otolith in the inner ear (Hughes et al. 2004; Hurle et al. 2003; Sollner et al., 2004). The protein is conserved in all vertebrates, and its biochemical function was first revealed by studying the phenotypes of two mutants, the *tilted* (*tlt*) and *mergulhador* (*mlh*) mice, which carry single-point mutations in the predicted transmembrane (TM) domains (*tlt*, Ala151Glu in TM3; *mlh*, Leu408Gln in TM9) of the *Otop1* gene. Both *tlt* and *mlh* homozygous mutant mice show non-syndromic vestibular disorders caused by the absence of otoconia crystals in the utricle and saccule (Hurle et al. 2003; Zhao et al. 2008b). Those mutations in *Otop1* do not appear to affect other inner ear organs, making *tlt* and *mlh* excellent tools to investigate how Otop1 participates in the development of otoconia and in what aspects the absence of otoconia impacts balance functions.

In fish, expression of *Otop1* is in both hair cells and supporting cells before otolith seeding, but is restricted in hair cells during otolith growth (Hurle et al. 2003; Sollner et al. 2004). In mice, *Otop1* exhibits complementary mRNA expression pattern with Oc90 in the developing otocyst, and high Otop1 protein level is visible in the gelatinous membrane overlying the sensory epithelium, suggesting that it may be integral to the membrane vesicles released into the gelatinous layer (Hurle et al. 2003). However, a more recent study by Kim and colleagues using a different antibody (Kim et al. 2010) demonstrated that Otop1 is expressed in the extrastriolar epithelia of the utricle and saccule, and is specifically localized in the apical end of the supporting cells and a subset of transitional cells. They also found that the *tlt* and *mlh* mutations of Otop1 change the subcelluar localization of the mutant protein, and may underlie its function in otoconia development (Kim et al. 2011).

Both *in vitro* and *ex vivo* studies demonstrated that one of the functions of Otop1 is to modulate intra- and extracellular Ca2+ concentrations by specifically inhibiting purinergic receptor P2Y, depleting of endoplasmic reticulum Ca2+ stores and mediating influx of extracellular Ca2+ (Hughes et al. 2007; Kim et al. 2010). Under normal conditions, the concentration of Ca2+ in the mammalian endolymph is much lower than that in the perilymph and other extracellular fluids, and is insufficient to support normal growth of otoconia. Hence, Otop1 may serve as the indispensible Ca2+ source that supports otoconia mineralization.

Moreover, Otop1 may also regulate the secretion of components required for otoconia formation. In zebrafish, Otop1 was shown to affect the secretion of *starmaker*, a protein essential for otolith formation, in the sensory epithelia (Sollner et al. 2004).
