**3.3 PMCA2 is a critical source of Ca2+ for CaCO3 formation**

Calmodulin-sensitive plasma membrane Ca2+-ATPases (PMCAs) are vital regulators of otoconia formation by extruding Ca2+ from hair cells and thereby maintaining the appropriate Ca2+ concentration near the plasma membrane. There are four isoforms of mammalian PMCA (PMCA1-4) encoded by four distinct genes and each of them undergoes

Proteins Involved in Otoconia Formation and Maintenance 13

transepithelial transport of bicarbonate ions to the endolymph, and affect carbon

Studies suggest that TRPVs may also play an important part in fluid homeostasis of the inner ear. All TRPVs (TRPV1-6) are expressed in vestibular and cochlear sensory epithelia (Ishibashi et al. 2008; Takumida et al. 2009). In addition, TRPV4 is also present in the endolymphatic sac and presumably acts as an osmoreceptor in cell and fluid volume regulation (Kumagami et al. 2009). Both TRPV5 and TRPV6 are found in vestibular semicircular canal ducts (Yamauchi et al. 2010). In pendrin-deficient mice, the acidic vestibular endolymphatic pH is thought to inhibit the acid-sensitive TRPV5/6 calcium channels and lead to a significantly higher Ca2+ concentration in the endolymph, which may be another factor causing the formation of abnormal otoconia crystals (Nakaya et al. 2007). However, direct evidence has yet to be presented on whether TRPV-deficiency will lead to otoconia

**4. The roles of anchoring proteins in the pathogenesis of otoconia-related** 

The inner ear acellular membranes, namely the otoconial membranes in the utricule and saccule, the cupula in the ampulla, and the tectorial membrane in the cochlea, cover their corresponding sensory epithelia, have contact with the stereocilia of hair cells and thus play crutial role in mechanotransduction. In the utricle and saccule, otoconia crystals are attached to and partially embedded in a honeycomb layer above a fibrous meshwork, which are collectively called otoconial membranes, and are responsible for the site-specific anchoring of otoconia. Disruption of the otoconial membrane structure may cause the detachment and

The acellular structures of the inner ear consist of collagenous and non-collagenous glycoproteins and proteoglycans. Several types of collagen, including type II, IV, V and IX, have been identified in the mammalian tectorial membrane (Richardson et al. 1987; Slepecky et al. 1992). In the otoconial membranes, however, otolin is likely the main collagenous component. As to the noncollagenous constituents, three glycoproteins, otogelin, α-tectorin and β-tectorin, have been identified in the inner ear acellular membranes in mice to date (Cohen-Salmon et al. 1997; Legan et al. 1997). The proteoglycan in mouse otoconia is keratin

Otogelin is a glycoprotein that is present and restricted to all acellular membranes of the inner ear (Cohen-Salmon et al. 1997). At early embryonic stages, otogelin is produced by the supporting cells of the sensory epithelia of the developing vestibule and cochlea, and presents a complementary distribution pattern with Myosin VIIA, a marker of hair cells and precursors (El-Amraoui et al. 2001). At adult stages, otogelin is still expressed in the vestibular supporting cells, but become undetectable in the cochlear cells. Otogelin may be required for the attachment of the otoconial membranes and consequently site-specific anchoring of otoconia crystals. Dysfunction of otogelin in either the *Otog* knockout mice or



**3.6 Transient receptor potential vanilloids (TRPVs) may also regulate endolymph** 

In addition to CA, HCO3-

**homeostasis** 

abnormalities.

**imbalance and dizziness/vertigo** 

dislocation of otoconia and thus vestibular disorders.

sulfate proteoglycan (KSPG) (Xu et al. 2010).

incorporation into otoliths (Tohse and Mugiya 2001).

alternative exon splicing in two regions (Keeton et al. 1993). All four PMCAs are expressed in the mammalian cochlea and extrude Ca2+ from hair cell stereocilia, whereas PMCA2a, a protein encoded by *Atp2b2* gene, is the only PMCA isoform present in vestibular hair bundles (Crouch and Schulte 1996; Dumont et al. 2001; Furuta et al. 1998; Yamoah et al. 1998). Null mutation in *Atp2b2* results in the absence of otoconia and subsequent balance deficits (Kozel et al. 1998), underpinning the importance of PMCA2 in otoconial genesis.

#### **3.4 Pendrin regulates endolymph pH, composition and volume**

Pendrin, encoded by *Slc26a4*, is an anion transporter which mediates the exchange of Cl-, I-, OH-, HCO3-, or formate, across a variety of epithelia (Scott et al. 1999; Scott and Karniski 2000). In the inner ear, pendrin is primarily expressed in the endolymphatic duct and sac, the transitional epithelia adjacent to the macula of the utricle and saccule, and the external sulcus of the cochlea (Everett et al. 1999). Pendrin is critical for maintaining the appropriate anionic and ionic composition and volume of the endolymphatic fluid, presumably due to HCO3 secretion. Mutations in human *SLC26A4* are responsible for Pendred syndrome, a genetic disorder which causes early hearing loss in children (Dai et al. 2009; Luxon et al. 2003). Studies using an *Slc26a4* knockout mouse model have revealed that pendrin dysfunction can cause an enlargement and acidification of inner ear membrane labyrinth and thyroid at embryonic stages, leading to deafness, balance disorders and goiter similar to the symptoms of human Pendred syndrome (Everett et al. 2001; Kim and Wangemann 2010; Kim and Wangemann 2011). The mice have much lower endolymphatic pH, resulting in the formation of giant crystals with reduced numbers in both the utricle and saccule (Everett et al. 2001; Nakaya et al. 2007). Recently, Dror et al. have also demonstrated that a recessive missense mutation within the highly conserved region of *slc26a4* results in a mutant pendrin protein with impaired transport activity. This mutant mouse has severely abnormal mineral composition, size and shape of otoconia, i.e., giant CaCO3 crystals in the utricle at all ages, giant CaOx crystals in the saccule of older adults, and ectopic giant stones in the crista (Dror et al. 2010). Therefore, pendrin participates in otoconia formation through providing HCO3-, which is essential for forming CaCO3 crystals and for buffering the endolymphatic pH. Pendrin can also buffer pH through other anions such as formate.

#### **3.5 Carbonic anhydrase (CA) provides HCO3 and maintains appropriate pH for otoconia formation and maintenance**

CA catalyzes the hydration of CO2 to yield HCO3- and related species, and is thus thought to be important for otoconia formation by producing HCO3 and keeping appropriate endolymph pH. CA is widely present in the sensory and non-sensory epithelia of the inner ear (Lim et al. 1983; Pedrozo et al. 1997), especially the developing endolymphatic sac of mammalian embryos contain high levels of CA. Administration of acetazolamide, a CA inhibitor, in the latter tissue can decrease the luminal pH and HCO3 - concentration (Kido et al. 1991; Tsujikawa et al. 1993). Injection of acetazolamide into the yolk sac of developing chick embryos alters and inhibits normal otoconial morphogenesis (Kido et al. 1991). Activation/deactivation of macular CA under different gravity is associated with changes in otolith sizes in fish (Anken et al. 2004). Immunohistochemstry shows that CAII is coexpressed with pendrin in the same cells in the endolymphatic sac, suggesting that those two proteins may cooperate in maintaining the normal function of the endolymphatic sac (Dou et al. 2004), which is an important tissue for endolymph production.

alternative exon splicing in two regions (Keeton et al. 1993). All four PMCAs are expressed in the mammalian cochlea and extrude Ca2+ from hair cell stereocilia, whereas PMCA2a, a protein encoded by *Atp2b2* gene, is the only PMCA isoform present in vestibular hair bundles (Crouch and Schulte 1996; Dumont et al. 2001; Furuta et al. 1998; Yamoah et al. 1998). Null mutation in *Atp2b2* results in the absence of otoconia and subsequent balance deficits (Kozel et al. 1998), underpinning the importance of PMCA2 in otoconial genesis.

Pendrin, encoded by *Slc26a4*, is an anion transporter which mediates the exchange of Cl-, I-,

2000). In the inner ear, pendrin is primarily expressed in the endolymphatic duct and sac, the transitional epithelia adjacent to the macula of the utricle and saccule, and the external sulcus of the cochlea (Everett et al. 1999). Pendrin is critical for maintaining the appropriate anionic and ionic composition and volume of the endolymphatic fluid, presumably due to

 secretion. Mutations in human *SLC26A4* are responsible for Pendred syndrome, a genetic disorder which causes early hearing loss in children (Dai et al. 2009; Luxon et al. 2003). Studies using an *Slc26a4* knockout mouse model have revealed that pendrin dysfunction can cause an enlargement and acidification of inner ear membrane labyrinth and thyroid at embryonic stages, leading to deafness, balance disorders and goiter similar to the symptoms of human Pendred syndrome (Everett et al. 2001; Kim and Wangemann 2010; Kim and Wangemann 2011). The mice have much lower endolymphatic pH, resulting in the formation of giant crystals with reduced numbers in both the utricle and saccule (Everett et al. 2001; Nakaya et al. 2007). Recently, Dror et al. have also demonstrated that a recessive missense mutation within the highly conserved region of *slc26a4* results in a mutant pendrin protein with impaired transport activity. This mutant mouse has severely abnormal mineral composition, size and shape of otoconia, i.e., giant CaCO3 crystals in the utricle at all ages, giant CaOx crystals in the saccule of older adults, and ectopic giant stones in the crista (Dror et al. 2010). Therefore, pendrin participates in otoconia formation through providing HCO3-, which is essential for forming CaCO3 crystals and for buffering the endolymphatic pH.

**-**

CA catalyzes the hydration of CO2 to yield HCO3- and related species, and is thus thought to be important for otoconia formation by producing HCO3- and keeping appropriate endolymph pH. CA is widely present in the sensory and non-sensory epithelia of the inner ear (Lim et al. 1983; Pedrozo et al. 1997), especially the developing endolymphatic sac of mammalian embryos contain high levels of CA. Administration of acetazolamide, a CA inhibitor, in the latter tissue can decrease the luminal pH and HCO3- concentration (Kido et al. 1991; Tsujikawa et al. 1993). Injection of acetazolamide into the yolk sac of developing chick embryos alters and inhibits normal otoconial morphogenesis (Kido et al. 1991). Activation/deactivation of macular CA under different gravity is associated with changes in otolith sizes in fish (Anken et al. 2004). Immunohistochemstry shows that CAII is coexpressed with pendrin in the same cells in the endolymphatic sac, suggesting that those two proteins may cooperate in maintaining the normal function of the endolymphatic sac

 **and maintains appropriate pH for** 

, or formate, across a variety of epithelia (Scott et al. 1999; Scott and Karniski

**3.4 Pendrin regulates endolymph pH, composition and volume** 

Pendrin can also buffer pH through other anions such as formate.

(Dou et al. 2004), which is an important tissue for endolymph production.

**3.5 Carbonic anhydrase (CA) provides HCO3**

**otoconia formation and maintenance** 

OH-, HCO3-

HCO3-

#### **3.6 Transient receptor potential vanilloids (TRPVs) may also regulate endolymph homeostasis**

Studies suggest that TRPVs may also play an important part in fluid homeostasis of the inner ear. All TRPVs (TRPV1-6) are expressed in vestibular and cochlear sensory epithelia (Ishibashi et al. 2008; Takumida et al. 2009). In addition, TRPV4 is also present in the endolymphatic sac and presumably acts as an osmoreceptor in cell and fluid volume regulation (Kumagami et al. 2009). Both TRPV5 and TRPV6 are found in vestibular semicircular canal ducts (Yamauchi et al. 2010). In pendrin-deficient mice, the acidic vestibular endolymphatic pH is thought to inhibit the acid-sensitive TRPV5/6 calcium channels and lead to a significantly higher Ca2+ concentration in the endolymph, which may be another factor causing the formation of abnormal otoconia crystals (Nakaya et al. 2007). However, direct evidence has yet to be presented on whether TRPV-deficiency will lead to otoconia abnormalities.
