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stereocilia organization (reviewed and suggested by Ref. 47). The first possible molecular function for myosin VI is as a transporter of cargo through the cell and the second function is to generate cell expansion force (Fig. 8). These molecular functions may act in several biological processes where myosin VI is required, i.e., pseudocleavage in embryo blastoderm, sperm individualization, and border cell migration in Drosophila, and stereocilia development in mice. In the hair cell, myosin VI may function to transport plasma membrane, thereby stabilizing the stereocilia by transporting a component that strengthens the stereocilia rootlets. This movement would occur toward actin filaments minus ends in the stereocilia rootlets. A second possible role is in generating expansion force. This is manifested as myosin VI pushing (expansion force) between adjacent stereocilia rootlets, maintaining the stereocilia in place.

Figure 8 Two proposed functions for myosin VI, based on mutant studies in Drosophila, C. elegans, and mice. (A) Myosin VI may act as a motor to transport cargo, in this case plasma membrane, toward the minus ends of actin filaments lying near the stereocilia rootlets. (B) Myosin VI may act to generate a cell expansion force, by pushing on actin filaments and causing them to slide; this ‘‘pushing’’ force will allow stereocilia to remain in their proper location. (Adapted with permission from Ref. 47.)

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VII. CONCLUDING REMARKS

Myosin VI clearly plays a crucial role in the auditory system, as mutations in this gene lead to both human and mouse hearing loss. A missense mutation in myosin VI leads to progressive hearing loss in humans, transmitted as a dominant trait, while in the Snell’s waltzer mouse model, both a frameshift mutation in myosin VI and a chromosomal inversion (with loss of putative myosin VI regulatory sequences) lead to congenital deafness, inherited in a recessive mode. Studies of model systems are providing critical information about the function of myosin VI. A number of proteins that interact with myosin VI have been identified recently [e.g., GLUT1CBP (48); DOC-2/ DAB2 (49); SAP97 (50)]. The identification of interacting molecules, coupled with phenotypic analyses and localization studies, has provided insight into myosin VI function in endocytosis and vesicle tra cking. Once more molecules are identified, we will begin to obtain a global picture of the network that myosin VI is involved in, particularly as it relates to auditory and vestibular dysfunction.

ACKNOWLEDGMENTS

The authors wish to thank the many collaborators and laboratory members who have contributed to work described in this chapter. We would like to thank Margaret Titus and Tama Sobe for reviewing the chapter. Research in the K.B.A. laboratory is supported by the European Commission (QLG2- 1999-00988), the NIH/Fogarty International Center Grant 1 R03 TW0110801, the Israel Science Foundation, the F.I.R.S.T. Foundation of the Israel Academy of Sciences and Humanities, the Israel Ministry of Health, and the Israel Ministry of Science, Culture, and Sport.

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18.Novak KD, Titus MA. The myosin I SH3 domain and TEDS rule phosphorylation site are required for in vivo function. Mol Biol Cell 1998; 9:75–88.

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21.Kellerman KA, Miller KG. An unconventional myosin heavy chain gene from Drosophila melanogaster. J Cell Biol 1992; 119:823–834.

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24.Avraham KB, Hasson T, Sobe T, Balsara B, Testa JR, Skvorak AB, Morton CC, Copeland NG, Jenkins NA. Characterization of unconventional MYO6, the human homologue of the gene responsible for deafness in Snell’s waltzer mice. Hum Mol Genet 1997; 6:1225–1231.

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16

K+-Channel Gene KCNQ4

Paul Coucke

University Hospital Ghent, Ghent, Belgium

Patrick J. Willems

GENDIA, Antwerp, Belgium

I.SUMMARY

Mutations in the K+-channel gene KCNQ4 are responsible for the most frequent form of nonsyndromic autosomal dominant hearing loss, a ecting at least nine families originating from the Netherlands (4 families), the United States (2 families), Belgium, France, and Japan. The KCNQ4-associated hearing loss is nonsyndromic, sensorineural, and progressive. It starts between the age of 5 and 15 years with high-tone loss, and deteriorates gradually over several decades a ecting also the middle and lower tones. By the age of 70 most patients have severe to profound hearing loss. The hearing impairment is completely penetrant, whereas intraand interfamilial variability is small. The KCNQ4 gene represents the DFNA2 locus on chromosome 1p34, and encodes a K+ channel that is probably involved in the K+ homeostasis in the cochlea, which is essential for normal hearing.

II.THE DFNA2 LOCUS

Until 1994 only one locus (DFNA1) for nonsyndromic hearing loss (HL) had been localized on the human genome. However, in that year three more loci (DFNA2, DFNB1, and DFNB2) were mapped, marking the start of a successful hunt for genes involved in human HL (for review, see Refs. 1–4). The DFNA2 locus was originally mapped to chromosome 1p32 by a genome

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screen in two extended families with an autosomal dominant progressive HL originating from Indonesia and the United States (5). Afterward the original chromosomal location was corrected for 1p34 (6). Seven additional families with a similar HL were later linked to the DFNA2 locus. This includes a family from Belgium (7), a family from the Netherlands (7,8), a second Dutch family (7,9), a second U.S. family (10), a third Dutch family (11,12), a Japanese family (13), and a fourth Dutch family (14; Van Camp et al., unpublished results).

Surprisingly, the DFNA2 locus was found to represent more than one gene responsible for autosomal dominant nonsyndromic progressive HL starting in the high frequencies. The gap junction protein GJB3, which encodes connexin 31 and is located in the DFNA2 linkage region, was shown to cause HL in two small Chinese families (15). Soon thereafter, mutations in the K+-channel gene KCNQ4 were identified in a small French family (16), the U.S. 1, Belgian, Dutch 1 and 2 families (17), the U.S. 2 family (10), the Dutch 3 family (12), the Japanese family (13), and the Dutch 4 family (Van Camp et al., unpublished results) (Table 1). However, in the original Indonesian family (5,7) no mutation in the KCNQ4 or GJB3 gene has yet been found despite intensive mutation analysis. This suggests that a third and unknown gene responsible for postlingual autosomal dominant HL is located in the DFNA2 region (18). The cluster of connexin genes located in the linkage region of the Indonesian family represents good candidate genes for the HL in this family (19). Another unexplained observation is the digenic inheritance pattern of a Swedish family showing both suggestive linkage to the DFNA2 locus (lod score: 2.7) and significant linkage to the DFNA12

Table 1 KCNQ4 Mutations in DFNA2 Hearing Loss

Family	Origin	Mutation	Type	Location	Ref.
1	French	G285S	Missense	Pore (exon 6)	16
2	Belgian	211del13	Deletion	Amino terminus	17
				(exon 1)
3	USA 1	G285C	Missense	Pore (exon 6)	17
4	Dutch 1	W276S	Missense	Pore (exon 5)	17
5	Dutch 2	G321S	Missense	S6 (exon 7)	17
6	USA 2	L281S	Missense	Pore (exon 6)	10
7	Dutch 3	L274H	Missense	Pore (exon 5)	12
8	Japanese	W276S	Missense	Pore (exon 5)	13
9	Dutch 4	W276S	Missense	Pore (exon 5)	Van Camp et al.,

unpublished results

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locus (lod score: 3.9) (20). Although neither of these lod scores definitively proves linkage, the concept of digenic inheritance is supported by the observation that the HL is more severe in individuals having both diseaseassociated haplotypes (21). DFNA12 HL can be caused by mutations in the alpha-tectorin (22), but it is hard to imagine an interaction between alphatectorin, on one hand, and KCNQ4 or connexin 31, on the other hand. Mutation analysis of these three genes in the Swedish family has not yet been reported, and is necessary to prove the digenic inheritance.

III.THE KCNQ4 GENE

A.Isolation and Localization

The KCNQ4 gene was identified by Kubisch et al. (16) when screening a human retina cDNA library with a cDNA clone from another member of the KCNQ gene family (KCNQ3). The gene was mapped to chromosome 1p34 by FISH, and found to be located in the immediate vicinity of the D1S432 marker (16). As the latter marker is located in the DFNA2 linkage region (5,7), the KCNQ4 gene was a good positional candidate gene for DFNA2.

B.Structure

The KCNQ4 gene is a member of a family of K+-channel subunits characterized by six transmembrane domains that anchor the protein in the cell membrane, and a single pore loop that forms the ion-specificity filter of the channel (for review, see Ref. 23). The pore loop is located between the fifth and sixth transmembrane domain, and encoded by exons 5 and 6. The fourth transmembrane domain functions as a voltage sensor. Both the short aminoand the long carboxy-terminus are located inside the cell. The KCNQ4 gene contains 14 exons encoding for 695 amino acids with a predicted molecular weight of 77 kDa (16).

C.Tissue Expression

KCNQ4 is expressed in the cochlea, the vestibular system, and the central auditory pathway (Fig. 1). In the mouse cochlea, KCNQ4 expression is found in the basal membrane of the outer hair cells, but not in the inner hair cells (24). However, Beisel et al. (25) found expression in both the inner and the outer hair cells in rat cochlea. These authors also showed that the KCNQ4 expression in inner hair cells and the spiral sensory neurons

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Figure 1 (A) The organ of Corti. KCNQ4 is expressed in the hair cells of the organ of Corti, which act as mechanoelectrical transducers transforming the mechanical movements of their stereocilia into electrical signals transmitted through the cochlear nerve to the auditory cortex. The mechanical vibrations of the endolymph produced by sound lead to deflection of the stereocilia, resulting in influx of K+ ions. These ions leave the hair cells probably through K+ channels formed by the KCNQ4 gene. They then recirculate to the spiral limbus and stria vascularis, where they are secreted back into the endolymph through K+ channels formed by KCNQ1/KCNE1. K+ homeostasis is essential for normal hearing. (B) Sound oscillations cause deflection of the stereocilia of the hair cells, leading to influx of K+ ions, resulting in depolarization of the hair cells. This leads to entry of Ca2+ ions, leading to neurotransmitter release from synaptic vesicles, which activates the acoustic nerve. Repolarization of the hair cell occurs when the K+ ions leave the hair cell through K+ channels at the basolateral side of the cell. The K ions than recirculate to the endolymph through connexins expressed in the membranes of the supporting cells. (From N Engl J Med 2000; 342:1101–1109.)

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Figure 1 Continued.

decreased from the base to the apex of the cochlea, whereas the expression in outer hair cells increases from base to apex. The KCNQ4 gene is also expressed in the vestibular organ (in type 1 vestibular hair cells and their a erent neurons), and in many nuclei of the central auditory pathway (24).

D.Function

Based on the selective expression of the KCNQ4 channel in the outer hair cells and the type 1 vestibular hair cells, Jentsch and colleges suggested that KCNQ4 generates the K+-selective ‘‘leak’’ current [termed I (K+,n) current in outer hair cells and I (K+,L) current in type 1 vestibular hair cells] (16,24). However, this was questioned by Trussell (26). Furthermore, the observation that KCNQ4 is also expressed in inner hair cells, in which this leak current is not present, argues against this hypothesis. The function of the KCNQ4 channel, therefore, is not yet completely clear. As KCNQ4 is specifically expressed at the basolateral side of hair cells (Fig. 1), it might