Much is known about the mechanotransducer (MT) channels mediating transduction in hair cells of the vertrbrate inner ear. With the use of isolated preparations, it is experimentally feasible to deliver precise mechanical stimuli to individual cells and record the ensuing transducer currents. This approach has shown that small (1-100 nm) deflections of the hair-cell stereociliary bundle are transmitted via interciliary tip links to open MT channels at the tops of the stereocilia. These channels are cation-permeable with a high selectivity for Ca(2+); two channels are thought to be localized at the lower end of the tip link, each with a large single-channel conductance that increases from the low- to high-frequency end of the cochlea. Ca(2+) influx through open channels regulates their resting open probability, which may contribute to setting the hair cell resting potential in vivo. Ca(2+) also controls transducer fast adaptation and force generation by the hair bundle, the two coupled processes increasing in speed from cochlear apex to base. The molecular intricacy of the stereocilary bundle and the transduction apparatus is reflected by the large number of single-gene mutations that are linked to sensorineural deafness, especially those in Usher syndrome. Studies of such mutants have led to the discovery of many of the molecules of the transduction complex, including the tip link and its attachments to the stereociliary core. However, the MT channel protein is still not firmly identified, nor is it known whether the channel is activated by force delivered through accessory proteins or by deformation of the lipid bilayer.

Sound stimuli elicit movement of the stereocilia that make up the hair bundle of cochlear hair cells, putting tension on the tip links connecting the stereocilia and thereby opening mechanotransducer (MT) channels. Tmc1 and Tmc2, two members of the transmembrane channel–like family, are necessary for mechanotransduction. To assess their precise role, we recorded MT currents elicited by hair bundle deflections in mice with null mutations of Tmc1, Tmc2, or both. During the first postnatal week, we observed a normal MT current in hair cells lacking Tmc1 or Tmc2; however, in the absence of both isoforms, we recorded a large MT current that was phase-shifted 180°, being evoked by displacements of the hair bundle away from its tallest edge rather than toward it as in wild-type hair cells. The anomalous MT current in hair cells lacking Tmc1 and Tmc2 was blocked by FM1-43, dihydrostreptomycin, and extracellular Ca2+ at concentrations similar to those that blocked wild type. MT channels in the double knockouts carried Ca2+ with a lower permeability than wild-type or single mutants. The MT current in double knockouts persisted during exposure to submicromolar Ca2+, even though this treatment destroyed the tip links. We conclude that the Tmc isoforms do not themselves constitute the MT channel but are essential for targeting and interaction with the tip link. Changes in the MT conductance and Ca2+ permeability observed in the absence of Tmc1 mutants may stem from loss of interaction with protein partners in the transduction complex.

Vibration of the stereociliary bundles activates calcium-permeable mechanotransducer (MT) channels to initiate sound detection in cochlear hair cells. Different regions of the cochlea respond preferentially to different acoustic frequencies, with variation in the unitary conductance of the MT channels contributing to this tonotopic organization. Although the molecular identity of the MT channel remains uncertain, two members of the transmembrane channel–like family, Tmc1 and Tmc2, are crucial to hair cell mechanotransduction. We measured MT channel current amplitude and Ca2+ permeability along the cochlea’s longitudinal (tonotopic) axis during postnatal development of wild-type mice and mice lacking Tmc1 (Tmc1−/−) or Tmc2 (Tmc2−/−). In wild-type mice older than postnatal day (P) 4, MT current amplitude increased ∼1.5-fold from cochlear apex to base in outer hair cells (OHCs) but showed little change in inner hair cells (IHCs), a pattern apparent in mutant mice during the first postnatal week. After P7, the OHC MT current in Tmc1−/− (dn) mice declined to zero, consistent with their deafness phenotype. In wild-type mice before P6, the relative Ca2+ permeability, PCa, of the OHC MT channel decreased from cochlear apex to base. This gradient in PCa was not apparent in IHCs and disappeared after P7 in OHCs. In Tmc1−/− mice, PCa in basal OHCs was larger than that in wild-type mice (to equal that of apical OHCs), whereas in Tmc2−/−, PCa in apical and basal OHCs and IHCs was decreased compared with that in wild-type mice. We postulate that differences in Ca2+ permeability reflect different subunit compositions of the MT channel determined by expression of Tmc1 and Tmc2, with the latter conferring higher PCa in IHCs and immature apical OHCs. Changes in PCa with maturation are consistent with a developmental decrease in abundance of Tmc2 in OHCs but not in IHCs.

Proteins other than TMC1 and TMC2 must contribute to the pore of the mechanotransducer channel of cochlear hair cells; an external vestibule subject to disruption in Tmc mutants may influence the channel’s properties.

Kim SH, Kim KX, Raveendran NN, Wu T, Pondugula SR, Marcus DC. Regulation of ENaC-mediated sodium transport by glucocorticoids in Reissner's membrane epithelium. 's membrane epithelium forms much of the barrier that produces and sustains the large ionic differences between cochlear endolymph and perilymph. We have reported that Reissner's membrane contributes to normal cochlear function by absorbing Na ϩ from endolymph via amiloridesensitive channels in gerbil inner ear. We used mouse Reissner's membrane to 1) identify candidate genes involved in the Na ϩ transport pathway, 2) determine whether their level of expression was regulated by the synthetic glucocorticoid dexamethasone, and 3) obtain functional evidence for the physiological importance of these genes. Transcripts were present for ␣-, ␤-, and ␥-subunits of epithelial Na ϩ channel (ENaC); corticosteroid receptors GR (glucocorticoid receptor) and MR (mineralocorticoid receptor); GR agonist regulator 11␤-hydroxysteroid dehydrogenase (HSD) type 1 (11␤-HSD1); Na ϩ transport control components SGK1, Nedd4-2, and WNKs; and K ϩ channels and Na ϩ -K ϩ -ATPase. Expression of the MR agonist regulator 11␤-HSD2 was not detected. Dexamethasone upregulated transcripts for ␣-and ␤-subunits of ENaC (ϳ6-and ϳ3-fold), KCNK1 (ϳ3-fold), 11␤-HSD1 (ϳ2-fold), SGK1 (ϳ2-fold), and WNK4 (ϳ3-fold). Transepithelial currents from the apical to the basolateral side of Reissner's membrane were sensitive to amiloride (IC50 ϳ0.7 M) and benzamil (IC50 ϳ0.1 M), but not EIPA (IC50 ϳ34 M); amilorideblocked transepithelial current was not immediately changed by forskolin/IBMX. Currents were reduced by ouabain, lowered bath Na ϩ concentration (from 150 to 120 mM), and K ϩ channel blockers (XE-991, Ba 2ϩ , and acidification from pH 7.4 to 6.5). Dexamethasone-stimulated current and gene expression were reduced by mifepristone, but not spironolactone. These molecular, pharmacological, and functional observations are consistent with Na ϩ absorption by mouse Reissner's membrane, which is mediated by apical ENaC and/or other amiloride-sensitive channels, basolateral Na ϩ -K ϩ -ATPase, and K ϩ -permeable channels and is under the control of glucocorticoids. These results provide an understanding and a molecular definition of an important transport function of Reissner's membrane epithelium in the homeostasis of cochlear endolymph.