[show abstract][hide abstract] ABSTRACT: Immunocytochemical studies have shown that protocadherin-15 (PCDH15) and cadherin-23 (CDH23) are associated with tip links, structures thought to gate the mechanotransducer channels of hair cells in the sensory epithelia of the inner ear. The present report describes functional and structural analyses of hair cells from Pcdh15(av3J) (av3J), Pcdh15(av6J) (av6J) and Cdh23(v2J) (v2J) mice. The av3J and v2J mice carry point mutations that are predicted to introduce premature stop codons in the transcripts for Pcdh15 and Cdh23, respectively, and av6J mice have an in-frame deletion predicted to remove most of the 9th cadherin ectodomain from PCDH15. Severe disruption of hair-bundle morphology is observed throughout the early-postnatal cochlea in av3J/av3J and v2J/v2J mice. In contrast, only mild-to-moderate bundle disruption is evident in the av6J/av6J mice. Hair cells from av3J/av3J mice are unaffected by aminoglycosides and fail to load with [(3)H]-gentamicin or FM1-43, compounds that permeate the hair cell's mechanotransducer channels. In contrast, hair cells from av6J/av6J mice load with both FM1-43 and [(3)H]-gentamicin, and are aminoglycoside sensitive. Transducer currents can be recorded from hair cells of all three mutants but are reduced in amplitude in all mutants and have abnormal directional sensitivity in the av3J/av3J and v2J/v2J mutants. Scanning electron microscopy of early postnatal cochlear hair cells reveals tip-link like links in av6J/av6J mice, substantially reduced numbers of links in the av3J/av3J mice and virtually none in the v2J/v2J mice. Analysis of mature vestibular hair bundles reveals an absence of tip links in the av3J/av3J and v2J/v2J mice and a reduction in av6J/av6J mice. These results therefore provide genetic evidence consistent with PCDH15 and CDH23 being part of the tip-link complex and necessary for normal mechanotransduction.
PLoS ONE 01/2011; 6(4):e19183. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: We assessed the involvement of harmonin-b, a submembranous protein containing PDZ domains, in the mechanoelectrical transduction machinery of inner ear hair cells. Harmonin-b is located in the region of the upper insertion point of the tip link that joins adjacent stereocilia from different rows and that is believed to gate transducer channel(s) located in the region of the tip link's lower insertion point. In Ush1c (dfcr-2J/dfcr-2J) mutant mice defective for harmonin-b, step deflections of the hair bundle evoked transduction currents with altered speed and extent of adaptation. In utricular hair cells, hair bundle morphology and maximal transduction currents were similar to those observed in wild-type mice, but adaptation was faster and more complete. Cochlear outer hair cells displayed reduced maximal transduction currents, which may be the consequence of moderate structural anomalies of their hair bundles. Their adaptation was slower and displayed a variable extent. The latter was positively correlated with the magnitude of the maximal transduction current, but the cells that showed the largest currents could be either hyperadaptive or hypoadaptive. To interpret our observations, we used a theoretical description of mechanoelectrical transduction based on the gating spring theory and a motor model of adaptation. Simulations could account for the characteristics of transduction currents in wild-type and mutant hair cells, both vestibular and cochlear. They led us to conclude that harmonin-b operates as an intracellular link that limits adaptation and engages adaptation motors, a dual role consistent with the scaffolding property of the protein and its binding to both actin filaments and the tip link component cadherin-23.
Pflügers Archiv - European Journal of Physiology 09/2009; 459(1):115-30. · 4.87 Impact Factor
[show abstract][hide abstract] ABSTRACT: TRPML3 (mucolipin-3) belongs to one of the transient-receptor-potential (TRP) ion channel families. Mutations in the Trpml3 gene cause disorganization of the stereociliary hair bundle, structural aberrations in outer and inner hair cells and stria vascularis defects, leading to deafness in the varitint-waddler (Va) mouse. Here we refined the stereociliary localization of TRPML3 and investigated cochlear hair cell function in varitint-waddler (Va(J)) mice carrying the TRPML3<I362T/A419P> mutations. Using a TRPML3-specific antibody we detected a approximately 68 kDa protein with near-equal expression levels in cochlea and vestibule of wild-type and Va(J) mutants. At postnatal days 3 and 5, we observed abundant localization of TRPML3 at the base of stereocilia near the position of the ankle links. This stereociliary localization domain was absent in Va(J) heterozygotes and homozygotes. Electrophysiological recordings revealed reduced mechano-electrical transducer currents in hair cells from Va(J)/+ and Va(J)/Va(J) mice. Furthermore, FM1-43 uptake and [(3)H]gentamicin accumulation were decreased in hair cells in cultured organs of Corti from Va(J)/+ and Va(J)/Va(J) mice. We propose that TRPML3 plays a critical role at the ankle-link region during hair-bundle growth and that an adverse effect of mutant TRPML3 on bundle development and mechano-electrical transduction is the main cause of hearing loss in Va(J)/+ mutant mice. Outer hair cells of Va(J)/Va(J) mice additionally had depolarized resting potentials due to an inwardly rectifying leak conductance formed by the mutant channels, leading over time to hair-cell degeneration and contributing to their deafness. Our findings argue against TRPML3 being a component of the hair-cell transducer channel.
The Journal of Physiology 10/2008; 586(Pt 22):5403-18. · 4.38 Impact Factor
[show abstract][hide abstract] ABSTRACT: Homozygote varitint-waddler (Va) mice, expressing a mutant isoform (A419P) of TRPML3 (mucolipin 3), are profoundly deaf and display vestibular and pigmentation deficiencies, sterility, and perinatal lethality. Here we show that the varitint-waddler isoform of TRPML3 carrying an A419P mutation represents a constitutively active cation channel that can also be identified in native varitint-waddler hair cells as a distinct inwardly rectifying current. We hypothesize that the constitutive activation of TRPML3 occurs as a result of a helix-breaking proline substitution in transmembrane-spanning domain 5 (TM5). A proline substitution scan demonstrated that the inner third of TRPML3's TM5 is highly susceptible to proline-based kinks. Proline substitutions in TM5 of other TRP channels revealed that TRPML1, TRPML2, TRPV5, and TRPV6 display a similar susceptibility at comparable positions, whereas other TRP channels were not affected. We conclude that the molecular basis for deafness in the varitint-waddler mouse is the result of hair cell death caused by constitutive TRPML3 activity. To our knowledge, our study provides the first direct mechanistic link of a mutation in a TRP ion channel with mammalian hearing loss.
Proceedings of the National Academy of Sciences 01/2008; 104(49):19583-8. · 9.74 Impact Factor