Corné J. Kros

Development and properties of stereociliary link types in hair cells of the mouse cochlea

The hair bundles of outer hair cells in the mature mouse cochlea possess three distinct cell‐surface specializations: tip links, horizontal top connectors, and tectorial membrane attachment crowns. Electron microscopy was used to study the appearance and maturation of these link types and examine additional structures transiently associated with the developing hair bundle. At embryonic day 17.5 (E17.5), the stereocilia are interconnected by fine lateral links and have punctate elements distributed over their surface. Oblique tip links are also seen at this stage. By postnatal day 2 (P2), outer hair cell bundles have a dense cell coat, but have lost many of the lateral links seen at E17.5. At P2, ankle links appear around the base of the bundle and tectorial membrane attachment crowns are seen at the stereociliary tips. Ankle links become less apparent by P9 and are completely lost by P12. The appearance of horizontal top connectors, which persist into adulthood, occurs concomitant with this loss of ankle links. Treatment with the calcium chelator BAPTA or the protease subtilisin enabled these links to be further distinguished. Ankle links are susceptible to both treatments, tip links are only sensitive to BAPTA, and tectorial membrane attachment crowns are removed by subtilisin but not BAPTA. The cell‐coat material is partially sensitive to subtilisin alone, while horizontal top connectors resist both treatments. These results indicate there is a rich, rapidly changing array of different links covering the developing hair bundle that becomes progressively refined to generate the mature complement by P19. J. Comp. Neurol. 485:75–85, 2005.

Increase in efficiency and reduction in Ca2+ dependence of exocytosis during development of mouse inner hair cells

Developmental changes in the coupling between Ca2+ entry and exocytosis were studied in mouse inner hair cells (IHCs) which, together with the afferent endings, form the primary synapse of the mammalian auditory system. Ca2+ currents (ICa) and changes in membrane capacitance (ΔCm) were recorded using whole‐cell voltage clamp from cells maintained at body temperature, using physiological (1.3 mm) extracellular Ca2+. The magnitudes of both ICa and ΔCm increased with maturation from embryonic stages until postnatal day 6 (P6). Subsequently, ICa gradually declined to a steady level of about −100 pA from P13 while the Ca2+‐induced ΔCm remained relatively constant, indicating a developmental increase in the Ca2+ efficiency of exocytosis. Although the size of ICa changed during development, its activation properties did not, suggesting the presence of a homogeneous population of Ca2+ channels in IHCs throughout development. The Ca2+ dependence of exocytosis changed with maturation from a fourth power relation in immature cells to an approximately linear relation in mature cells. This change applies to the release of both a readily releasable pool (RRP) and a slower secondary pool of vesicles, implying a common release mechanism for these two kinetically distinct pools that becomes modified during development. The increased Ca2+ efficiency and linear Ca2+ dependence of mature IHC exocytosis, especially over the physiological range of intracellular Ca2+, could improve the high‐fidelity transmission of both brief and long‐lasting stimulation. These properties make the mature cell ideally suited for fine intensity discrimination over a wide dynamic range.

A helix-breaking mutation in TRPML3 leads to constitutive activity underlying deafness in the varitint-waddler mouse

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 TRPML3s 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.

TRPC3 and TRPC6 are essential for normal mechanotransduction in subsets of sensory neurons and cochlear hair cells

Summary Transient receptor potential (TRP) channels TRPC3 and TRPC6 are expressed in both sensory neurons and cochlear hair cells. Deletion of TRPC3 or TRPC6 in mice caused no behavioural phenotype, although loss of TRPC3 caused a shift of rapidly adapting (RA) mechanosensitive currents to intermediate-adapting currents in dorsal root ganglion sensory neurons. Deletion of both TRPC3 and TRPC6 caused deficits in light touch and silenced half of small-diameter sensory neurons expressing mechanically activated RA currents. Double TRPC3/TRPC6 knock-out mice also showed hearing impairment, vestibular deficits and defective auditory brain stem responses to high-frequency sounds. Basal, but not apical, cochlear outer hair cells lost more than 75 per cent of their responses to mechanical stimulation. FM1-43-sensitive mechanically gated currents were induced when TRPC3 and TRPC6 were co-expressed in sensory neuron cell lines. TRPC3 and TRPC6 are thus required for the normal function of cells involved in touch and hearing, and are potential components of mechanotransducing complexes.

Position-dependent patterning of spontaneous action potentials in immature cochlear inner hair cells

Spontaneous action potential activity is crucial for mammalian sensory system development. In the auditory system, patterned firing activity has been observed in immature spiral ganglion and brain-stem neurons and is likely to depend on cochlear inner hair cell (IHC) action potentials. It remains uncertain whether spiking activity is intrinsic to developing IHCs and whether it shows patterning. We found that action potentials were intrinsically generated by immature IHCs of altricial rodents and that apical IHCs showed bursting activity as opposed to more sustained firing in basal cells. We show that the efferent neurotransmitter acetylcholine fine-tunes the IHCs resting membrane potential (Vm), and as such is crucial for the bursting pattern in apical cells. Endogenous extracellular ATP also contributes to the Vm of apical and basal IHCs by triggering small-conductance Ca2+-activated K+ (SK2) channels. We propose that the difference in firing pattern along the cochlea instructs the tonotopic differentiation of IHCs and auditory pathway.

Mutations in protocadherin 15 and cadherin 23 affect tip links and mechanotransduction in mammalian sensory hair cells

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 Pcdh15av3J (av3J), Pcdh15av6J (av6J) and Cdh23v2J (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 [3H]-gentamicin or FM1-43, compounds that permeate the hair cells mechanotransducer channels. In contrast, hair cells from av6J/av6J mice load with both FM1-43 and [3H]-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.

Effects of intracellular stores and extracellular Ca2+ on Ca2+-activated K+ currents in mature mouse inner hair cells

Ca2+‐activated K+ currents were studied in inner hair cells (IHCs) of mature mice. IK,f, the large‐conductance Ca2+‐activated K+ current (BK) characteristic of mature IHCs, had a fast activation time constant (0.4 ms at −25 mV at room temperature) and did not inactivate during 170 ms. Its amplitude, measured at −25 mV, and activation time constant were similar between IHCs in the apical and basal regions of the cochlea. IK,f was selectively blocked by 30 nm IbTx but was unaffected by superfusion of Ca2+‐free solution, nifedipine or Bay K 8644, excluding the direct involvement of voltage‐gated Ca2+ channels in IK,f activation. Increasing the intracellular concentration of the Ca2+ chelator BAPTA from 0.1 mm to 30 mm reduced the amplitude of IK,f at −25 mV and shifted its activation by 37 mV towards more depolarized potentials. A reduction in the size of IK,f and a depolarizing shift of its activation were also seen when either thapsigargin and caffeine or ryanodine were added intracellularly, suggesting that IK,f is modulated by voltage‐dependent release from intracellular Ca2+ stores. Mature IHCs had a small additional Ca2+‐activated K+ current (IK(Ca)), activated by Ca2+ flowing through L‐type Ca2+ channels. This current was still present during superfusion of either IbTx (60 nm) or apamin (300 nm) but was abolished in Cs+‐based intracellular solution or during superfusion of 5 mm TEA, suggesting the presence of an additional BK‐channel type. Current clamp experiments at body temperature show that IK,f, but not IK(Ca), is essential for fast voltage responses of mature IHCs.

Tmc1 is necessary for normal functional maturation and survival of inner and outer hair cells in the mouse cochlea

The deafness (dn) and Beethoven (Bth) mutant mice are models for profound congenital deafness (DFNB7/B11) and progressive hearing loss (DFNA36), respectively, caused by recessive and dominant mutations of transmembrane cochlear‐expressed gene 1 (TMC1), which encodes a transmembrane protein of unknown function. In the mouse cochlea Tmc1 is expressed in both outer (OHCs) and inner (IHCs) hair cells from early stages of development. Immature hair cells of mutant mice seem normal in appearance and biophysical properties. From around P8 for OHCs and P12 for IHCs, mutants fail to acquire (dn/dn) or show reduced expression (Bth/Bth and, to a lesser extent Bth/+) of the K+ currents which contribute to their normal functional maturation (the BK‐type current IK,f in IHCs, and the delayed rectifier IK,n in both cell types). Moreover, the exocytotic machinery in mutant IHCs does not develop normally as judged by the persistence of immature features of the Ca2+ current and exocytosis into adulthood. Mutant mice exhibited progressive hair cell damage and loss. The compound action potential (CAP) thresholds of Bth/+ mice were raised and correlated with the degree of hair cell loss. Homozygous mutants (dn/dn and Bth/Bth) never showed CAP responses, even at ages where many hair cells were still present in the apex of the cochlea, suggesting their hair cells never function normally. We propose that Tmc1 is involved in trafficking of molecules to the plasma membrane or serves as an intracellular regulatory signal for differentiation of immature hair cells into fully functional auditory receptors.

Harmonin-b, an actin-binding scaffold protein, is involved in the adaptation of mechanoelectrical transduction by sensory hair cells.

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 links lower insertion point. In Ush1cdfcr-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.

TRPML3 mutations cause impaired mechano-electrical transduction and depolarization by an inward-rectifier cation current in auditory hair cells of varitint-waddler mice.

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 (VaJ) mice carrying the TRPML3 mutations. Using a TRPML3‐specific antibody we detected a ∼68 kDa protein with near‐equal expression levels in cochlea and vestibule of wild‐type and VaJ 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 VaJ heterozygotes and homozygotes. Electrophysiological recordings revealed reduced mechano‐electrical transducer currents in hair cells from VaJ/+ and VaJ/VaJ mice. Furthermore, FM1‐43 uptake and [3H]gentamicin accumulation were decreased in hair cells in cultured organs of Corti from VaJ/+ and VaJ/VaJ 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 VaJ/+ mutant mice. Outer hair cells of VaJ/VaJ 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.