Calcium entry into stereocilia drives adaptation of the mechanoelectrical transducer current of mammalian cochlear hair cells

Mechanotransduction in the auditory and vestibular systems depends on mechanosensitive ion channels in the stereociliary bundles that project from the apical surface of the sensory hair cells. In lower vertebrates, when the mechanoelectrical transducer (MET) channels are opened by movement of the bundle in the excitatory direction, Ca2+ entry through the open MET channels causes adaptation, rapidly reducing their open probability and resetting their operating range. It remains uncertain whether such Ca2+-dependent adaptation is also present in mammalian hair cells. Hair bundles of both outer and inner hair cells from mice were deflected by using sinewave or step mechanical stimuli applied using a piezo-driven fluid jet. We found that when cochlear hair cells were depolarized near the Ca2+ reversal potential or their hair bundles were exposed to the in vivo endolymphatic Ca2+ concentration (40 µM), all manifestations of adaptation, including the rapid decline of the MET current and the reduction of the available resting MET current, were abolished. MET channel adaptation was also reduced or removed when the intracellular Ca2+ buffer 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) was increased from a concentration of 0.1 to 10 mM. The findings show that MET current adaptation in mouse auditory hair cells is modulated similarly by extracellular Ca2+, intracellular Ca2+ buffering, and membrane potential, by their common effect on intracellular free Ca2+. Hearing and balance depend on the transduction of mechanical stimuli into electrical signals. This process depends on the opening of mechanoelectrical transducer (MET) channels located at the tips of the shorter of pairs of adjacent stereocilia (1), which are specialized microvilli-like structures that form the hair bundles that project from the upper surface of hair cells (2,3). Deflection of hair bundles in the excitatory direction (i.e., toward the taller stereocilia) stretches specialized linkages, the tip-links, present between adjacent stereocilia (3⇓–5), opening the MET channels. In hair cells from lower vertebrates, open MET channels reclose during constant stimuli via an initial fast adaptation mechanism followed by a much slower, myosin-based motor process, both of which are driven by Ca2+ entry through the channel itself (6⇓⇓⇓⇓⇓⇓–13). In mammalian auditory hair cells, MET current adaptation seems to be mainly driven by the fast mechanism (14⇓–16), although the exact process by which it occurs is still largely unknown. The submillisecond speed associated with the adaptation kinetics of the MET channels in rat and mouse cochlear hair cells (17, 18) indicates that Ca2+, to cause adaptation, has to interact directly with a binding site on the channel or via an accessory protein (16). However, a recent investigation on rat auditory hair cells has challenged the view that Ca2+ entry is required for fast adaptation, and instead proposed an as-yet-undefined mechanism involving a Ca2+-independent reduction in the viscoelastic force of elements in series with the MET channels (19). In the present study, we further investigated the role of Ca2+ in MET channel adaptation in mouse cochlear hair cells by deflecting their hair bundles using a piezo-driven fluid jet, which is believed to produce a more uniform deflection of the hair bundles (20⇓⇓–23) compared with the piezo-driven glass rod (19, 24)

Tmc1 point mutation affects Ca2+ sensitivity and block by dihydrostreptomycin of the mechanoelectrical transducer current of mouse outer hair cells

The transduction of sound into electrical signals depends on mechanically sensitive ion channels in the stereociliary bundle. The molecular composition of this mechanoelectrical transducer (MET) channel is not yet known. Transmembrane channel-like protein isoforms 1 (TMC1) and 2 (TMC2) have been proposed to form part of the MET channel, although their exact roles are still unclear. Using Beethoven (Tmc1Bth/Bth) mice, which have an M412K point mutation in TMC1 that adds a positive charge, we found that Ca2+ permeability and conductance of the MET channel of outer hair cells (OHCs) were reduced. Tmc1Bth/Bth OHCs were also less sensitive to block by the permeant MET channel blocker dihydrostreptomycin, whether applied extracellularly or intracellularly. These findings suggest that the amino acid that is mutated in Bth is situated at or near the negatively charged binding site for dihydrostreptomycin within the permeation pore of the channel. We also found that the Ca2+ dependence of the operating range of the MET channel was altered by the M412K mutation. Depolarization did not increase the resting open probability of the MET current of Tmc1Bth/Bth OHCs, whereas raising the intracellular concentration of the Ca2+ chelator BAPTA caused smaller increases in resting open probability in Bth mutant OHCs than in wild-type control cells. We propose that these observations can be explained by the reduced Ca2+ permeability of the mutated MET channel indirectly causing the Ca2+ sensor for adaptation, at or near the intracellular face of the MET channel, to become more sensitive to Ca2+ influx as a compensatory mechanism

TMC2 modifies permeation properties of the mechanoelectrical transducer channel in early postnatal mouse cochlear outer hair cells

The ability of cochlear hair cells to convert sound into receptor potentials relies on the mechanoelectrical transducer (MET) channels present in their stereociliary bundles. There is strong evidence implying that transmembrane channel-like protein (TMC) 1 contributes to the pore-forming subunit of the mature MET channel, yet its expression is delayed (∼>P5 in apical outer hair cells, OHCs) compared to the onset of mechanotransduction (∼P1). Instead, the temporal expression of TMC2 coincides with this onset, indicating that it could be part of the immature MET channel. We investigated MET channel properties from OHCs of homo- and heterozygous Tmc2 knockout mice. In the presence of TMC2, the MET channel blocker dihydrostreptomycin (DHS) had a lower affinity for the channel, when the aminoglycoside was applied extracellularly or intracellularly, with the latter effect being more pronounced. In Tmc2 knockout mice OHCs were protected from aminoglycoside ototoxicity during the first postnatal week, most likely due to their small MET current and the lower saturation level for aminoglycoside entry into the individual MET channels. DHS entry through the MET channels of Tmc2 knockout OHCs was lower during the first than in the second postnatal week, suggestive of a developmental change in the channel pore properties independent of TMC2. However, the ability of TMC2 to modify the MET channel properties strongly suggests it contributes to the pore-forming subunit of the neonatal channel. Nevertheless, we found that TMC2, different from TMC1, is not necessary for OHC development. While TMC2 is required for mechanotransduction in mature vestibular hair cells, its expression in the immature cochlea may be an evolutionary remnant

The acquisition of mechano-electrical transducer current adaptation in auditory hair cells requires myosin VI

Mutations in Myo6, the gene encoding the (F-actin) minus end-directed unconventional myosin, myosin VI, cause hereditary deafness in mice (Snell's waltzer) and humans. In the sensory hair cells of the cochlea, myosin VI is expressed in the cell bodies and along the stereocilia that project from the cells’ apical surface. It is required for maintaining the structural integrity of the mechanosensitive hair bundles formed by the stereocilia. In this study we investigate whether myosin VI contributes to mechano-electrical transduction. We report that Ca²+-dependent adaptation of the mechano-electrical transducer (MET) current, which serves to keep the transduction apparatus operating within its most sensitive range, is absent in outer and inner hair cells from homozygous Snell's waltzer mutant mice, which fail to express myosin VI. The operating range of the MET channels is also abnormal in the mutants, resulting in the absence of a resting MET current. We found that cadherin 23, a component of the hair bundle's transient lateral links, fails to be downregulated along the length of the stereocilia in maturing Myo6 mutant mice. MET currents of heterozygous littermates appear normal. We propose that myosin VI, by removing key molecules from developing hair bundles, is required for the development of the MET apparatus and its Ca²+-dependent adaptation

Eerste fase van de ontwikkeling van het successie model SUMO 1; verbetering van de vegetatiemodellering in de Natuurplanner

Voor de Natuurplanner van het Natuurplanbureau is het successiemodel SUMO ontwikkeld. Het vervangt de vegetatievoorspelling in het bodemmodel SMART2, waar SUMO een geontegreerd onderdeel van is. De biomassa wordt gemodelleerd voor vijf functionele typen (kruiden, dwergstruiken, struiken, pionierbomen en climaxbomen) en drie organen (wortels, tak/stam en blad). De biomassaontwikkeling is afhankelijk van de stikstof- en lichtbeschikbaarheid en het beheer. Successie (bijvoorbeeld bij het staken van beheer) vindt plaats op basis van veranderende biomassa per functioneel type. De biomassaontwikkeling en successie zijn goed te voorspellen op zowel regionale als landelijke schaal en vormen zo een goede aanvulling van de Natuurplanner

Coordinated calcium signalling in cochlear sensory and non‐sensory cells refines afferent innervation of outer hair cells

Outer hair cells (OHCs) are highly specialized sensory cells conferring the fine-tuning and high sensitivity of the mammalian cochlea to acoustic stimuli. Here, by genetically manipulating spontaneous Ca 2+ signalling in mice in vivo, through a period of early postnatal development, we find that the refinement of OHC afferent innervation is regulated by complementary spontaneous Ca 2+ signals originating in OHCs and non-sensory cells. OHCs fire spontaneous Ca 2+ action potentials during a narrow period of neonatal development. Simultaneously, waves of Ca 2+ activity in the non-sensory cells of the greater epithelial ridge cause, via ATP-induced activation of P2X 3 receptors, the increase and synchronization of the Ca 2+ activity in nearby OHCs. This synchronization is required for the refinement of their immature afferent innervation. In the absence of connexin channels, Ca 2+ waves are impaired, leading to a reduction in the number of ribbon synapses and afferent fibres on OHCs. We propose that the correct maturation of the afferent connectivity of OHCs requires experience-independent Ca 2+ signals from sensory and non-sensory cells

Presenile Alzheimer dementia characterized by amyloid angiopathy and large amyloid core type senile plaques in the APP 692 Ala => Gly mutation

Mutations at codons 717 and 670/671 in the amyloid precursor protein (APP) are rare genetic causes of familial Alzheimer's disease (AD). A mutation at codon 693 of APP has also been described as the genetic defect in hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D). We have reported a APP692Ala-->Gly (Flemish) mutation as a cause of intracerebral hemorrhage and presenile dementia diagnosed as probable AD in a Dutch family. We now describe the post-mortem examination of two demented patients with the APP692 mutation. The neuropathological findings support the diagnosis of AD. Leptomeningial and parenchymal vessels showed extensive deposition of A

Validatie van de modellen SMART2, SUMO 1, NUCOM en MOVE op site-, regionaal en national niveau

De bodem-vegetatiemodellen NUCOM, SMART2, SUMO 1 en MOVE zijn in dit onderzoek gevalideerd op drie schaalniveaus. NUCOM is een model dat de bodem- en vegetatieontwikkelingsprocessen simuleert op lokale en regionale schaal. SMART2 simuleert de bodemprocessen en is in principe schaalonafhankelijk. SUMO 1 simuleert de vegetatieontwikkeling op basis van simulaties door SMART2. MOVE voorspelt onder andere de kans op voorkomen van plantensoorten op basis van SMART2-uitkomsten. Op siteniveau, het Hulshorsterzand en Sellingen, bleken de modellen de situatie redelijk te kunnen voorspellen. Over het algemeen kwamen de biomassavoorspellingen van NUCOM iets beter overeen met de metingen dan de biomassavoorspellingen van SUMO. Op regionale schaal, de Veluwe, bleken SMART2, SUMO 1 en NUCOM de pH redelijk te voorspellen. De N-beschikbaarheid is niet gevalideerd aan de hand van metingen, maar alleen tussen de modellen onderling vergeleken. SMART2 voorspelt een kleinere range dan SUMO 1. NUCOM voorspelde voor de bossen, graslanden en heidevelden zonder beheer een hogere N-beschikbaarheid dan de twee andere modellen, terwijl er met NUCOM met beheer alleen voor heidevelden een lagere N-beschikbaarheid werd voorspeld. De door MOVE voorspelde kans op voorkomen van plantensoorten op basis van de modeluitkomsten van SMART2-SUMO 1 en NUCOM is positief gecorreleerd met de werkelijke presentie van soorten. De voorspelbaarheid van aanwezige soorten is echter vrij gering. Mogelijke oorzaken hiervan zijn een te hoge gesimuleerde N-beschikbaarheid door de simulatiemodellen, en/of een slechte omzetting naar de Ellenberg-indicatorwaarde voor nutriëntenrijkdom, die als invoer voor MOVE dient. Deze laatste oorzaak speelt waarschijnlijk de grootste rol hierbij. Op nationaal niveau zijn alleen SMART2 SUMO 1 getest. De voorspelde pH is vergeleken met een van vegetatieopnamen afgeleide pH. Voor beide modellen geldt dat de overeenkomst redelijk goed was tussen beide en de afgeleide pH