A large-conductance calcium-selective mechanotransducer channel in mammalian cochlear hair cells
ABSTRACT Sound stimuli are detected in the cochlea by opening of hair cell mechanotransducer (MT) channels, one of the few ion channels not yet conclusively identified at a molecular level. To define their performance in situ, we measured MT channel properties in inner hair cells (IHCs) and outer hair cells (OHCs) at two locations in the rat cochlea tuned to different characteristic frequencies (CFs). The conductance (in 0.02 mM calcium) of MT channels from IHCs was estimated as 260 pS at both low-frequency and mid-frequency positions, whereas that from OHCs increased with CFs from 145 to 210 pS. The combination of MT channel conductance and tip link number, assayed from scanning electron micrographs, accounts for variation in whole-cell current amplitude for OHCs and its invariance for IHCs. Channels from apical IHCs and OHCs having a twofold difference in unitary conductance were both highly calcium selective but were distinguishable by a small but significant difference in calcium permeability and in their response to lowering ionic strength. The results imply that the MT channel has properties possessed by few known candidates, and its diversity suggests expression of multiple isoforms.
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- "These generated ensemble averages synchronized to the displacement step and single peaks in the amplitude histogram (Fig. 8, B and D). The mean single-channel conductances were significantly different between apex and base (62 and 101 pS, respectively; P < 0.02; Table 1), consistent with a tonotopic gradient in conductance as seen in the turtle (Ricci et al., 2003) and rat (Beurg et al., 2006). No significant difference was seen between wild type and heterozygotes, or between wild type and Tmc2/ (Table 1). "
ABSTRACT: 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 Ca(2+) at concentrations similar to those that blocked wild type. MT channels in the double knockouts carried Ca(2+) with a lower permeability than wild-type or single mutants. The MT current in double knockouts persisted during exposure to submicromolar Ca(2+), 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 Ca(2+) permeability observed in the absence of Tmc1 mutants may stem from loss of interaction with protein partners in the transduction complex.The Journal of General Physiology 10/2013; 142(5). DOI:10.1085/jgp.201311068 · 4.57 Impact Factor
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- "As in other hair cell preparations (Beurg et al. 2006, 2010; Ricci et al. 1998), the MT current amplitude increased ϳ50% (mean increase ϭ 1.56 Ϯ 0.3, N ϭ 18 SHCs) when the Ca 2ϩ concentration around the hair bundle was reduced from that in perilymph (2.5 mM) to 0.24 mM, which is the concentration reported for endolymph (Sauer et al. 1999). This is indicative of a relief of block of the channel by external Ca 2ϩ , which is regarded as a permeant blocker. "
ABSTRACT: The avian auditory papilla contains two classes of sensory receptor, tall hair cells (THCs) and short hair cells (SHCs), the latter analogous to mammalian outer hair cells with large efferent but sparse afferent innervation. Little is known about the tuning, transduction or electrical properties of SHCs. To address this problem, we made patch clamp recordings from hair cells in an isolated chicken basilar papilla preparation at 33°C. We found SHCs are electrically tuned by a Ca(2+)-activated K(+) current, their resonant frequency varying along the papilla in tandem with that of the THCs, which also exhibit electrical tuning. The tonotopic map for THCs was similar to maps previously described from auditory nerve fiber measurements. SHCs also possess an A-type K(+) current, but electrical tuning was observed only at resting potentials positive to 45 mV where the A-current is inactivated. We predict the resting potential in vivo is ~ 40 mV, depolarized by a standing inward current through mechanotransducer (MT) channels having a resting open probability ~0.26. The resting open probability stems from a low endolymphatic Ca(2+) concentration (0.24 mM) and high intracellular mobile Ca(2+) buffer, estimated from perforated patch recordings as equivalent to 0.5 mM BAPTA. The high buffer concentration was confirmed by quantifying parvalbumin-3 and calbindinD-28K using calibrated post-embedding immunogold labeling, demonstrating over 1 mM calcium-binding sites. Both proteins displayed an apex-to-base gradient matching that in the MT current amplitude, which increased exponentially along the papilla. Stereociliary bundles also labeled heavily with antibodies against the Ca(2+) pump isoform PMCA2a.Journal of Neurophysiology 01/2013; 109(8). DOI:10.1152/jn.01028.2012 · 3.04 Impact Factor
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- "To date, biophysical evidence of similarities between candidate channels and the hair cell native channel have not been used to support or negate potential candidacy, with none of the above candidates having biophysical properties similar to the native hair cell (either previously known data or those presented herein). Single-channel data suggest the hair cell MET channel has multiple subtypes such that homomeric expression systems used for comparing biophysical properties of potential candidate channels may not provide data similar to those for the native channel despite the tested component being a channel subunit (Beurg et al. 2006; Ricci et al. 2003). The most recent candidates are not TRP channels; TMC1 and 2 (Kawashima et al. 2011) are viable channel candidates despite the lack of evidence to support their ability to act as ion channels. "
ABSTRACT: Mechanoelectric transducer (MET) channels, located near stereocilia tips, are opened by deflecting the hair bundle of sensory hair cells. Defects in this process result in deafness. Despite this critical function, the molecular identity of MET channels remains a mystery. Inherent channel properties, particularly those associated with permeation, provide the backbone for the molecular identification of ion channels. Here, a novel channel rectification mechanism is identified, resulting in a reduced pore size at positive potentials. The apparent difference in pore dimensions results from Ca(2+) binding within the pore, occluding permeation. Driving force for permeation at hyperpolarized potentials is increased because Ca(2+) can more easily be removed from binding within the pore due to the presence of an electronegative external vestibule that dehydrates and concentrates permeating ions. Alterations in Ca(2+) binding may underlie tonotopic and Ca(2+)-dependent variations in channel conductance. This Ca(2+)-dependent rectification provides targets for identifying the molecular components of the MET channel.Journal of Neurophysiology 02/2012; 107(9):2408-20. DOI:10.1152/jn.01178.2011 · 3.04 Impact Factor