Article

Mechano-Electrical Transduction Currents in Isolated Vestibular Hair Cells of the Chick

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Abstract

Properties of a mechano-electrical transduction channel were studied in enzymatically dissociated chick vestibular hair cells by using a whole-cell recording variation of the patch voltage-clamp technique. The apical hair bundle was stimulated by a glass rod which moved along a one-dimensional axis when stimulated by either a triangular or a trapezoidal command voltage. The motion of the glass rod was monitored optically using a photodiode. In response to triangular stimuli, the hair cell generated a current of triangular wave form with occasional step-like spiky or zigzag-appearing events. Control experiments confirmed that the current was generated only when the hair bundle was displaced towards the tallest stereocilium. The mechano-sensitive current was blocked by streptomycin and by neomycin. The blockage by streptomycin was clearly voltage dependent: the reduction of the current became larger with hyperpolarization of the membrane. This suggests that the positively charged antibiotic molecules plug the mechanically gated channels. From the evidence presented in 3 and 4 above, the mechano-sensitive current recorded here was identified as the mechano-electrical transduction (m-e.t.) current. The permeability of the m-e.t. channel to various monovalent cations was determined from reversal potential measurements. Since a CsCl-EGTA intracellular medium was used, all the permeabilities were calculated relative to PCs. The sequence of permeabilities was Li greater than Na greater than or equal to K greater than or equal to Rb greater than Cs greater than choline greater than TMA greater than TEA. External Ca ions were indispensable for the recording of transduction current and Sr ions could replace Ca ions without loss of the transduction activity. The minimum [Ca]o for stable generation of the m-e.t. current was 20 microM in Cs saline. The addition of 50-200 microM-Ca to the isotonic Ba saline could maintain the m-e.t. current. The m-e.t. current was observed in isotonic Ca and in Sr salines. Isotonic Ba, Mg and Mn salines were enriched with 1-2 mM-Ca in order to generate the m-e.t. current. The permeabilities of the divalent cations relative to Cs were calculated from the reversal potentials, and the sequence of permeabilities among divalent cations was Ca greater than Sr greater than Ba greater than Mn greater than Mg. Step-like m-e.t. currents were observed in Cs saline. The smallest step amplitude with clear resolution had a conductance of 49.7 +/- 4.5 pS (mean +/- S.D., n = 7 cells). This is likely to be an elementary m-e.t. channel conductance.(ABSTRACT TRUNCATED AT 400 WORDS)

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... The cytoplasmic concentration of Ca 2þ in most cells is held at ~100 nM, determined in the long term as the balance between two factors, the rates of influx and of efflux of the ion. For the hair cell, the principal routes of entry are through Ca 2þ permeable MET channels in the hair bundle ( Beurg et al., 2006;Ohmori, 1985) and voltage-sensitive Ca 2þ (Cav1.3) channels around the basolateral aspect of the hair cells ( Brandt et al., 2003). ...
... A large (silent) transducer current is therefore constantly flowing into OHCs. The MET channels show a non-selective cation permeability, with Ca 2þ being most permeable, about 5 time more permeable than Na þ ( Beurg et al., 2006;Ohmori, 1985). High speed imaging has been used to estimate the fraction of the MET current carried by Ca 2þ as about 0.15 with bundles exposed to perilymph, and 0.002 when exposed to endolymph (Beurg et al., 2010) containing only 20e40 mM Ca 2þ (Gill and Salt, 1997). ...
Article
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Ototoxicity, noise overstimulation, or aging, can all produce hearing loss with similar properties, in which outer hair cells (OHCs), principally those at the high-frequency base of the cochlea, are preferentially affected. We suggest that the differential vulnerability may partly arise from differences in Ca²⁺ balance among cochlear locations. Homeostasis is determined by three factors: Ca²⁺ influx mainly via mechanotransducer (MET) channels; buffering by calcium-binding proteins and organelles like mitochondria; and extrusion by the plasma membrane CaATPase pump. We review quantification of these parameters and use our experimentally-determined values to model changes in cytoplasmic and mitochondrial Ca²⁺ during Ca²⁺ influx through the MET channels. We suggest that, in OHCs, there are two distinct micro-compartments for Ca²⁺ handling, one in the hair bundle and the other in the cell soma. One conclusion of the modeling is that there is a tonotopic gradient in the ability of OHCs to handle the Ca²⁺ load, which correlates with their vulnerability to environmental challenges. High-frequency basal OHCs are the most susceptible because they have much larger MET currents and have smaller dimensions than low-frequency apical OHCs.
... The physiological properties of this elusive channel provide a fingerprint for screening candidates . The transduction channel is a nonselective cation channel with high permeability to Ca 2+ (P Ca /P Na = 5–20) [3,4,5]. Although many divalent cations are permeant, they are also channel blockers: the channel can be blocked by Ca 2+ (IC50 = 1 mM), Mg 2+ , La 3+ (4 μm), and Gd 3+ (3 μm) [6,7,8]. ...
... The single channel conductance varies considerably, ranging from about 80 to 150 pS in 2–3 mM extracellular Ca 2+ , and is roughly twice that in low Ca 2+ [6,9,10,11,12]. Some organic cations are also permeant blockers, such as amiloride (IC50 = 50 μm [13]), the fluorescent dye FM1-43 (2 μm [14]) and the antibiotic dihydrostreptomycin (10‐70 μM [4,15,16]). The block is voltage dependent indicating that these cations block within the pore, part way along the transmembrane electric field [14,17] . ...
Article
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Members of the TRP superfamily of ion channels mediate mechanosensation in some organisms, and have been suggested as candidates for the mechanotransduction channel in vertebrate hair cells. Some TRP channels can be ruled out based on lack of an inner ear phenotype in knockout animals or pore properties not similar to the hair-cell channel. Such studies have excluded Trpv4, Trpa1, Trpml3, Trpm1, Trpm3, Trpc1, Trpc3, Trpc5, and Trpc6. However, others remain reasonable candidates. We used data from an RNA-seq analysis of gene expression in hair cells as well as data on TRP channel conductance to narrow the candidate group. We then characterized mice lacking functional Trpm2, Pkd2, Pkd2l1, Pkd2l2 and Pkd1l3, using scanning electron microscopy, auditory brainstem response, permeant dye accumulation, and single-cell electrophysiology. In all of these TRP-deficient mice, and in double and triple knockouts, mechanotransduction persisted. Together with published studies, these results argue against the participation of any of the 33 mouse TRP channels in hair cell transduction.
... Many proteins comprising the tip links and their attachments ( Fig. 1 B) have been discovered by the effects of specific mutations, such as those arising in Usher syndrome (6), but the identity of the channel is still controversial. In cochlear hair cells, the MET channel is cation selective with high permeability for Ca 2þ (4,7). Its most notable biophysical properties are its large single-channel conductance of 100 pS or more (4,8), and ultrafast activation kinetics (9,10), probably in the microsecond range (11). ...
... The wild-type MET channel is a cation channel with high selectivity for Ca 2þ , which is several times more permeant than monovalent cations Na þ , K þ , or Cs þ (7,8). Owing to the developmental changes in Ca 2þ selectivity occurring during the first two neonatal weeks (24), the effects of knockouts on this channel property have been less easy to interpret. ...
Article
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Transmembrane channel-like protein isoform-1 (TMC1) has emerged over the past five years as a prime contender for the mechano-electrical transducer (MET) channel in hair cells of the inner ear. TMC1 is thought to have a six-transmembrane domain structure reminiscent of some other ion-channel subunits, and is targeted to the tips of the stereocilia in the sensory hair bundle, where the MET channel is located. Moreover, there are TMC1 mutations linked to human deafness causing loss of conventional MET currents, hair cell degeneration, and deafness in mice. Finally, mutations of Tmc1 can alter the conductance and Ca2+ selectivity of the MET channels. For several reasons though, it is unclear that TMC1 is indeed the MET channel pore: 1) in other animals or tissues, mutations of TMC family members do not directly affect cellular mechanosensitivity; 2) there are residual manifestations of mechanosensitivity in hair cells of mouse Tmc1:Tmc2 double knockouts; 3) there is so far no evidence that expression of mammalian Tmc1 generates a mechanically sensitive ion channel in the plasma membrane when expressed in heterologous cells; and 4) there are other proteins, such as TMIE and LHFPL5, which behave similarly to TMC1, their mutation also leading to loss of MET current and deafness. This review will present these disparate lines of evidence and describes recent work that addresses the role of TMC1.
... The sensory mechanoelectrical transduction (MET) channel in hair cells is localized near the tips of stereocilia at the base of the tip link filament that connects a shorter stereocilium to its next taller neighbor (Figures 1A,B; Pickles et al., 1984;Beurg et al., 2008). Deflection of the hair bundle towards the tallest stereocilia leads to an increase in the MET channel open probability, while deflections in the opposite direction decrease channel open probability (Figures 2A,B; Hudspeth and Corey, 1977;Ohmori, 1985;Crawford et al., 1989;Kros et al., 1992;Nicolson et al., 1998). Tip links thus connect stereocilia in the direction of their greatest mechanical sensitivity. ...
... from a mM to a µM concentration (Corey and Hudspeth, 1983;Ohmori, 1985;Ricci et al., 2003;Pan et al., 2012). ...
Article
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Hair cells in the inner ear convert mechanical stimuli provided by sound waves and head movements into electrical signal. Several mechanically evoked ionic currents with different properties have been recorded in hair cells. The search for the proteins that form the underlying ion channels is still in progress. The mechanoelectrical transduction (MET) channel near the tips of stereociliary in hair cells, which is responsible for sensory transduction, has been studied most extensively. Several components of the sensory mechanotransduction machinery in stereocilia have been identified, including the multi-transmembrane proteins tetraspan membrane protein in hair cell stereocilia (TMHS)/LHFPL5, transmembrane inner ear (TMIE) and transmembrane channel-like proteins 1 and 2 (TMC1/2). However, there remains considerable uncertainty regarding the molecules that form the channel pore. In addition to the sensory MET channel, hair cells express the mechanically gated ion channel PIEZO2, which is localized near the base of stereocilia and not essential for sensory transduction. The function of PIEZO2 in hair cells is not entirely clear but it might have a role in damage sensing and repair processes. Additional stretch-activated channels of unknown molecular identity and function have been found to localize at the basolateral membrane of hair cells. Here, we review current knowledge regarding the different mechanically gated ion channels in hair cells and discuss open questions concerning their molecular composition and function.
... In the adult mammalian cochlea, the 100 or so stereocilia within a hair bundle are located in 3 rows of decreasing height (Lim, 1980). Transducer channels are located at the tips of the stereocilia (Hudspeth, 1982;Ohmori, 1985;Kroese, Das and Hudspeth, 1989) and are suggested to be gated by the tension in attached, elastic elements termed gating springs (Hudspeth, 1989). The morphological correlate of these gating springs is thought to be tip-links, fine, filamentous links that run from the tops of the shorter stereocilia to the side of adjacent, taller stereocilia (Pickles, Comis and Osborne, 1984). ...
... Assuming the single channel conductance of MET channels is 112 pS (Géléoc, Lennan, Richardson and Kros, 1997), adult mammalian OHCs posess 8 functioning channels per hair bundle. Alternatively, a single channel conductance of 50 pS (Ohmori, 1985), predicts that hair bundles posess 20 functioning channels. These calculations suggest that adult mammalian OHCs have a ratio of MET channels to tip-links somewhere between 1:8 and 1:3. ...
Thesis
This thesis investigates three characteristics of outer hair cells (OHCs) of the adult mammalian cochlea. The first investigation compared the basolateral membrane K+ channel expression of turn 4 (T4) and turn 1 (T1) OHCs, cells that respond to low and high frequency sound respectively. The second and third studies investigated two further aspects of T4 OHCs, the equivalent concentration of endogenous Ca2+ buffer and the characteristics of the mechanoelectric transduction (MET) current. OHCs in situ were recorded from using the whole-cell patch-clamp technique. For the first study, the kinetics, pharmacology and Ca2+ sensitivity of T4 and T1 OHC K+ channels were investigated. This work demonstrated that T4 OHCs express at least three types of K+ channels, termed Ikss, IkCa and lkT4. In contrast, T1 OHCs express different ion channels that exhibit faster onset kinetics, different pharmacology and an insensitivity to raised intracellular Ca2+ concentrations compared to those channels expressed in T4. These T1 channels have been termed IkT1 and Ik,n. The characteristics of these ion channels are discussed in relation to the particular sound frequency to which the OHC best responds. The equivalent concentration of endogenous Ca2+ buffer in T4 OHCs was investigated by using the time constant of current onset as a tool to compare the effects of various concentrations of BAPTA, introduced into the cell via the patch pipette, with those of the endogenous buffer, assayed using the perforated-patch technique. The concentration of endogenous Ca2+ buffer was found to be equivalent to the Ca2+ binding capacity of 2.1 mM BAPTA. This value converts to a Ca2+ binding ratio of 10,500. These results indicate that OHCs posess an enormous Ca2+ buffering capacity, have a low free [Ca2+]i and a huge pool of bound Ca2+ within their cytosol. Finally, the biophysical properties of the MET current of T4 OHCs were investigated. The few recordings obtained were variable but indicated that MET currents in situ are small (60 pA), limited in their onset kinetics only by the kinetics of the fluid-jet stimulus and run-down over a period of 20 minutes in the whole-cell recording configuration. These currents were found to be physiologically effective in activating the motile response of the OHC.
... Electrophysiological studies of hair cells reveal that the MET channel is a large conductance (150-300 pS in low Ca 2+ ) and cation-selective channel (Beurg et al., 2006;Corey and Hudspeth, 1979;GéléocGeleoc et al., 1997;Kros et al., 1992;Ohmori, 1985) that is permeable to relatively large organic compounds, including quaternary ammonium ions, FM1-43 and aminoglycoside antibiotics (Alharazneh et al., 2011;Gale et al., 2001;Marcotti et al., 2005;Meyers et al., 2003;Ohmori, 1985). Although the molecular identity and structure of the MET channel remains elusive, a growing body of evidence supports the hypothesis that the transmembrane channel-like 1 and 2 (TMC1 and TMC2) proteins are pore-forming subunits of the MET channel. ...
... Electrophysiological studies of hair cells reveal that the MET channel is a large conductance (150-300 pS in low Ca 2+ ) and cation-selective channel (Beurg et al., 2006;Corey and Hudspeth, 1979;GéléocGeleoc et al., 1997;Kros et al., 1992;Ohmori, 1985) that is permeable to relatively large organic compounds, including quaternary ammonium ions, FM1-43 and aminoglycoside antibiotics (Alharazneh et al., 2011;Gale et al., 2001;Marcotti et al., 2005;Meyers et al., 2003;Ohmori, 1985). Although the molecular identity and structure of the MET channel remains elusive, a growing body of evidence supports the hypothesis that the transmembrane channel-like 1 and 2 (TMC1 and TMC2) proteins are pore-forming subunits of the MET channel. ...
Article
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The hair cell mechanotransduction (MET) channel complex is essential for hearing, yet its molecular identity and structure remain elusive. The transmembrane channel–like 1 (TMC1) protein localizes to the site of the MET channel, interacts with the tip-link responsible for mechanical gating, and genetic alterations in TMC1 alter MET channel properties and cause deafness, supporting the hypothesis that TMC1 forms the MET channel. We generated a model of TMC1 based on X-ray and cryo-EM structures of TMEM16 proteins, revealing the presence of a large cavity near the protein-lipid interface that also harbors the Beethoven mutation, suggesting that it could function as a permeation pathway. We also find that hair cells are permeable to 3 kDa dextrans, and that dextran permeation requires TMC1/2 proteins and functional MET channels, supporting the presence of a large permeation pathway and the hypothesis that TMC1 is a pore forming subunit of the MET channel complex.
... Such a result is consistent with the afferent current developing in proportion to the accumulation of any of the vesicular contents or ion species. Similarly, with hair cell hyperpolarization conductances that were on at rest were deactivated (Fig. 2C, upper traces), and at -120 mV the residual conductance of < 1 nS in the steady state was a leak to 0 mV, representing either the hair cell transducer (Ohmori, 1985) or the seal conductance. Under these conditions, we expect little Ca 2+ flux into the hair cell, and that both vesicle fusion and potassium efflux into the cleft will be blocked. ...
Article
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Key points: In the synaptic cleft between type I hair cells and calyceal afferents, K(+) ions accumulate as a function of activity, dynamically altering the driving force and permeation through ion channels facing the synaptic cleft. High-fidelity synaptic transmission is possible due to large conductances that minimize hair cell and afferent time constants in the presence of significant membrane capacitance. Elevated potassium maintains hair cells near a potential where transduction currents are sufficient to depolarize them to voltages necessary for calcium influx and synaptic vesicle fusion. Elevated potassium depolarizes the postsynaptic afferent by altering ion permeation through hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, and contributes to depolarizing the afferent to potentials where a single EPSP (quantum) can generate an action potential. With increased stimulation, hair cell depolarization increases the frequency of quanta released, elevates [K(+) ]cleft and depolarizes the afferent to potentials at which smaller and smaller EPSPs would be sufficient to trigger APs. Abstract: Fast neurotransmitters act in conjunction with slower modulatory effectors that accumulate in restricted synaptic spaces found at giant synapses such as the calyceal endings in the auditory and vestibular systems. Here, we used dual patch-clamp recordings from turtle vestibular hair cells and their afferent neurons to show that potassium ions accumulating in the synaptic cleft modulated membrane potentials and extended the range of information transfer. High-fidelity synaptic transmission was possible due to large conductances that minimized hair cell and afferent time constants in the presence of significant membrane capacitance. Increased potassium concentration in the cleft maintained the hair cell near potentials that promoted the influx of calcium necessary for synaptic vesicle fusion. The elevated potassium concentration also depolarized the postsynaptic neuron by altering ion permeation through hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. This depolarization enabled the afferent to reliably generate action potentials evoked by single AMPA-dependent EPSPs. Depolarization of the postsynaptic afferent could also elevate potassium in the synaptic cleft, and would depolarize other hair cells enveloped by the same neuritic process increasing the fidelity of neurotransmission at those synapses as well. Collectively, these data demonstrate that neuronal activity gives rise to potassium accumulation, and suggest that potassium ion action on HCN channels can modulate neurotransmission, preserving the fidelity of high-speed synaptic transmission by dynamically shifting the resting potentials of both presynaptic and postsynaptic cells.
... Gentamicin is thought to operate by accumulating in hair cells and also interfering with calcium dependent receptors in the plasma membrane by competitive inhibition [89,90] . Aminoglycosides can also interfere with hair cell secondary cell messengers and the integrity of the cell membrane [91] . ...
... The endolymphatic Ca 2+ concentration is critical for normal auditory function. (Tanaka et al. 1980;Ohmori, 1985). Long-standing untreated hypoparathyroidism appears to be associated with a high incidence of sensorineural hearing loss (Ikeda, 1987a). ...
... As the only cochlear PMCA2 pump exposed to endolymph is that of the stereocilia [24,69], this may provide a clue as to why, in some cases, mutations in the gene of the PMCA2 pump potentiated the deafness phenotype induced by co-existing mutations of cadherin-23 forming the tip link of MET channels, which is compromised below 1 μM Ca 2+ endolymphatic concentration [3]. Indeed, a number of in vitro studies have shown that the generation of MET currents, for instance in chickens, requires a minimum of 20 μM extracellular Ca 2+ and is not elicited at 10 μM [53]. The dfw mutation lowers cochlear endolymphatic Ca 2+ concentration from 23 μM to about 6 μM [71], supporting the conclusion that a diminished Ca 2+ removal from the hair cell affects MET currents and adaptation [2,25,55,73]. ...
Article
Hair cells of the inner ear detect sound stimuli, inertial or gravitational forces by deflection of their apical stereocilia. A small number of stereociliary cation-selective mechanotransduction (MET) channels admit K+and Ca2+ions into the cytoplasm promoting hair cell membrane depolarization and, consequently, neurotransmitter release at the cell basolateral pole. Ca2+influx into the stereocilia compartment is counteracted by the unusual w/a splicing variant of plasma-membrane calcium-pump isoform 2 (PMCA2) which, unlike other PMCA2 variants, increases only marginally its activity in response to a rapid variation of the cytoplasmic free Ca2+concentration ([Ca2+]c). Missense mutations of PMCA2w/a cause deafness and loss of balance in humans. Mouse models in which the pump is genetically ablated or mutated show hearing and balance impairment, which correlates with defects in homeostatic regulation of stereociliary [Ca2+]c, decreased sensitivity of mechanotransduction channels to hair bundle displacement and progressive degeneration of the organ of Corti. These results highlight a critical role played by the PMCA2w/a pump in the control of hair cell function and survival, and provide mechanistic insight into the etiology of deafness and vestibular disorders.
... Sequence alignment showed both CmTMC1 and MuTMC2 are highly conserved with human TMC1 and TMC2, respectively [140]. Accordingly, like the arrangement of stereocilia in descending heights observed in mammals, birds and reptiles possess hair cells with stereocilia distributed similarly and also tip links to transmit forces [133][134][135][136][137][138]. Therefore, studying the functional properties of CmTMC1 and MuTMC2 also provides insights into the properties of their mammalian orthologs. ...
Chapter
The ability of living organisms to detect mechanical force originates from mechanotransduction ion channels, which convert membrane tension into electrical or chemical signals that are transmitted to the brain. A variety of studies on touch and sound perception in both vertebrates and invertebrates have broadened our understanding of mechanotransduction and identified promising candidates for mechanotransduction ion channels. Here, we discussed the physiological properties of mechanotransduction ion channels in hearing and touch, the identification of their molecular entities, and recent structural studies providing insights to their gating mechanisms in force sensing. We present an updated review of the evidence supporting several candidates, including NOMPC, Brv1, and TMC channels, as mechanotransduction ion channels and highlight their qualifications satisfying the specific criteria proposed for a mechanotransducer.
... In fact, even the inactivating dfw mutation only lowers the endolymph Ca 2+ concentration to about 6 μM [ 28 ]. However, a number of in vitro studies have shown that the generation of MET currents, for instance, in chickens, requires a minimum of 20 μM extracellular Ca 2+ and is not elicited at 10 μM [ 23 ]. A diminished Ca 2+ removal from the hair cell affects MET currents and adaptation, the process by which hair cells continuously readjust their sensitivity to the ciliary bundle displacements [ 34 , 35 , 62 , 63 ]. ...
Chapter
Sensory hair cells of the inner ear detect sound stimuli, inertial or gravitational forces. These mechanical inputs cause deflection of the cell stereociliary bundle and activate a small number of cation-selective mechano-transduction (MET) channels that admit K+ and Ca2+ ions into the cytoplasm. Stereociliary Ca2+ levels are homeostatically regulated by an unusual splicing isoform (w/a) of plasma membrane calcium-pump isoform 2 (PMCA2w/a), ablation or missense mutations of which cause deafness and loss of balance in humans and mice. At variance with other PMCA2 isoforms, PMCA2w/a expressed in CHO transfectants increases only marginally its activity in response to a rapid increase of the cytoplasmic free Ca2+ concentration ([Ca2+]c). In this expression system, deafness-related mutations of PMCA2w/a decrease the pump ability to extrude Ca2+ both at steady state and in response to a [Ca2+]c rise. Consistent with these findings, mouse strains in which the pump is genetically ablated or mutated show hearing impairment correlated with defects in homeostatic regulation of stereociliary Ca2+, decreased sensitivity of the MET channels to hair bundle displacement, and morphological abnormalities in the organ of Corti. These results highlight a critical role played by PMCA2w/a in the control of hair cell function and survival and provide mechanistic insight into the etiology of deafness and vestibular disorders.
... Under fortunate conditions, this leaves enough intact tip links to allow electrical recording from one or a few channels at a time, and thus measurement of the channel conductance. When the extracellular Ca 2þ concentration was subsequently raised to about 3 mM, the channel conductance measured was on the order of 100 picosiemens (100 pS) (Ohmori 1985;Crawford et al. 1991;Beurg et al. 2006). In contrast, most voltage-gated ion channels have conductances in the range of 10-30 pS (Hille 2001). ...
Article
Synopsis: Sensory hair cells are specialized secondary sensory cells that mediate our senses of hearing, balance, linear acceleration, and angular acceleration (head rotation). In addition, hair cells in fish and amphibians mediate sensitivity to water movement through the lateral line system, and closely related electroreceptive cells mediate sensitivity to low-voltage electric fields in the aquatic environment of many fish species and several species of amphibian.Sensory hair cells share many structural and functional features across all vertebrate groups, while at the same time they are specialized for employment in a wide variety of sensory tasks. The complexity of hair cell structure is large, and the diversity of hair cell applications in sensory systems exceeds that seen for most, if not all, sensory cell types. The intent of this review is to summarize the more significant structural features and some of the more interesting and important physiological mechanisms that have been elucidated thus far. Outside vertebrates, hair cells are only known to exist in the coronal organ of tunicates. Electrical resonance, electromotility, and their exquisite mechanical sensitivity all contribute to the attractiveness of hair cells as a research subject.
... The physiological properties of this elusive channel provide a fingerprint for screening candidates. The transduction channel is a nonselective cation channel with high permeability to Ca 2+ (P Ca /P Na = 5-20) [3,4,5]. Although many divalent cations are permeant, they are also channel blockers: the channel can be blocked by Ca 2+ (IC50 = 1 mM), Mg 2+ , La 3+ (4 μm), and Gd 3+ (3 μm) [6,7,8]. ...
Article
Full-text available
Mechanotransducer channels at the tips of sensory stereocilia of inner ear hair cells are gated by the tension of 'tip links' interconnecting stereocilia. To ensure maximal sensitivity, tip links are tensioned at rest, resulting in a continuous influx of Ca2+ into the cell. Here, we show that this constitutive Ca2+ influx, usually considered as potentially deleterious for hair cells, is in fact essential for stereocilia stability. In the auditory hair cells of young postnatal mice and rats, a reduction in mechanotransducer current, via pharmacological channel blockers or disruption of tip links, leads to stereocilia shape changes and shortening. These effects occur only in stereocilia that harbor mechanotransducer channels, recover upon blocker washout or tip link regeneration and can be replicated by manipulations of extracellular Ca2+ or intracellular Ca2+ buffering. Thus, our data provide the first experimental evidence for the dynamic control of stereocilia morphology by the mechanotransduction current.
... Fluid shear stress has been shown to trigger microvilli formation through Ca 2+ influx through mechanosensitive activation of the Ca 2+ -selective transient receptor potential cation channel vanilloid 6 (TRPV6), which is expressed in intestinal epithelial cells (Bianco et al., 2006;Miura et al., 2015). Likewise, mechanical stimuli to deflect stereocilia result in opening of MET channels, giving rise to an electrical output (Ohmori, 1985). Finally, both microvilli and stereocilia have to maintain tension generated by the formation of actin-based protrusions on the apical cell membrane. ...
Article
Full-text available
Cells of transporting epithelia are characterized by the presence of abundant F-actin-based microvilli on their apical surfaces. Likewise, auditory hair cells have highly reproducible rows of apical stereocilia (giant microvilli) that convert mechanical sound into an electrical signal. Analysis of mutations in deaf patients has highlighted the critical components of tip links between stereocilia, and related structures that contribute to the organization of microvilli on epithelial cells have been found. Ezrin/radixin/moesin (ERM) proteins, which are activated by phosphorylation, provide a critical link between the plasma membrane and underlying actin cytoskeleton in surface structures. Here, we outline recent insights into how microvilli and stereocilia are built, and the roles of tip links. Furthermore, we highlight how ezrin is locally regulated by phosphorylation, and that this is necessary to maintain polarity. Localized phosphorylation is achieved through an intricate coincidence detection mechanism that requires the membrane lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and the apically localized ezrin kinase, lymphocyte-oriented kinase (LOK, also known as STK10) or Ste20-like kinase (SLK). We also discuss how ezrin-binding scaffolding proteins regulate microvilli and how, despite these significant advances, it remains to be discovered how the cell polarity program ultimately interfaces with these processes.
... We then investigated whether the MET current in Piezo1 +/À mice was affected by the aminoglycoside dihydrostreptomycin (DHS), a large polycation known to block (Ohmori 1985;Kroese et al. 1989;Ricci 2002;Marcotti et al. 2005) and permeate (Marcotti et al. 2005) the MET channel in vertebrate hair cells. Using 50 Hz sinusoidal stimulation, we recorded the MET current by stepping the membrane between À161 and +99 mV in 20 mV increments from OHCs of both Piezo1 +/+ and Piezo1 +/À mice before (Fig. 3A, B, left panels) and during the superfusion of 10 lmol/L extracellular DHS (Fig. 3A, B, right panels). ...
Article
Full-text available
The mechanoelectrical transducer (MET) channels located at the stereocilia tip of cochlear hair cells are crucial to convert the mechanical energy of sound into receptor potentials, but the identity of its pore-forming subunits remains uncertain. Piezo1, which has been identified in the transcriptome of mammalian cochlear hair cells, encodes a transmembrane protein that forms mechanosensitive channels in other tissues. We investigated the properties of the MET channel in outer hair cells (OHCs) of Piezo1 mice (postnatal day 6-9). The MET current was elicited by deflecting the hair bundle of OHCs using sinewave and step stimuli from a piezo-driven fluid jet. Apical and basal OHCs were investigated because the properties of the MET channel vary along the cochlea. We found that the maximal MET current amplitude and the resting open probability of the MET channel in OHCs were similar between Piezo1(+/-) haploinsufficient mice and wild-type littermates. The sensitivity to block by the permeant MET channel blocker dihydrostreptomycin was also similar between the two genotypes. Finally, the anomalous mechano-gated current, which is activated by sheer force and which is tip-link independent, was unaffected in OHCs from Piezo1(+/-) haploinsufficient mice. Our results suggest that Piezo1 is unlikely to be a component of the MET channel complex in mammalian cochlear OHCs.
... The currents associated with SAC NSC are blocked by the pharmacological agents gadolinium (Gd 3+ ) 55 and streptomycin. 46,56 Use of these agents can either prevent 4,19 or have no effect on 48,53,57,58 the SFR. These directly conflicting findings are because of limitations with a number of these experiments. ...
Article
When exposed to an abrupt stretch, cardiac muscle exhibits biphasic active force enhancement. The initial, instantaneous, force enhancement is well explained by the Frank‐Starling mechanism. However, the cellular mechanisms associated with the second, slower, phase remain contentious. This review explores hypotheses regarding this ‘slow force response’ with the intention of clarifying some apparent contradictions in the literature. The review is partitioned into three sections. The first section considers pathways that modify the intracellular calcium handling to address the role of the sarcoplasmic reticulum in the mechanism underlying the slow force response. The second section focuses on extracellular calcium fluxes and explores the identity and contribution of the stretch‐activated, non‐specific, cation channels as well as signalling cascades associated with G‐protein coupled receptors. The final section introduces promising candidates for the mechanosensor(s) responsible for detecting the stretch perturbation. This article is protected by copyright. All rights reserved.
... The biophysical properties of the MET channel essential for hearing have been extensively characterized. Among other properties, these channels are cationic selective and have a relatively large conductance (150-300 pS in low Ca 2+ ) 4,5,6,7,8,9,10 . Remarkably, large fluorescent molecules such as FM1-43 and Texas Red-labeled aminoglycosides are permeant blockers of the MET channel, resulting in their accumulation in the hair cell body that can be visualized using fluorescence microscopy 11,12,13,14 . ...
Article
The hair cell mechanotransduction (MET) channel plays an important role in hearing. However, the molecular identity and structural information of MET remain unknown. Electrophysiological studies of hair cells revealed that the MET channel has a large conductance and is permeable to relatively large fluorescent cationic molecules, including some styryl dyes and Texas Red-labeled aminoglycoside antibiotics. In this protocol, we describe a method to visualize and evaluate the uptake of fluorescent dextrans in hair cells of the organ of Corti explants that can be used to assay for functional MET channels. We found that 3 kDa Texas Red-labeled dextran specifically labels functional auditory hair cells after 1-2 h incubation. In particular, 3 kDa dextran labels the two shorter stereocilia rows and accumulates in the cell body in a diffuse pattern when functional MET channels are present. An additional vesicle-like pattern of labeling was observed in the cell body of hair cells and surrounding supporting cells. Our data suggest that 3 kDa Texas-Red dextran can be used to visualize and study two pathways for cellular dye uptake; a hair cell-specific entry route through functional MET channels and endocytosis, a pattern also available to larger dextran.
... In the first step in auditory transduction, sound-induced motion within the cochlea culminates in opening of mechanoelectrical transducer (MET) channels in the stereociliary (hair) bundle of each hair cell (Hudspeth and Corey, 1977;Ohmori, 1985;Crawford et al., 1989;Fettiplace and Kim, 2014). Despite insight into the likely identity of the channel protein as an isoform of the transmembrane channel-like family (TMC1; Kawashima et al., 2011;Pan et al., 2018), the molecular organization of the channel is still uncertain. ...
Article
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Although mechanoelectrical transducer (MET) channels have been extensively studied, uncertainty persists about their molecular architecture and single-channel conductance. We made electrical measurements from mouse cochlear outer hair cells (OHCs) to reexamine the MET channel conductance comparing two different methods. Analysis of fluctuations in the macroscopic currents showed that the channel conductance in apical OHCs determined from nonstationary noise analysis was about half that of single-channel events recorded after tip link destruction. We hypothesized that this difference reflects a bandwidth limitation in the noise analysis, which we tested by simulations of stochastic fluctuations in modeled channels. Modeling indicated that the unitary conductance depended on the relative values of the channel activation time constant and the applied low-pass filter frequency. The modeling enabled the activation time constant of the channel to be estimated for the first time, yielding a value of only a few microseconds. We found that the channel conductance, assayed with both noise and recording of single-channel events, was reduced by a third in a new deafness mutant, Tmc1 p.D528N. Our results indicate that noise analysis is likely to underestimate MET channel amplitude, which is better characterized from recordings of single-channel events.
... Thus, MT channels in the resting state are in a region of greatest sensitivity to the force, i.e., a typical 0.5-μm deflection of the hair bundle is able to evoke a maximal response. Electrophysiological characterization revealed that MT channels are nonselective cation channels with high permeability to Ca 2+ (79). Since the endolymph solution bathing the hair bundle contains high K + and low Ca 2+ (approximately 140 mM K + and 20-40 μM Ca 2+ ), the predominant MT current in vivo is carried by K + . ...
Article
Sound-induced mechanical stimuli are detected by elaborate mechanosensory transduction (MT) machinery in highly specialized hair cells of the inner ear. Genetic studies of inherited deafness in the past decades have uncovered several molecular constituents of the MT complex, and intense debate has surrounded the molecular identity of the pore-forming subunits. How the MT components function in concert in response to physical stimulation is not fully understood. In this review, we summarize and discuss multiple lines of evidence supporting the hypothesis that transmembrane channel-like 1 is a long-sought MT channel subunit. We also review specific roles of other components of the MT complex, including protocadherin 15, cadherin 23, lipoma HMGIC fusion partner-like 5, transmembrane inner ear, calcium and integrin-binding family member 2, and ankyrins. Based on these recent advances, we propose a unifying theory of hair cell MT that may reconcile most of the functional discoveries obtained to date. Finally, we discuss key questions that need to be addressed for a comprehensive understanding of hair cell MT at molecular and atomic levels. Expected final online publication date for the Annual Review of Biophysics, Volume 50 is May 6, 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... Moreover, endolymph acidification may also inhibit the pH-sensitive transient receptor potential subfamily V (TRPV) members TRPV5 and TRPV6, which are also expressed in different epithelial cell types in the inner ear, including the endolymphatic sac (39)(40)(41)(42). Inhibition of TRPV5 and TRPV6 may explain the pathological increase in endolymphatic Ca 2þ (40), which is thought to disturb hair cell function (43,44), and thus may play an important role in the pathogenesis of cochleovestibular symptoms in LVAS mouse models (40). ...
Article
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Hypothesis: Epithelial ion transport pathologies of the endolymphatic sac (ES) are associated with large vestibular aqueduct syndrome (LVAS). Background: LVAS is defined by the pathognomonic features of a widened bony vestibular aqueduct (VA) and an enlarged ES. The underlying cause of its associated cochleovestibular symptoms remains elusive. Disturbances in epithelial ion transport in the enlarged ES, affecting inner ear fluid regulation, were proposed as a possible pathophysiology. However, although respective epithelial ion transport pathologies have been demonstrated in the enlarged ES from transgenic LVAS mouse models, these pathologies have not been investigated in human LVAS cases. Methods: Histological and immunohistochemical analysis of the enlarged ES epithelium in postmortem temporal bones from two individuals with a clinical diagnosis of LVAS. Results: The enlarged ES epithelium demonstrated an overall atypical epithelial differentiation and a lack of the immunolocalization of signature ion transport proteins. Notably, in both cases, a rudimentary branch of the ES with a typically differentiated ES epithelium was present. Conclusions: The described cellular and molecular pathologies of the enlarged ES in humans provide evidence of epithelial transport pathology as one potential cause of cochleovestibular symptoms in LVAS. The present findings also emphasize the clinical relevance of already established LVAS mouse models.
... The precise functional consequences of positive selection in mammalian Tmc1, and their effect on mammalian-specific auditory functions, remain to be determined. Nonetheless, we wonder whether evolutionary changes in TMC1 may account for some of the reported differences in the biophysical properties of mechanosensory transduction in HCs of vertebrates such as, zebrafish, turtles, frogs, chickens, mice, and rats (Chou et al., 2017;Corey and Hudspeth, 1983;Crawford et al., 1989;Kennedy et al., 2003;Kros et al., 1992;Ohmori, 1985). ...
Article
Full-text available
Auditory sensation in the mammalian inner ear requires mechanosensitive hair cells and an elaborate array of molecules that form the sensory transduction machinery. Several components of this machine have been identified, yet how the ensemble functions in harmony to enable the sense of hearing has not been clarified. We review recent evidence supporting the role of key transduction molecules and focus on TMC1 and TMC2, pore-forming proteins at the heart the mechanosensory device. We consider how vertebrate TMCs changed during evolution and how they function in concert with other components of the transduction apparatus. We also examine the role of TMCs in non-mammalian systems to gain insight into common themes for TMC function and properties unique to mammalian hair cell TMCs. Although the tempo of discovery has accelerated, important questions remain which will need to be addressed for a comprehensive understanding of mechanosensory transduction in the mammalian inner ear.
... Hair cell function in the cochlea and the vestibular system is calcium (Ca 2+ )-dependent (Tanaka et al. 1980;Ohmori 1985;Kozel et al. 1998) and requires the endolymphatic Ca 2+ concentration ([Ca 2+ ] endolymph ) to be tightly controlled at a level that is unusually low (0.017-0.133 mmol/l (Salt et al. 1989)) for an extracellular fluid (e.g., [Ca 2+ ] blood plasma = 1.2 mmol/l (Diem and Lentner 1970)). To maintain this physiological [Ca 2+ ] endolymph , numerous epithelial sites within the membranous labyrinth are engaged in Ca 2+ transport across the endolymph-perilymph barrier, utilizing Ca 2+permeable channels (e.g., transient receptor potential cation channel subfamily V members [TRPV channels] (Takumida et al. 2005;Wangemann et al. 2007;Nakaya et al. 2007;Ishibashi et al. 2008)), ion exchangers (e.g., sodium-calcium exchangers [NCX] (Oshima et al. 1997;Yamauchi et al. 2010)), and Ca 2+ ATPases (e.g., plasma membrane calcium ATPase [PMCA] (Crouch and Schulte 1995;Ågrup et al. 1999)). ...
Article
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An exceptionally low calcium (Ca²⁺) concentration in the inner ear endolymph ([Ca²⁺]endolymph) is crucial for proper auditory and vestibular function. The endolymphatic sac (ES) is believed to critically contribute to the maintenance of this low [Ca²⁺]endolymph. Here, we investigated the immunohistochemical localization of proteins that are presumably involved in the sensing and transport of extracellular Ca²⁺ in the murine ES epithelium. Light microscopic and fluorescence immunolabeling in paraffin-embedded murine ES tissue sections (male C57BL/6 mice, 6–8 weeks old) demonstrated the presence of the calcium-sensing receptor CaSR, transient receptor potential cation channel subtypes TRPV5 and TRPV6, sarco/endoplasmic reticulum Ca²⁺-ATPases SERCA1 and SERCA2, Na⁺/Ca²⁺ exchanger NCX2, and plasma membrane Ca²⁺ ATPases PMCA1 and PMCA4 in ES epithelial cells. These proteins exhibited (i) membranous (apical or basolateral) or cytoplasmic localization patterns, (ii) a proximal-to-distal labeling gradient within the ES, and (iii) different distribution patterns among ES epithelial cell types (mitochondria-rich cells (MRCs) and ribosome-rich cells (RRCs)). Notably, in the inner ear membranous labyrinth, CaSR was exclusively localized in MRCs, suggesting a unique role of the ES epithelium in CaSR-mediated sensing and control of [Ca²⁺]endolymph. Structural loss of the distal ES, which is consistently observed in Meniere’s disease, may therefore critically disturb [Ca²⁺]endolymph and contribute to the pathogenesis of Meniere’s disease.
... The MET channels are thought to be located at the lower ends of extracellular tip links connecting adjacent stereocilia (1)(2)(3). These channels are cation channels with high permeability to Ca 2+ (4)(5)(6). There has been much controversy over the molecular underpinning of the channel, but the current view is that the transmembrane channellike protein isoforms 1 and 2 (TMC1 and TMC2) play a pivotal role in MET channel function (7,8). ...
Article
Full-text available
Mechanoelectrical transducer (MET) currents were recorded from cochlear hair cells in mice with mutations of transmembrane channel-like protein TMC1 to study the effects on MET channel properties. We characterized a Tmc1 mouse with a single-amino-acid mutation (D569N), homologous to a dominant human deafness mutation. Measurements were made in both Tmc2 wild-type and Tmc2 knockout mice. By 30 d, Tmc1 pD569N heterozygote mice were profoundly deaf, and there was substantial loss of outer hair cells (OHCs). MET current in OHCs of Tmc1 pD569N mutants developed over the first neonatal week to attain a maximum amplitude one-third the size of that in Tmc1 wild-type mice, similar at apex and base, and lacking the tonotopic size gradient seen in wild type. The MET-channel Ca ²⁺ permeability was reduced 3-fold in Tmc1 pD569N homozygotes, intermediate deficits being seen in heterozygotes. Reduced Ca ²⁺ permeability resembled that of the Tmc1 p M412K Beethoven mutant, a previously studied semidominant mouse mutation. The MET channel unitary conductance, assayed by single-channel recordings and by measurements of current noise, was unaffected in mutant apical OHCs. We show that, in contrast to the Tmc1 M412K mutant, there was reduced expression of the TMC1 D569N channel at the transduction site assessed by immunolabeling, despite the persistence of tip links. The reduction in MET channel Ca ²⁺ permeability seen in both mutants may be the proximate cause of hair-cell apoptosis, but changes in bundle shape and protein expression in Tmc1 D569N suggest another role for TMC1 apart from forming the channel.
... Des canaux de mécanotransduction (MT) ont été localisés par imagerie calcique à l'extrémité des stéréocils, mais ils sont absents sur la rangée la plus haute (Beurg et al., 2009) (Fig.4B). Ils forment de larges pores cationiques non sélectifs avec une grande perméabilité pour le K + et le Ca 2+ (Ohmori et al., 1985). La déflexion de la touffe ciliaire vers les stéréocils les plus longs, conduit à une augmentation de la probabilité d'ouverture des canaux de MT, alors que les déflections dans le sens opposé, diminuent cette probabilité d'ouverture (Gillespie and Müller, 2009). ...
Thesis
L'encodage du signal acoustique en impulsions nerveuses se réalise au niveau des synapses à ruban des cellules ciliées internes (CCI) de la cochlée. Une dépolarisation déclenche l'exocytose des vésicules synaptiques suite à l'activation des canaux calciques CaV1.3 et à l'action d'un senseur calcique particulier, l'otoferline, une grande protéine se composant d'un domaine transmembranaire en C-terminal et de six domaines C2 (A-F) pouvant lier le Ca2+ et les phospholipides. Afin de caractériser le rôle de ces différents domaines C2, nous avons utilisé des vecteurs viraux (AAV) permettant l'expression de formes raccourcies de l'otoferline (mini-Otof) in vivo dans les CCI de souris dépourvues d'otoferline (Otof -/-). Nous montrons que les mini-Otof contenant les domaines C2-EF, C2-DEF ou C2-ACEF sont suffisantes pour restaurer l'exocytose rapide des CCI Otof -/-, sans toutefois restaurer l'audition car le recrutement des vésicules synaptiques reste altéré. Nous révélons pour la première fois la présence d'une endocytose ultra-rapide (t < 20 ms) dynamine- et otoferline-dépendante, une fonction certainement essentielle à l'homéostasie membranaire des CCI. L'expression des mini-Otof C2-EF et C2-DEF a également permis de restaurer partiellement la composante rapide de l'inactivation du courant calcique des CCI, celle-ci étant absente chez les souris Otof -/-. Cette inactivation rapide est réalisée par les isoformes courtes Cav1.3S qui ont leur partie C-terminale régulatrice tronquée, contrairement aux isoformes longues Cav1.3L dépourvues d'inactivation. Afin de différencier les rôles spécifiques de ces isoformes dans le cycle des vésicules synaptiques, nous avons utilisé la technologie CRISPR-Cas9, nous permettant d'éditer spécifiquement la partie C-terminale régulatrice des canaux Cav1.3L. Nos résultats montrent que les souris CRISPR-Cav1.3L présentent une surdité sévère expliquée au niveau des CCI par un défaut de recrutement vésiculaire aux zones actives, alors que les Cav1.3S inaltérés contrôlent la fusion rapide des vésicules synaptiques.
Chapter
Studies have demonstrated that hypertensive left ventricular hypertrophy (LVH) simultaneously exhibits physiological and pathological aspects, suggesting that LVH is not a simple adaptation to an increased external load. Many investigators have extensively studied the activation of a novel program of gene expression in the heart induced by pressure overload (i.e., the transient induction of immediate early genes and the reexpression of fetal-type genes of both contractile and noncontractile proteins), which has been shown to be important as an adaptation to maintain cardiac function. Using an in vitro model system of mechanical stress-induced cardiac hypertrophy, we have demonstrated that the external stimuli imposed on cardiomyocytes are transduced into the nucleus through activation of the protein kinase cascade of phosphorylation: passive stretch of cultured cardiomyocytes stimulates the activation of protein kinase C, which leads to the successive phosphorylation of the mitogenactivated protein (MAP) kinase cascade including MAP kinase kinase kinase, MAP kinase, kinase, MAP kinase, and S6 kinase. Furthermore, we have demonstrated that the endogenous angiotensin II (Ang II) partially mediates mechanical stretch-induced cardiomyocyte hypertrophy, indicating that the cardiac tissue renin-angiotensin system plays an important role in the formation of hypertensive LVH. The precise mechanisms by which the sustained increase in external load leads to the impairment of cardiac function remain unclear, although impairment of diastolic function has been detected during the early stage of load-induced hypertrophy when there is no sign of reduced contractility. Using an in vivo model of hypertensive LVH, we noted previously that the depressed function of sarcoplasmic reticulum and the accumulation of interstitial collagen fibers may be responsible for the impaired diastolic function in stressed hearts. Here we review recent work on mechanical stress-induced cardiac hypertrophy and failure, especially bringing into focus the molecular mechanisms by which external load induces cellular hypertrophy of cardiac myocytes and impairment of the diastolic properties of the myocardium.
Article
The subsurface cytoskeletal lattice of guinea pig outer hair cells: structure and development.
Chapter
Conversion of mechanical signals into electrical signals is one of the oldest problems faced by living forms. Many simple unicellular organisms possess a mechano-sensing mechanism, which with growing complexity has become elaborated to detect a wide variety of specialized stimuli. This chapter will describe recent work on the biophysics of the interconversion of mechanical and electrical energy in hair cells, the specialized cells of the acoustico-lateralis system of vertebrates. This system includes the cochlea, the organ of hearing; the semicircular canals, the organs concerned with the detection of angular acceleration; the saccule and utricle of the vestibular system, specialized organs concerned with the detection of linear acceleration. In some lower vertebrates,the system also includes the lateral line, the organ concerned with detection of pressure waves in an aquatic environment.
Chapter
The cochlea is a mechanosensitive organ that perceives sounds. Hair cells with mechanosensitive transducer channels are the sensory cells of hearing. The stria vascularis generates specific electrochemical gradients of potassium and an endocochlear potential that are needed for the mechanoelectrical transduction of hair cells. Spiral ganglion neurons form the VIIIth cranial nerve and conduct action potentials. So far, little is known about the effects of cytokines/chemokines on the cochlear physiology in auditory perception. Recent reports have clarified the involvements of cytokines in the death of cochlear cells in various cochlear injuries. On the other hand, neurotrophic factors play key roles in the development and maintenance of spiral ganglion neurons. This review summarizes what is currently known about the involvement of cytokines in cochlear pathophysiology.
Chapter
In den 50er und 60er Jahren ermöglichte die Verbreitung objektiver Methoden zur Messung der Augenbewegungen und der Aktivität der peripheren und zentralen Neuronen die intensive Erforschung des vestibulookulären Reflexes. Seit Mitte der siebziger Jahre konnten durch die Untersuchung des vestibulookulomotorischen Systems weitere wesentliche Fortschritte erzielt werden. Nicht zuletzt deshalb zählt es heute zu einem der meist erforschten neurophysiologischen Systeme.
Chapter
The initial step in hair cell transduction involves coordinated flexion of an array of microvilli, the hair bundle, which gates a membrane conductance enabling a transducer current to flow into the cell (Hudspeth & Corey, 1977; Ohmori, 1985). Although the ionic selectivity of the transducer conductance is known (Corey & Hudspeth, 1979; Ohmori, 1985), the events leading to activation of the channel are not fully understood, and there is no consensus even on the precise form of relationship between bundle displacement and transducer current. Here we present some observations on the transduction process in turtle hair cells measured both directly in isolated cells under voltage clamp and indirectly from receptor potentials in the intact ear.
Chapter
Hair cells of the inner ear in many lower vertebrates behave like electrical resonators so that the transducer current develops a receptor potential that is maximal for a narrow range of frequencies around resonance (Crawford and Fettiplace, 1981). The resonance is thought to arise from potassium channels in the baso-lateral membrane of the cell (Crawford and Fettiplace, 1981; Lewis and Hudspeth, 1983), and it is abolished by tetraethylammonium ions (TEA). Since different hair cells have different resonant frequencies to cover the auditory range, this implies that some membrane property changes from cell to cell. We have investigated the source of the variability by recording the principal ionic currents in isolated cochlear hair cells of the turtle (Pseudemys scripta elegans). The preparation is particularly suitable on two counts: the ear shows a wide range of resonant frequencies (30 Hz to 700 Hz), and the hair cells are tonotopically organized along the basilar membrane, so that cells of different frequency can be selected for investigation (Crawford and Fettiplace, 1980).
Chapter
The hair cells in the cochlea are divided into two morphologically distinct populations: a single row of inner hair cells (IHC) and three or four rows of outer hair cells (OHC). On the basis of differences in morphology, IHCs and OHCs have been attributed with different roles in mechano-electric transduction in the cochlea. The aim of this paper is to review recent studies on the electrophysiological properties of hair cells in the mammalian cochlea to see if there is any basis for this suggestion.
Chapter
If a maintained displacement is imposed upon the ciliary bundle of a hair cell, many of the transducer channels that initially open in response to the stimulus eventually close. This process of adaptation of the transducer current has been investigated in several preparations, by delivering test steps to the bundle following a conditioning step to induce adaptation. The results of such experiments have shown that even after the transducer current has declined extensively from its initial peak value, the transducer channels can still be re-opened by further displacement of the cell’s ciliary bundle towards the kinocilium (Corey & Hudspeth. 1983a,b; Eatock, Corey & Hudspeth, 1987; Assad, Hacohen & Corey, 1989; Crawford, Evans & Fettiplace, 1989). There is therefore no evidence that transducer channels enter a true inactivated state.
Chapter
The sensory cell of the auditory, vestibular and lateral line organs is the hair cell, a highly specialised neuroepithelial cell designed to detect small perturbations applied to the stereocilia projecting from its apical membrane. The mechanical displacements induced by a sound stimulus may be only a few nanometres and this presents technical problems in both measurement and conceptualisation. In the case of the inner ear, the threshold for hearing has been measured to correspond to distortions of cochlear structures of about 0.3nm. How these small displacements are sensed, from the structure of the mechanosensing channel to the mechanics of the embedding tissue within the cochlea, to the neural encoding employed by the auditory pathway is the subject of auditory research. The purpose of this chapter is to highlight some topics which recur in this field. These are the facts. The fantasies are the models which attempt to summarize some of the experimental observations.
Chapter
One of the most intensely debated questions arising from the study of cellular responses to physical forces is how cells sense these mechanical stimuli and convert them into electrical, chemical or biochemical responses, the process known as mechanotransduction. Systems in which mechanotransduction is essential include the senses of hearing and touch as well as baroreception, the ability of the body to perceive blood pressure. In all three cases, mechanical forces move or deform specialized sensory structures such as hair cells (hearing); Pacinian corpuscles, Meissner’s corpuscles, and free nerve endings (touch); or baroreceptors. These physical stimuli lead to altered membrane potential, which in turn triggers the initiation of action potentials. In these examples it is widely accepted that the mechanosensitive elements are ion channels whose probability of being open is affected by physical deformation (reviewed by French1 and Sachs2,3). Once a physical stimulus is converted to an electrochemical signal, the transfer and amplification of the signal follows classic signal transduction pathways often utilized by agonists.
Article
Full-text available
Significance Geckos are lizards capable of vocalization and can detect frequencies up to 5 kHz, but the mechanism of frequency discrimination is incompletely understood. The gecko’s auditory papilla has a unique arrangement over the high-frequency zone, with rows of mechanically sensitive hair bundles covered with gelatinous sallets. Lower-frequency hair cells are tuned by an electrical resonance employing Ca ²⁺ -activated K ⁺ channels, but hair cells tuned above 1 kHz probably rely on a mechanical resonance of the sallets. The resonance may be boosted by an electromotile force from hair bundles found to be evoked by changes in hair cell membrane potential. This unusual mechanism operates independently of mechanotransduction and differs from mammals which amplify the mechanical input using the motor protein prestin.
Chapter
Various membrane-bound proteins function to exchange ions across the membrane. Some of these proteins are called a carrier since ionic transport must be coupled with the transport of another substances or it requires metabolic energy. The other transporting protein is called a channel and serves as a rapid and a large scale ion transport across the membrane. The channel is opened and closed by the inherent gate. Ions transported are selected by its filter function. The transport is fast and many thousands of ions are carried in milliseconds, and achieves the unitary conductance of 10 to 100 pS. The gate is the most unique function of the channel, and depending on its nature the channel becomes either voltage-dependent, agonist-dependent or achieves an energy-transducing function. The mechanoreceptor cell is therefore a cell equipped with an ion channel whose gate is mechanically opened and closed. The ionic flow regulated in this manner generates membrane-potential changes and triggers other voltage-dependent channel events. Ca channels are activated by depolarizing, transducing potentials, allow Ca ions to flow into the cell for synaptic transmission, and initiate higher order of information processing.
Chapter
The acousticolateral system of vertebrates includes the lateral-line organs, vestibular apparatus, and cochlea. Integration of these organs into a single system is based on the similarity of their functional and morphological characteristics, innervation, and embryonal development. The specialized receptors for the perception of weak electric fields in the lateral-line organs of some lower aquatic vertebrates may also be considered part of the acousticolateral system.
Chapter
The conversion of the mechanical stimulus of sound into an electrical signal by hair cells of the cochlea is the central event in hearing. Their bundles of actin-rich stereocilia bear the proteins essential for hearing at their tips. Filamentous tip links, composed of the cadherins PCDH15 and CDH23, stretch between the tips of adjacent stereocilia. Deflection of the bundle increases tension in tip links, which pull open mechanically gated ion channels that include TransMembrane Channel-like 1 (TMC1) and TMC2. Channel opening allows entry of the receptor current that can depolarize hair cells with a submillisecond time constant, generating a neural signal. Adaptation, mediated by at least two different mechanisms, relaxes the receptor current in milliseconds even with maintained bundle deflection. Different adaptation mechanisms cause the bundle to exert force, powering mechanical feedback to amplify the mechanical stimulus and tune the response to a certain frequency. Mammalian cochlear hair cells also show a voltage-dependent motility mediated by SLC26A5 (prestin) in the lateral wall, which contributes to amplification and tuning. Although much is understood about hair cell function, remaining questions include the molecular structure of the transduction complex, the nature of fast adaptation, and how motility of different components is integrated to produce amplification and tuning.
Chapter
Sound pressure fluctuations striking the ear are conveyed to the cochlea, where they vibrate the basilar membrane on which sit hair cells, the mechanoreceptors of the inner ear. Recordings of hair cell electrical responses have shown that they transduce sound via submicrometer deflections of their hair bundles, which are arrays of interconnected stereocilia containing the mechanoelectrical transducer (MET) channels. MET channels are activated by tension in extracellular tip links bridging adjacent stereocilia, and they can respond within microseconds to nanometer displacements of the bundle, facilitated by multiple processes of Ca2+-dependent adaptation. Studies of mouse mutants have produced much detail about the molecular organization of the stereocilia, the tip links and their attachment sites, and the MET channels localized to the lower end of each tip link. The mammalian cochlea contains two categories of hair cells. Inner hair cells relay acoustic information via multiple ribbon synapses that transmit rapidly without rundown. Outer hair cells are important for amplifying sound-evoked vibrations. The amplification mechanism primarily involves contractions of the outer hair cells, which are driven by changes in membrane potential and mediated by prestin, a motor protein in the outer hair cell lateral membrane. Different sound frequencies are separated along the cochlea, with each hair cell being tuned to a narrow frequency range; amplification sharpens the frequency resolution and augments sensitivity 100-fold around the cell's characteristic frequency. Genetic mutations and environmental factors such as acoustic overstimulation cause hearing loss through irreversible damage to the hair cells or degeneration of inner hair cell synapses. © 2017 American Physiological Society. Compr Physiol 7:1197-1227, 2017.
Chapter
In early time, the coarse signals, such as cochlear microphonics and summating potentials, were recorded by electrocochleography by placing a metal electrode on the round window, which reflected the sound-induced electrical responses mostly mediated by hair cells. However, these signals are mainly a summated response from a group of hair cells. The direct evidence came from the intracellular recording of hair cells in the tail lateral line of mudpuppy Necturus maculosus (Harris et al., Science 167(3914):76–79, 1970). Similarly, auditory response from the cochlear hair cells was probed by intracellular recording in guinea pig (Russell and Sellick, Nature 267(5614):858–860, 1977; J Physiol 284:261–290, 1978). In isolated bullfrog saccule tissue, the mechanotransduction (MET) current was recorded in hair cells, which provided the first evidence that the deflection of hair bundle induced receptor potential change of hair cells (Hudspeth and Corey, Proc Natl Acad Sci USA 74(6):2407–2411, 1977). Nevertheless, whole-cell patch clamp was applied to hair cells to achieve a detailed information of the MET channel including some single-channel behaviour (Ohmori, J Physiol 359:189–217, 1985). From then on, researchers have studied most of the biophysical properties of the channel systematically by electrophysiology, pharmacology, and optical imaging without knowing the molecular identity of the MET channel. Now several important questions have been tackled in this chapter, including the following: Where does the channels localize in the hair cells? What kind of ions do the channels pass through? How are the channels activated and then adapted? How many channels are there opened per tip link? What are the single-channel properties?
Conference Paper
Cochlear hair cell stereocilia move semi-independently, shaping the force transfer to mechanoelectrical transduction (MET) channels, as indicated by the MET current response. Semi-independent movement of stereocilia was evoked by stimulating inner hair cell (IHC) bundles from acutely dissected rat cochlea with stiff probes ranging in size from 1 to 10 µm. MET current responses were recorded using whole-cell patch-clamp electrophysiology. Small probes directly displaced stereocilia they contacted, and recruited adjacent stereocilia depending on stimulus magnitude. We inferred that the recruitment of stereocilia resulted in less uniform and less synchronous movement. Step displacements using smaller probes resulted in smaller current responses (from 1 nA for large probes to 0.3 nA for small, p <.0001), slower rate of current activation, as measured from the linear portion (from 4 nA/ms to 1 nA/ms, p <.0001), slower time constants of adaptation, as measured from double exponential fits from peak to steady state current (fast component: from 0.6 to 1.2 ms, p =.004; slow component: from 8 ms to 12 ms, p =.001) and less complete adaptation (from 95% to 30%, p <.0001). These results indicate that the mechanical properties of less coherent bundles greatly affect force transfer to MET channels as indicated by the electrical response of the cell. Thus, outer hair cells (OHCs), with their bundles embedded in the tectorial membrane, may exhibit synchronous MET activation and therefore time-dependent adaptation where fast adaptation provides a high pass filter. Hair cells with free standing bundles, like inner hair cells (IHC), may exhibit more asynchronous MET activation and adaptation, in which case adaptation would not provide this additional filter.
Article
Transmembrane channel-like (TMC) 1 and 2 are required for the mechanotransduction of mouse inner ear hair cells and localize to the site of mechanotransduction in mouse hair cell stereocilia. However, it remains unclear whether TMC1 and TMC2 are indeed ion channels and whether they can sense mechanical force directly. Here we express TMC1 from the green sea turtle (CmTMC1) and TMC2 from the budgerigar (MuTMC2) in insect cells, purify and reconstitute the proteins, and show that liposome-reconstituted CmTMC1 and MuTMC2 proteins possess ion channel activity. Furthermore, by applying pressure to proteoliposomes, we demonstrate that both CmTMC1 and MuTMC2 proteins can indeed respond to mechanical stimuli. In addition, CmTMC1 mutants corresponding to human hearing loss mutants exhibit reduced or no ion channel activity. Taken together, our results show that the CmTMC1 and MuTMC2 proteins are pore-forming subunits of mechanosensitive ion channels, supporting TMC1 and TMC2 as hair cell transduction channels.
Chapter
Sound signal is transduced to electrical signal in hair cells, transmitted across synapse to auditory nerve fibers (ANFs), and along the ascending pathway to the cochlear nucleus in the brainstem, then to the midbrain, and through the auditory thalamus to the auditory cortex. In every step, auditory signal has temporal structures representing the sound. I will discuss in this chapter two fundamental attributes of hair cell transduction: (1) how the receptor potential is generated in hair cells, and (2) how the hair cell receptor potential is transmitted across synapse to the auditory nerve fiber.
Article
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Objective: Benign paroxysmal positional vertigo (BPPV) is a common cause of peripheral vertigo in the general population. We investigated the role of 25-hydroxy (25- OH) vitamin D deficiency in BPPV by comparing 25-OH vitamin D levels in healthy controls and in patients with BPPV. Methods: 25-OH vitamin D levels of 125 patients with idiopathic BPPV who were diagnosed at our clinic between January 2018 and September 2018 and 101 healthy controls without vertigo were compared statistically. Results: In this study, vitamin D deficiency was detected in patients diagnosed with BPPV, but there was no statistically significant difference with the control group. Conclusion: The prevalence of the vitamin D deficiency is very high in our population. Despite the major studies in the literature, vitamin D deficiency was not related to BPPV as a result of this research. Keywords: Benign paroxysmal positional vertigo, 25-hydroxy vitamin D, peripheral vertigo
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Inner-ear sensory hair cells convert mechanical stimuli from sound and head movements into electrical signals during mechanotransduction. Identification of all molecular components of the inner-ear mechanotransduction apparatus is ongoing; however, there is strong evidence that TMC1 and TMC2 are pore-forming subunits of the complex. We present molecular dynamics simulations that probe ion conduction of TMC1 models built based on two different structures of related TMEM16 proteins. Unlike most channels, the TMC1 models do not show a central pore. Instead, simulations of these models in a membrane environment at various voltages reveal a peripheral permeation pathway that is exposed to lipids and that shows cation permeation at rates comparable to those measured in hair cells. Furthermore, our analyses suggest that TMC1 gating mechanisms involve protein conformational changes and tension-induced lipid-mediated pore widening. These results provide insights into ion conduction and activation mechanisms of hair-cell mechanotransduction channels essential for hearing and balance.
Chapter
Electrophysiological characterization of inner ear hair cell properties including assessment of voltage-dependent and mechanosensitive currents has provided invaluable information about their development and maturation in several animal models. Beyond the basic understanding of hair cell properties, electrophysiological investigations combined with the use of different mouse models, pharmacological tools, and exogenous gene expression systems such as those driven by viral vectors have been essential in providing insights into the functional role of various proteins expressed in hair cells, many of which are associated with deafness and/or balance deficits. This chapter provides detailed methods designed to optimize recordings of voltage-dependent and mechanosensitive currents in sensory hair cells of the auditory and vestibular organs of the mammal.
Chapter
Auditory perception in mammals starts from the ear, the periphery part of the auditory system. The cochlear hair cells use their hair bundle to convert sound-induced mechanical vibration into receptor potential and then utilize ribbon synapse to convey the electrical signals to ascending spiral ganglion neurons. This biological process is called auditory transduction that is the first step and also the most essential part of hearing sensation. However, to achieve a competent processing capability of acoustic cues in large dynamics, high fidelity, and wide range, many astonishing molecular design and amazing biophysical assembly have been applied for the cochlear hair cells to fulfil the auditory transduction.
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1. The extracellular patch clamp method, which first allowed the detection of single channel currents in biological membranes, has been further refined to enable higher current resolution, direct membrane patch potential control, and physical isolation of membrane patches. 2. A description of a convenient method for the fabrication of patch recording pipettes is given together with procedures followed to achieve giga-seals i.e. pipette-membrane seals with resistances of 10(9) - 10(11) omega. 3. The basic patch clamp recording circuit, and designs for improved frequency response are described along with the present limitations in recording the currents from single channels. 4. Procedures for preparation and recording from three representative cell types are given. Some properties of single acetylcholine-activated channels in muscle membrane are described to illustrate the improved current and time resolution achieved with giga-seals. 5. A description is given of the various ways that patches of membrane can be physically isolated from cells. This isolation enables the recording of single channel currents with well-defined solutions on both sides of the membrane. Two types of isolated cell-free patch configurations can be formed: an inside-out patch with its cytoplasmic membrane face exposed to the bath solution, and an outside-out patch with its extracellular membrane face exposed to the bath solution. 6. The application of the method for the recording of ionic currents and internal dialysis of small cells is considered. Single channel resolution can be achieved when recording from whole cells, if the cell diameter is small (less than 20 micrometer). 7. The wide range of cell types amenable to giga-seal formation is discussed.
Article
It is now almost one hundred years since Sidney Ringer (1882) described the importance of Ca2+ in the maintenance of frog heart contractility. Subsequent to this observation, it has been increasingly recognized that Ca2+ plays a critical and central role in a multitude of biological events at both the intra-and extracellular levels (Duncan, 1976; Kretsinger, 1976a; Table 1). However, Ca2+ distribution across the cell membrane is far from equilibrium, since if the resting membrane potential (~-60mV) were equal to the Ca2+ equilibrium potential, then the intracellular Ca2+ activity should be some 100-fold greater than the extracellular activity. This is quite clearly not so and although accurate measurements of free ionized intracellular Ca2+ concentrations have not been made in many systems the concensus of evidence firmly indicates that [Ca int2+] < 10-7M (Baker, 1972; 1976; Blaustein, 1974; Reuter, 1973). Such a low intracellular Ca2+ concentration accords with the binding constants of Ca2+ for those intracellular proteins whose activity is known to be modulated by Ca2+ (pKD values ~ 6–7; Kretsinger, 1976a,b) and indicates the “trigger” functipn of an increased intracellular Ca2+ concentration (Heilbrunn, 1956).
Article
1. The lateral line organ ofNecturus maculosus was stimulated with water vibrations, and the degree of synchronization between stimulus and afferent activity was related to the ionic composition of the external solution. 2. The mechano-sensitivity was a function of the Ca++ concentration of the external medium (Fig. 2). The organs were insensitive to vibrations in Ca++ free solution (containing Ca-chelating agents) (Fig. 5), whereas the sensitivity leveled off at a maximum value for concentrations above about 1 mM Ca++. The effect of Sr++ was similar to Ca++ (Fig. 4). K+ and Na+ also enhanced the mechano-sensitivity, but the effect of these ions was much less than for Ca++ (Fig. 3). 3. The mechano-sensitivity was suppressed by Mg++, Co++ and La+++ (Figs. 7, 8), the order of effectiveness being La+++ Co++> Mg++. The suppression decreased with increasing Ca++ concentration, suggesting that the effect of Ca++ is competitively blocked by these ions. The mechano-sensitivity was also suppressed by low pH (Fig. 9). 4. The different ions tested in the present study affected the generation of hair cell receptor potentials, and it is suggested that the inward depolarizing receptor current of hair cells in mudpuppy lateral line organs is mainly carried by Ca++.
Article
1. The lateral line organ ofNecturus maculosus was stimulated with extracellular sine wave current, and the effect on the afferent activity was compared to the effect of mechanical vibration. Both mechanical and electrical stimulation caused phase locking between stimulus and afferent spikes (Figs. 2, 3). 2. Intracellular recordings were made from the three main cell types of the neuromast: hair cells, supporting cells and afferent nerve terminals. Mechanical stimulation evoked receptor potentials of less than 1 mV in the hair cells. These potentials were synchronized with the stimulus (Fig. 6E), and mechanical stimulation caused a corresponding synchronization of action potentials in the afferent nerve terminals (Fig. 6A). The supporting cells were insensitive to vibrations (Fig. 6C). 3. Intracellular injection of sine wave electrical current into hair cells caused synchrony between stimulus and afferent spikes (Fig. 6F), whereas even ten times more intense current was insufficient to cause such synchrony if the afferent nerve terminals were injected directly (Fig. 6B). Supporting cells were insensitive to electrical current stimulation (Fig. 6D). 4. The effective intracellular current injections in the hair cells caused membrane potential changes which overlapped in magnitude with the naturally occurring receptor potentials. The conclusion is therefore that the mechanically evoked receptor potentials in hair cells have a direct effect on the transmitter release, and the potentials are not an epiphenomenon caused by the secretory activity of the hair cells.
Article
Siliconized, glass micropipets whose tips were filled with oil were used to obtain small (Gerrhonotus multicarinatus), and 8 skates (Raja erinacea). Samples of cerebrospinal fluid and seawater were also obtained for skates. Electron probe microanalysis was used to measure the concentrations of the following elements in each sample: K, Na, Cl, Ca, Mg, P, S. The Na and K concentrations in cat perilymph (Fig. 1 and Table 2) agree with previous estimates (Table 4) while endolymph samples show relatively low Na and high K concentrations. From a comparison of our results with previous work (Table 3), we infer that contamination of endolymph samples with perilymph is relatively low in our study, and that no large species difference in endolymph content is indicated by present data available for mammals. Our results show that Cl concentration is higher and Ca and Mg concentrations are lower in endolymph than in perilymph. The composition of perilymph in cats and alligator lizards is roughly the same (Figs. 1 and 2, Table 2). Uncontaminated endolymph samples in lizards were apparently difficult to obtain, although the compositions of a few samples suggest that endolymph K concentration is high and Na concentration is low. In skates the concentration of Na is nearly the same in the two inner ear lymphs (Fig. 3 and Table 2), in contrast to the roughly hundredfold ratio of perilymph to endolymph Na concentrations found in the higher vertebrates. The element composition of perilymph is correlated with the composition of seawater in which the skates were kept, whereas the endolymph composition shows no such correlation.
Article
The ototoxicities of eight aminoglycoside antibiotics and fragments were measured quantitatively by cochlear perfusion in the guinea pig. Perilymphatic spaces were perfused for 1 hr with ‘artificial perilymph’ containing 10 mM drug, during which time continuous measurements of cochlear microphonic potentials were made. Kanamycin B and neomycin B caused the most rapid decline of cochlear microphonic potentials, followed by gentamicin C1a ∼- ribostamicin > kanamycin A ∼ G-418. Neamine and methylneobiosamine did not show significant effects. The same drugs were tested for their interaction with monomolecular films of polyphosphoinositides, and relative binding constants were determined. Neomycin B and kanamycin B had the highest affinities to the lipids, followed by the other drugs in the order as seen for toxicity. The correlation between the in situ and in vitro actions of the drugs was r = 0.9. These results support the hypothesis that binding to polyphosphoinositides plays an important role in the decrease of the cochlear microphonic potentials. Furthermore, the good correlation between the drug actions in the two test systems suggests that an in vitro assay may be possible for the assessment of aminoglycoside ototoxicity.
Article
Impedance and potential measurements have been made on a number of artificial membranes. Impedance changes were determined as functions of current and of the composition of the environmental solutions. It was shown that rectification is present in asymmetrical systems and that it increases with the membrane potential. The behavior in pairs of solutions of the same salt at different concentrations has formed the basis for the studies although a few experiments with different salts at the same concentrations gave results consistent with the conclusions drawn. A theoretical picture has been presented based on the use of the general kinetic equations for ion motion under the influence of diffusion and electrical forces and on a consideration of possible membrane structures. The equations have been solved for two very simple cases; one based on the assumption of microscopic electroneutrality, and the other on the assumption of a constant electric field. The latter was found to give better results than the former in interpreting the data on potentials and rectification, showing agreement, however, of the right order of magnitude only. Although the indications are that a careful treatment of boundary conditions may result in better agreement with experiment, no attempt has been made to carry this through since the data now available are not sufficiently complete or reproducible. Applications of the second theoretical case to the squid giant axon have been made showing qualitative agreement with the rectification properties and very good agreement with the membrane potential data.
Article
Vertebrate hair cells, the primary receptors of auditory, vestibular and lateral-line organs, occur in epithelia which separate fluids of differing ionic composition. The apical surfaces of hair cells, on which the mechanosensitive hair bundles are situated, face a high-K+ fluid (termed endolymph in the inner ear); the basolateral surfaces instead contact fluid (perilymph or a related substance) of a composition similar to that of other extracellular fluids1-3. The universal occurrence of high-K+ fluid on the apical surfaces of hair cells in vertebrates has been taken as evidence that it is important for the transduction process, in particular that it relates to the ionic specificity4 of the conductance change5 underlying the receptor potential. There is, however, conflicting experimental evidence regarding this specificity. K+ has generally been thought to carry the receptor current, as replacement of endolymph with perilymph in the guinea pig cochlea abolishes the extracellularly recorded microphonic potential6. Yet microphonic potentials, as well as intracellular receptor potentials, have been recorded in other preparations when the apical surfaces of the hair cells faced instead a high-Na+ saline, and thus when the electrochemical gradient for K+ was near zero5,7. Ca2+ has also been proposed to carry the receptor current8, but its concentration is quite low in endolymph3, particularly that of the mammalian cochlea9. We present evidence here that the receptor current in a vertebrate hair cell is carried in vivo by K+, but that the transduction channel is in fact nonspecific, being permeable to Li+, Na+, K+, Rb+, Cs+, Ca2+, and at least one small organic cation.
Article
This chapter discusses the evolution and function of calcium-binding proteins. Muscle calcium-binding parvalbumin (MCBP) amino acid sequences have been determined for carp, pike, frog, and coelocanth, and rabbit. The troponin “trimer” consists of tropomyosin-binding component (TnT, MW 37,000), an inhibitory component (TnI, MW 23,000), and a calcium-binding component (TnC, MW 17,846). TnT binds to tropomyosin either free or complexed with actin. In addition, the TnC-TnI complex, which is quite stable, binds strongly to actin-tropomyosin in the absence of Ca2+. Consistent with the model, the affinity of troponin for the actin-tropomyosin complex is reduced in the presence of calcium. TnI alone or as the TnI-TnT complex has a weak inhibitory effect on actomyosin ATPase. TnC binds to neither actin nor tropomyosin. Phosphofructokinase catalyzes the phosphorylation of fructose 6-phosphate, thereby forming fructose 1,6-diphosphate. The chapter discusses the mitochondrial enzymes with possible calcium involvement. The activity of kynurenine aminotransferase in intact mitochondria is increased threefold by the addition of 10-3 M CaCl2; however, calcium has no effect on the solubilized mitochondrial enzyme. These responses, which are hardly unusual for membrane enzymes, are interpreted in terms of enhanced translocation of the substrate, α-ketoglutarate, by Ca2+. Other intracellular enzymes that binds calcium is also discussed.
Article
1. A three-electrode voltage clamp method was used to investigate the effects of Ba and Sr ions on the inwardly rectifying K conductance of resting frog sartorius muscle fibres. 2. When Ba2+ (0.01-5 mM) was added to the control (115 mM-K+) solution the inward currents recorded during hyperpolarizing voltage steps turned off exponentially with time as the blockade by Ba2+ developed. Outward currents showed no time-dependence. 3. Ba2+ ions reduced both the instantaneous and the steady-state values of currents recorded on hyperpolarization. The blockade was potential-dependent, steady-state currents being increasingly reduced with increasing hyperpolarization. 4. The concentration-effect relation for the blockade of instantaneous currents by Ba2+ could be fitted assuming 1:1 binding of Ba2+ to a receptor, with the block being proportional to the number of Ba2+-filled receptors. The apparent dissociation constant at the holding potential (-5 mV) was 0.65 mM. Concentration-effect relations were shifted along the concentration axis to lower concentrations by hyperpolarization. The apparent dissociation constant was reduced e-fold for a 16.8 mV change in potential. 5. Increasing the [Ba]o increased the rate of onset of the blockade at a given potential. 6. The rate of onset of the blockade had a high temperature dependence (Q10 = 3.15 +/- 0.08). 7. When [K]o was doubled to 230 mM, under conditions where [K]i was also doubled, [Ba]o had to be raised approximately fourfold to produce the same degree and rate of onset of blockade. Similarly, when [K]o was decreased, the degree and rate of onset of blockade were increased for a given [Ba]o. 8. The blockade could be readily removed by removal of Ba2+ from the bathing solution. In addition the blockade which develops on hyperpolarization is removed exponentially on return to the holding potential. 9. The blockade which exists at the holding potential may be removed by a depolarizing prepulse. 10. Sr causes a similar potential-dependent blockade to that by Ba2+, but is around 400 times less effective. 11. The results have been fitted with a model assuming that the permeability mechanism is an aqueous pore with a site which binds one Ba2+ ion or two K+ ions. The site must have affinity for Ba2+ and a low affinity for K+.
Article
1. The blocking effects of Ba+ and H+ on the inward K current during anomalous rectification of the giant egg membrane of the starfish, Mediaster aequalis, were studied using voltage clamp techniques. 2. External Ba2+ at a low concentration (10--100 micron) suppresses the inward K current; the extent of suppression, expressed as the ratio of currents with and without Ba2+, can be described by a conventional bimolecular adsorption isotherm, K/(K + [Ba2+]o), K being an apparent dissociation constant. 3. The dissociation constant, K, decreases as the membrane potential V becomes more negative and can be expressed by K(V) = K(0) exp (zmuFV/RT), where K(0) is the K at V = 0, z is the charge of the blocking ion, and mu is a parameter for the membrane potential dependence of Ba2+ blockage. The value of mu ranges between 0.64 and 0.68. 4. Upon a sudden change in membrane potential the change in the blocking effect of Ba2+ follows first order kinetics; the forward rate constant is membrane-potential-dependent whereas the backward constant is potential-independent. 5. The blocking effect of Ba2+ appears to be independent of the activation of K channels during anomalous rectification. 6. The blocking effect of Ba2+ depends on V alone, in contrast to the activation of the K channel during anomalous rectification which depends on V--VK. 7. In these respects, the effect of Ba2+ is equivalent to the introduction of inactivation into the anomalous rectification. 8. SI2+ and Ca2+ show small but observable blocking effects only at much higher concentrations (about 10--20 mM). 9. The inward K current is suppressed when the external pH is reduced below 6.0. The blocking effect of H+ shows no significant potential dependence. The concentration dependence suggests that three H+ ions simultaneously titrate the acidic groups of each channel (pK = 5.3--5.4). 10. The implications of these results are discussed in terms of molecular models of the potassium channel of anomalous rectification and possible mechanisms of K channel inactivation.
Article
The papillae basilares of three species of turtles and four species of snakes were studied by SEM. The papillae of turtles are relatively large among reptiles and are characterized by a long, horizontal middle section resting on a wide basilar membrane. Both terminal ends of the papilla extend onto the surrounding limbus in the form of a forked or “T”-shaped end or as a curved, “hook”-like process. Details vary with the species. In the three species of turtles studied, there were between 1,100 and 1,400 hair cells on a papilla. The tectorial membrane covering the horizontal portion of the papilla is heavy in appearance and tightly attached to the kinocilial bulbs. The terminal ends of the papilla are covered by a thin gelatinous material. In addition, a mat-like tectorial network covers the supporting cells and extends from the microvilli of the supporting cells to the overlying tectorial membrane. All hair cells are unidirectionally and abneurally oriented. The supporting cell surfaces form a large part of the papilla and, thus, hair cell density is low. The papillae of the two boid snake species studied are moderately long among snakes and contain a moderate number of hair cells (574 in Epicrates and 710–780 in Constrictor). Papillar form is elongate, ovoid, or canoe-shaped. The tectorial membrane may be either highly fenestrated or moderately dense and covers all but a few of the terminal hair cells. A tectorial-like mat covers all but a few of the terminal hair cells. Most hair cells are unidirectionally and abneurally oriented. A few terminal cells in boids may show reverse orientation. Hair cell density is similar to that of turtles.
Article
IONIC calcium is important in many membrane functions and the possibility of it being involved in the cochlear transduction processes is supported by the scanty evidence available1. In the analogous lateral line organ of submammalian species, the mechanosensitivity of the hair cells is related directly to the external calcium concentration at their specialised receptor poles2. There is also evidence of interaction with monovalent cation effects2,3. In mammals, the cochlear duct is filled with endolymph, the unusual constitution of which (in the rat d.c. potential + 88 mV, K+ 148 mM, Na+ 0.84 mM) is seemingly maintained entirely by active transport mechanisms4 and is generally conceded to be necessary for normal transduction. We report here that the cochlear endolymph calcium occurs almost entirely in the non-complexed state at a concentration of 3×10-5 M in the rat. This is substantially below the usual extracellular concentration, and must influence the function of the receptor pole membranes and might well affect the role of calcium in the transduction process in the cochlea. The total magnesium concentration we measured was 1×10-5 M.
Article
1. Intracellular recordings were made from inner hair cells in the first turn of the guinea-pig cochlea, the recording sites being confirmed by the injection of Procion yellow dye and subsequent histology. 2. The receptor potential, in response to a pure tone burst, consisted of an AC response which followed the wave form of the stimulus and was analogous to the extracellularly recorded cochlear microphonic and a depolarizating DC response which followed the envelope of the tone burst and was analogous to the extracellularly recorded summating potential. 3. The DC response was broadly tuned at high sound pressure having a maximal amplitude of 27 mV at a sound pressure level of ca. 100 db; however the bandwidth of the response was reduced at lower sound pressure level. Isoamplitude curves for the DC response were indistinguishable from the threshold curves for auditory nerve fibres. 4. The AC response was tuned in a similar fashion to the DC response except that it was attenuated at 6-9 db/octave with respect to the DC response. It is suggested that this difference was due to the effect of membrane capacitance and resistance on the AC response. In contrast the extracellularly recorded AC component was not subject to this attenuation. 5. The total resistance and capacitance in three cells were found to be 46-61 Momega and 7.8-15.8 muF respectively. 6. Intracellular resistance changes were measured during sound stimulation, the resistance change being proportional to the DC receptor potential, indicating constant current flow through the hair cell. The current varied between 0.37 and 0.81 nA between cells. The time constant for seven cells was found to lie between 0.31 and 0.76 msec. 7. A map of the basilar membrane showing position of hair cells against characteristic frequency corresponded to the cut-off frequencies of the basilar membrane mechanical measurements and the innervation sites of spiral ganglion cells.
Article
Sensory cells in the pigeon's basilar papilla can be divided into three groups on the basis of shape, cuticular plate dimensions, and innervation. Tall hair cells are columnar with deep cuticular plates and are found over the superior cartilaginous plate from proximal to distal end of papilla; they cover the entire papilla at distal tip. Large cochlear and small efferent nerve endings terminate on their basal ends. Short hair cells are pitcher-shaped, with flattened cuticular plates and are found only on the proximal two-thirds of the free basilar membrane. They have small cochlear and large efferent nerve terminals. Transitional cells are present in between. The efferent nerve fibers, as revealed by the AChE stain, form extensive networks from which many branches are given off to the hair cells. The cochlear nerve fibers, as revealed by the Holmes silver stain, take transverse courses across the papilla with few branches before terminal ramification.Supported by N.I.H. grants Nos. NB 00966-13 and NS 08813-01.
The relationship between the high positive potential and the high potassium and low sodium concentrations within the endolymph has been investigated in the adult rat. Very small, (2 nl.) uncontaminated samples of cochlear endolymph and perilymph have been collected and the endolymph potential measured at the times of collection. The sodium and potassium contents of the samples were estimated by means of total emission, integrative flame spectrophotometry. In the course of the procedure a number of serious problems were encountered, in particular that arising from the extremely small sodium content of the endolymph. For their solution a number of technical improvements were required, including the development of a new type of burner. A measure of the sensitivity thus attained is provided by the finding that, using test samples containing 4$\cdot $8 $\times $ 10$^{-12}$ mequiv. (1$\cdot $1 $\times $ 10$^{-13}$ g) of sodium, the standard deviation of the analytical results was $\pm $ 8$\cdot $4 $\times $ 10$^{-13}$ mequiv. ($\pm $17$\cdot $6%). With a solution comparable in composition to endolymph the standard deviation was $\pm $6% for sodium and $\pm $1$\cdot $3% for potassium. The analytical results showed the values of the sodium and potassium concentrations in the endolymph to be 0$\cdot $91 and 154 mequiv./l. respectively. In perilymph these values were 138 and 6$\cdot $9 mequiv./l. The average endolymphatic potential was + 92 mV. During anoxia the positive endolymphatic potential was replaced by a negative potential reaching, on average, a maximum of -42 mV after 4$\frac{1}{2}$ min and thereafter slowly returning to zero. The principal ionic changes were a progressive increase in the endolymphatic sodium concentration from 3$\cdot $6 mequiv./l. after 2 min anoxia to 32 mequiv./l. after 30 min anoxia and an associated decrease in the endolymphatic potassium concentration to 116 mequiv./l. after 30 min anoxia. These results establish that the low sodium content of the endolymph is maintained by means of an active transport mechanism which is probably situated in the stria vascularis. It thus appears that the characteristic composition of the endolymph is due to the active transfer of sodium and chloride from and potassium into the cochlear duct and that the mechanisms concerned are highly dependent on oxidative metabolism. The possible inter-relationship of these mechanisms and the origin of the endolymphatic potential are briefly discussed but are considered to be still obscure.
Article
The basilar papilla of the lizard Calotes versicolor contains two different types of sensory cells. The ventral part of the organ is populated by the short-haired type A sensory cells whereas the dorsal part is populated by the long-haired type B sensory cells. The type A sensory cells are all unidirectionally oriented away from the cochlear nerve while the type B cells are bidirectionally oriented towards the midline of the organ. The ventral part of the basilar papilla, that is the type A sensory cell population, is covered by a tectorial membrane. Close to the base of the stereocilia in the sensory hair bundle a connective zone is seen linking them together. At the top of the kinocilium thin filaments are seen bridging the gap to the closest stereocilia, thus linking the kinocilium to the rest of the sensory hair bundle. Fine filaments are also seen linking the bulbous tip of the type A sensory cell kinocilium with the tectorial membrane.Copyright © 1974 S. Karger AG, Basel
Article
Experiments were performed on single, isolated muscle fibers of the crabCallinectes danae to investigate the effects of neomycin and streptomycin on membrane Ca++ conductance in relation to excitation-contraction coupling (ECC). Neomycin (2–8 mg/ml) hyperpolarizes the fiber and increases the effective membrane resistance. The antibiotic increases the threshold for, and reduces the amplitude of the graded membrane responses which result from activation of Ca++ conductance on membrane depolarization. Treatment of the fiber with either procaine or tetraethylammonium (TEA) converts the graded responses into regenerative Ca-spikes. Neomycin (2–4 mg/ml) increases spike threshold, reduces the rate of rise of the upstroke and shortens the duration of the action potentials. With higher neomycin concentrations (up to 8 mg/ml) the spikes are abolished. Streptomycin (8 mg/ml) is also capable of blocking Ca-spikes. As a consequence of their effects on membrane Ca++ conductance the antibiotics reduce the mechanical output associated with both graded responses and Ca-spikes. The data support the postulate that membrane Ca++ activation is an essential step in ECC in skeletal muscle.
Article
Breuer noted that tuft-like structures projected from the surface of the receptor epithelium. He attributed the receptor function to the shearing motion of these hairlike masses. With electron microscopy, two types of cilia are found in the tuft: (1) numerous stereocilia, which are positioned over a relatively ridged cuticular plate that is placed at the luminal end of the cell, and (2) a single kinocilium, which stands over a notch in this plate. Because all the kinocilia are on the same side of the tuft, Lowenstein and Wersall were able to relate this morphological polarity to the activity within the vestibular nerve. A force that was directed toward the kinocilium caused an increase in the activity of the vestibular nerve while a decrease in activity was seen when the force was directed away from the kinociliary side of the cell. The action of the ciliary hairs in the otolithic organs following a positional change shows that the sterocilia have a sliding motion at their distal ends. The kinocilia, however, are different in that the distal portion of the kinocilium, at least in the case of the frog, is attached to the adjacent stereocilia. For this reason, the kinocilium cannot slide at its distal end but it is displaced at its base. The cupular zone near the center of the crista would have minimal resistance and could allow for responses to small movements. On the other hand, during large endolymph changes, attachment of a cupula to the crista through the kinocilia and filaments holds the cupular base to this structure while the apex breaks away, thereby giving the classical swinging door appearance seen by Steinhausen, Dohlman, and other investigators.
Article
Microphonic potentials were recorded from the sacculus of goldfish with glass pipette electrodes to study the effects on the potentials of changes in the ionic composition of endo-and perilymph and of administered ouabain. Ions and drugs were applied with perfusion techniques and special attention was paid to the difference in effects between endo-and perilymphatic applications. The results obtained were as follows:1. No marked changes were found in the amplitude of microphonic potentials when the endolymph was replaced either with 120mm NaCl or with KCl.2. Microphonic potentials were reduced in size when the perilymphatic K+ concentration was raised, and this reduction was reversible when the solution returned to normal. The amplitude of microphonic potentials was linearly related to the logarithm of perilymphatic K+ concentrations in a range between 25 and 120mM.3. A marked reduction in the amplitude of microphonic potentials was produced when over 5×10-6g/ml of ouabain was applied to the perilymphatic space. An endolymphatic application of ouabain produced no effects even at much higher concentrations. This finding suggests the presence of an active extrusion mechanism of Na+ across the membrane at the basal part of hair cells. Functional differentiation between the membrane at the hair-bearing surface and at the basal part of hair cells and the functional significance of a high K+ concentration of the endolymph were discussed.
Article
Observations made with a scanning electron microscope confirm the binding of the stereocilia to a matchhead-like bulbous terminal at the apex of the kinocilium in frog saccular receptor cells. Since the kinocilium is shown to rest on a portion of the receptor cell that lacks the rigid cuticular base of the stereocilia, movenment of the ciliary ensemble results in a "plunging-like" effect of the kinocilium which produces a distension of the membranc at its base. This membrane distension is envisaged as bringing about the ionic conductance changes necessary for the production of a generator potential and, thus, for the transduction of movement into vestibular nerve activity.
Article
The effects of streptomycin and kanamycin on the goldfish's saccular microphonic potentials were examined following their local application with a perfusion technique. The results obtained were compared with those of other drugs.Streptomycin and kanamycin suppressed microphonics only when they were administered intraluminally to the sacculus but showed almost no effect when administered extraluminally.Metabolic inhibitors (cyanide, azide, DNP), quinine, and procaine also suppressed microphonics, but, unlike streptomycin and kanamycin, there was no difference in their effects if administered to the luminal side or to the extraluminal side. Namely, cyanide and azide at 5×10-4g/ml, DNP at 5×10-3g/ml, quinine at 1×10-4g/ml and procaine over 2×10-3g/ml suppressed microphonic potentials through either route of administration. Tetrodotoxin and salicylates showed no effect on microphonics.The site and the possible mechanism of action of streptomycin and kanamycin on hair cells were discussed by comparing their effects with those of other drugs.
Article
The ionic currents in enzymatically isolated vestibular hair cells of the chick were studied by a whole-cell-clamp variation of the patch voltage clamp, and by single channel recording. At membrane potentials more negative than -80 mV the hair cell showed anomalous rectification, and at potentials more positive than -40 mV large outward K currents were observed in normal saline. The outward K current decreased at large positive potentials, showing an N-shaped I-V relation. The outward K current was carried mostly through the Ca-activated K channel. K currents through the anomalous rectifier channel showed a decay in normal saline. This decay was eliminated reversibly in Na-free saline when the isotonic KCl-EGTA solution was used as the internal medium. However, a fast decay was still observed in Na-free high-K external solution when isotonic CaCl-EGTA was used as the internal medium. An increase in [K]o decreased the decay rate of the inward K current. The single-channel conductance of the anomalous rectifier channel was 50 pS in 160 mM-K saline and 23 pS in 40 mM-K saline. In 100 mM-Ca, -Sr and -Ba salines a large inward current was observed. At positive potentials the inward current carried by Ca and Sr ions showed significant decay; the current became outward at large positive potentials. Since the decay of the inward current was eliminated when 100 microM-quinine was added to the bathing medium, it was probably due to the activation of some Ca-activated K conductance which remained even with isotonic CsCl-EGTA internal medium. The activation kinetics of the Ca channel were studied in 100 mM-Ba solution at low temperatures (9-13 degrees C). From a comparison of the time constants of activation with the time constants of the tail currents, it was concluded that the Ca channel follows Hodgkin-Huxley-type m2 kinetics. A slow component that deviated from m2 kinetics was frequently observed at relatively large positive potentials. The steady-state fluctuations of Ba current showed a power density spectrum reasonably well fitted by a sum of two Lorentzian functions. The spectrum has a low-frequency component which indicates kinetics close to the macroscopic activation process of the Ca channel and a high-frequency component that indicates very fast flickering kinetics operating in the Ca channel.
Article
The relationship between calcium current and transmitter release was studied in squid giant synapse. It was found that the voltage-dependent calcium current triggers the release of synaptic transmitter in direct proportion to its magnitude and duration. Transmitter release occurs with a delay of approximately 200 mus after the influx of calcium. A model is presented which describes these relations formally.
Article
Acetylcholine (ACh) activates in the synaptic membrane of skeletal muscle an inward current composed of many elementary currents1,2. High resolution current measurements in adult frog muscle have shown that the elementary current is a pulse-like event of unit amplitude, indicating that ACh opens ion channels which have only two conductance states, fully open or closed3. We now present evidence for a third conductance state. In the membrane of uninnervated embryonic rat muscle we observe that ACh activates two independent classes of currents of different amplitude and average duration, apparently arising from two populations of ACh receptor (AChR) channels. The currents from both classes show, at low incidence, transitions between a main level and a sublevel of lower amplitude. From this we conclude that AChR channels in embryonic muscle adopt, in addition to a `main' conductance state, a `substate' of lower conductance.
Article
The transduction process of a vertebrate hair cell commences with the application of mechanical stimuli to the hair bundle, a cluster microvillous stereocilia and single axonemal kinocilium. In an effort to determine where within the hair bundle transduction occurs, I have measured extracellular potentials around the hair bundles of mechanically stimulated hair cells from the bullfrog's sacculus. Stimulus-dependent signals up to 17 microV in peak-to-peak amplitude have been found. These appear to be due to the flow of transduction current on the basis of their amplitude, phase, dependence on stimulus size and orientation, proportionality to membrane potential, and sensitivity to an ototoxic antibiotic. The responses are consistently larger near the top of the hair bundle than at its base, suggesting that the transduction apparatus lies at or near the distal ends of the stereocilia.
Article
1. The properties of the Ca channel in tissue cultured clonal cells (GH(3)) isolated from a rat anterior pituitary tumour were studied with the patch electrode voltage-clamp technique.2. To isolate the current through the Ca channel, the currents through the Na channel, the delayed K channel and the Ca(2+) induced K channel were minimized by replacing the external Na(+) with TEA(+) and adding EGTA to the K-free solution inside the patch electrode.3. The selectivity ratios through the Ca channel with different cations were 2.7 (Ba(2+)):1.6 (Sr(2+)):1.0 (Ca(2+)) and the m(2) form of the activation kinetics and the relationships between the time constant and the membrane potential were common to the three divalent cations.4. The amplitude of the Ba(2+) current increased linearly with [Ba(2+)](o) up to 25 mM and thereafter tended to show saturation.5. The current-voltage relation showed a positive shift along the voltage axis as [Ba(2+)](o) increased, probably due to the screening effect of Ba(2+) on the negative surface charges.6. The time constant of activation as a function of the membrane potential showed a parallel shift as [Ba(2+)](o) was increased, suggesting that the activation kinetics were independent of the permeant ion concentration.7. The time constant of the tail current was consistent with m(2) kinetics for opening and closing of the Ca channel.8. The extrapolated ;instantaneous' tail current rapidly increased as the activating membrane potential became more positive and reached an apparent saturation at membrane potentials substantially more positive than the potential that gave the maximum peak inward current, and suggested that the single channel has a sigmoidal current-voltage relationship.9. The power density spectrum obtained during the steady-state inward Ba(2+) current had a cut-off frequency which was nearly voltage independent; this is expected if the fluctuation of the current originates from m(2) activation kinetics.10. The results of noise analysis suggest that the amplitude of the single Ca channel current was about 0.2 pA at 25 mM-Ba(2+) and 0.7 pA at 100 mM-Ba(2+) for membrane potentials in the vicinity of the maximum inward current.
Article
In the mammalian central nervous system, glycine and gamma-aminobutyric acid (GABA) bind to specific and distinct receptors and cause an increase in membrane conductance to CI- (refs 5-7). Neurones in various regions of the nervous system show differential sensitivity to glycine and GABA; thus GABA and glycine receptors are spatially distinct from one another. However, on the basis of desensitization experiments on spinal cord neurones, it was suggested that the receptors for glycine and GABA may share the same CI- channel. We now report that in small membrane patches, isolated from the soma of spinal neurones, both receptor channels display several (multiple) conductance states. Two of the states are common to both receptor channels. However, the most frequently observed 'main conductance states' of the GABA and glycine receptor channels are different. Both channels display the same anion selectivity. We propose that one class of multistate CI- channel is coupled to either GABA or glycine receptors. The main conductance state adopted by this channel is determined by the receptor to which it is coupled.
Article
Single ventricular cells were enzymatically isolated from adult guinea-pig hearts (Isenberg & Klöckner, 1982). The patch-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981) was used to examine the conductance properties of an inward-rectifying K+ channel present in their sarcolemmal membrane. When the K+ concentration on the extracellular side of the patch was between 10.8 and 300 mM, inward current steps were observed at potentials more negative than the K+ equilibrium potential (EK). At more positive potentials no current steps were detectable, demonstrating the strong rectification of the channel. The zero-current potential extrapolated from the voltage dependence of the inward currents depends on the external K4 concentration [K+]o in a fashion expected for a predominantly K+-selective ion channel. It is shifted by 49 mV for a tenfold change in [K+]o. The conductance of the channel depends on the square root of [K+]o. In approximately symmetrical transmembrane K+ concentrations (145 mM-external K+), the single-channel conductance is 27 pS (at 19-23 degrees C). In normal Tyrode solution (5.4 mM-external K+) we calculate a single-channel conductance of 3.6 pS. The size of inward current steps at a fixed negative membrane potential V increases with [K+]o. The relation between step size and [K+]o shows saturation. Assuming a Michaelis-Menten scheme for binding of permeating K+ to the channel, an apparent binding constant of 210 mM is calculated for a membrane potential of -100 mV. For this potential the current at saturating [K+]o is estimated as 6.5 pA. The rectification of the single-channel conductance at membrane potentials positive to EK occurs within 1.5 ms of stepping the membrane potential from a potential of high conductance to one of low conductance. In addition to the main conductance state, the channel can adopt several substates of conductance. The main state could be the result of the simultaneous opening of four conducting subunits, each of which has a conductance of about 7 pS in 145 mM-external K+. The density of the inward-rectifying K+ channels in the ventricular sarcolemma is 0-10 channel/10 micron2 of surface membrane; the average of twenty-eight patches was 1 channel/1.8 micron2. It is concluded that the inward-rectifying K+ channels mediate the resting K+ conductance of ventricular heart muscle and the current termed IK1 in conventional voltage-clamp experiments.
Article
Properties of mechanoelectrical transduction were studied at the single-cell level by applying a whole-cell recording variation of the patch-clamp technique to dissociated vestibular hair cells of chicks. The hair bundle was directly stimulated by a glass rod, and transduction currents were recorded from the cell body. After a triangular movement of the stimulating probe, the transduction current was generated stepwise between discrete levels of amplitude. The minimum step amplitude was -1.8 pA at -27 mV in Na-containing normal saline.
Article
An important function of the peripheral auditory system is the resolution of complex sounds into their constituent frequency components. It is well established that each mechanoreceptive hair cell of the cochlea is maximally sensitive to a particular frequency of sound, but the mechanisms by which this sharp frequency selectivity is achieved are still controversial. The complex mechanical and hydrodynamic properties of the receptor organs and of the hair cells themselves are certainly involved. However, in at least one auditory organ, the turtle cochlea, frequency tuning is greatly enhanced by the electrical properties of the hair-cell membrane; each cell in this organ behaves as an electrical resonator tuned to a narrow band of frequencies. Using the 'Gigaseal', whole-cell recording technique, we have investigated the biophysical basis of similar resonant behaviour in enzymatically isolated hair cells from the bullfrog sacculus. We report here the identification of three voltage- and ion-dependent conductances which may contribute to the electrical tuning mechanism: a non-inactivating calcium conductance, an A-type K+ conductance, and a Ca2+-activated K+ conductance.
Article
Movements of the stiff sensory hairs, the stereocilia, which extend outwards from the apical surface of the hair cells in the auditory organ, are thought to have a key role in the process of transduction of sound energy into electrical impulses. Each stereocilium is supported by a paracrystalline axial bundle of actin filaments, which have identical polarities and are extensively cross-linked1-4. Recent immunohistochemical observations on whole mounts of the guinea pig organ of Corti indicated myosin-like immunoreactivity in association with hair cell stereocilia5. Considering the steric situation, it is, however, difficult to imagine any functional interaction between the bundled actin filaments and the assumed myosin molecules. Here we report that in semi-thin, transverse sections of quick-frozen, freeze-dried and plastic-embedded guinea pig organ of Corti, myosin-like immunostaining was restricted to the apical cytoplasm of hair cells and was not detected along the stereocilia. With respect to the immunohistochemical distribution of actin, myosin and the muscular Z-line protein, alpha-actinin, the apex of hair cells strongly resembles the intestinal epithelial brush border6-7.
Article
The aminoglycoside antibiotic, gentamicin (GM), depressed the plateau phase and shortened the duration of the action potential in guinea pig papillary muscle. Its effect on the membrane currents was studied by a single sucrose gap voltage clamp method. The slow inward current (is) was remarkably diminished by GM with little change in its time course, in the voltage-dependency of the steady-state inactivation and activation or in its reversal potential. The maximal amplitude of is, obtained by subtracting the Co2+-resistant current, was reduced to 57% by 0.1 mmol/l GM and almost reduced to zero by 1 mmol/l GM. The efficacy of GM in inhibiting (is) was reduced by increasing the external Ca2+ concentration from 1.8 to 5.4 or 10.8 mmol/l, but not by the application of adrenaline. The time-dependent outward current (ik) was also decreased by GM but only at higher concentrations. It is proposed that the depressant action of GM on is was due to a blockade of slow channels, whereby GM may have dislocated Ca from the binding sites at slow channels on the external surface of the membrane.
Article
Piezoelectric bimorph elements are versatile and inexpensive electromechanical transducers which may be used to construct fast mechanical stimulators and finely controlled micromanipulators. The mechanical stimulators described provide continuously graded displacements ranging from nanometers to about a millimeter, or forces equivalent to 0-7 g. Appropriately designed units can produce small step displacements complete within 100 microseconds. Micromanipulators are described which generate 3-dimensional motion under remote electrical control and which enable positioning within a few tenths of a micrometer. They are sufficiently stable to hold glass microelectrodes for cell penetration or probes for microdissection. The two significant drawbacks of bimorph elements are mechanical resonance and continued movement following displacement, or 'creep", but methods have been developed to compensate for these. A number of methods are available to measure motion of bimorphs with spatial resolution of 10 nm and temporal resolution of 2 microseconds in favorable situations.
Article
A voltage clamp study has been performed in the presynaptic terminal of the squid stellate ganglion. After blockage of the voltage-dependent sodium and potassium conductances, an inward calcium current is demonstrated. Given a step-depolarization pulse, this voltage- and time-dependent conductance has an S-shaped onset. At the "break" of the voltage step, a rapid tail current is observed. From these results a kinetic model is generated which accounts for the experimental results and predicts for the time course and amplitude a possible calcium entry during presynaptic action potentials.
Article
1. Intracellular recordings were made from single cochlear hair cells in the isolated half-head of the turtle. Receptor potentials were recorded while the ear was stimulated with high-intensity tones in order to examine the cochlear non-linearities which shape the hair cell responses.2. The size of a hair cell's voltage response to a tone burst was reduced, abolished and then reversed by steady depolarizing currents of increasing strength. The average current needed to produce reversal was about 0.3 nA, the reversal potential being close to zero with respect to the scala tympani.3. Short current pulses injected on the peaks and dips of the receptor potential showed that the membrane resistance and time constant were decreased on the depolarizing phase of the receptor potential. These changes were not due to non-linearity in the hair cell's current-voltage curve in the absence of acoustic stimulation. The results are consistent with the idea that the transducer causes the cell to depolarize by increasing the membrane conductance to ions with an equilibrium potential close to zero.4. Saturated receptor potentials from poorly tuned cells exhibited a pronounced asymmetry, with the maximum depolarizing excursion being several times the maximum hyperpolarizing excursion. This asymmetry was not seen in sharply tuned cells. It is proposed that the asymmetry is present in the transducer conductance change and in sharply tuned cells is reduced in the receptor potential by subsequent filtering.5. For high sound pressures which produced close to a saturated response, the hair cell voltage wave form displayed a number of non-linear features dependent upon the frequency of stimulation relative to the characteristic frequency (c.f.). The most prominent feature occurred at very low frequencies where the potential exhibited damped oscillations on the depolarizations and hyperpolarizations; these ;ringing frequencies' lay above and below the c.f. of the cell respectively.6. The ;ringing frequencies' varied with the c.f. of the cell but for a given cell were largely independent of the frequency of stimulation. The ;ringing frequencies' could be changed by injecting steady currents into the cell during acoustic stimulation; depolarizing currents increased the ringing frequencies and hyperpolarizing currents decreased the frequencies.7. The hair cell's response to a continuous test tone at the c.f. of the cell could be suppressed by simultaneous addition of a second tone whose sound presure was comparable to, or greater than, the test tone. The degree of suppression varied with the intensity and frequency of the second tone, and was maximal close to the c.f. of the cell. The sound pressure required to produce a constant suppression as a function of frequency was sharply tuned, and the tuning of the suppression showed similarities to the frequency selectivity of two-tone suppression described in the auditory nerve.8. An attempt was made to reconstruct the main features of the receptor potential at high intensities.
Article
Summary The hearing organ in the lizard, the basilar papilla, is an oblong organ situated in the central opening of the surrounding limbus. The hair cells of the basilar papilla inCalotes versicolor consist of two different types. The type A sensory cells have short hair bundles whose arrangement resembles that of organ pipes, and are situated in the ventral part of the organ. The type B sensory cells have tall, whisk-like hair bundles and are situated in the dorsal part of the basilar papilla. The type A sensory cells are unidirectionally orientated, whereas the type B cells are orientated towards the central sulcus in the papilla. Between the stereocilia, quite close to their base, there is a thin network of interconnecting fibres. Another type of connection is found between the kinocilium and the five adjacent stereocilia. These fibres, however, are situated close to the tips of the relevant cilia. The ventral part of the basilar papilla, i.e., the type A cell population, is covered by a tectorial membrane. Between the microvilli of the supporting cells and the tectorial membrane a network of thin interconnecting filaments is seen. This totally encloses the hair bundles, thus causing them to stand in tubular formations between the sensory epithelium and the tectorial membrane.
Article
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The sensory hairs and tectorial membrane of the basilar papilla in the lizard Calote8 ver8color
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BXGGER-SJoBXCK, D. & WERSXLL, J. (1973). The sensory hairs and tectorial membrane of the basilar papilla in the lizard Calote8 ver8color. Journal of Neurocytology 2, 329-350