Membrane Of Outer Hair Cells Research Articles

Distribution of Ca2+ channels on cochlear outer hair cells revealed by fluorescent dihydropyridines.

Physiological evidence has shown that cochlear outer hair cells (OHC) possess L-type voltage-dependent Ca2+ channels through which Ca2+ enters the OHC during depolarization. Their subcellular distribution has, however, remained unclear. In this study, the distribution of L-type Ca2+ channels on the basolateral plasma membrane of OHC has been demonstrated by the use of a laser scanning confocal microscope (LSCM) and a fluorescent probe DMBODIPY-DHP. The fluorescent staining pattern on the basolateral wall is nonuniform, suggesting a heterogeneous distribution of the channels in the plasma membrane. Direct imaging of intracellular Ca2+ visualized in real time by means of the LSCM and the fluorescent Ca2+ probe fluo 3 revealed temporal and spatial integration of Ca2+ movements and Ca2+ channel distribution. Exposure to high-K+ solution induced heterogeneity in the subcellular increase in the intracellular Ca2+ concentration. These results suggest that the heterogeneous distribution of L-type Ca2+ channels on the basolateral membrane might induce heterogeneous intracellular Ca2+ distribution during electrical activity in the OHC.

Postnatal expression of the facilitated glucose transporter, GLUT 5, in gerbil outer hair cells

The fifth isoform from the family of facilitative glucose transporters (GLUT5) was identified in the gerbil cochlea by light and electron microscopic immunohistochemistry. The immunostaining procedure employed a polyclonal antibody raised against a synthetic peptide representing the carboxy terminus of human GLUT 5. Immunoreactive GLUT 5 was abundant in the basolateral plasma membrane of outer hair cells (OHCs) in the mature gerbil cochlea. No immunostaining was observed in any other site in the cochlea or vestibular system. During postnatal development of the gerbil inner ear, OHCs were first observed to express GLUT 5 in a punctate pattern at 10 days after birth. Immunostaining intensity increased gradually and developed a linear pattern along the entire OHC basolateral plasmalemma between 10 and 16 days after birth when it approached adult levels. The developmental expression of GLUT 5 corresponded with the appearance of glycogen in OHCs and structural maturation of the organ of Corti. This time period also coincided with the onset and rapid maturation of auditory function in the gerbil. GLUT 5 apparently supports OHC function by facilitating uptake of glucose which enables these cells to sustain a high level of metabolic activity in a relatively anaerobic environment.

A calcium-activated nonselective cationic channel in the basolateral membrane of outer hair cells of the guinea-pig cochlea.

The patch-clamp technique was used to investigate ion channels in the basolateral perilymph-facing membrane of freshly isolated outer hair cells (OHCs) from the guinea-pig cochlea. These sensory cells probably determine, via their motile activity, the fine tuning of sound frequencies and the high sensitivity of the inner ear. A Ca(2+)-activated nonselective cationic channel was found in excised inside-out membrane patches. The current/voltage relationship was linear with a unit conductance of 26.3 +/- 0.3 pS (n = 15) under symmetrical inger conditions. The channel excluded anions (PNa/PCl = 18 where PNa/PCl denotes the relative permeability of Na to Cl); it was equally permeant to the Na+ and K+ ions and exhibited a low permeability to N-methyl-D-glucamine and Ba2+ or Ca2+. Channel opening required a free Ca2+ concentration of about 10(-6) mol/l on the internal side of the membrane and the open probability (Po) was maximal at 10(-3) mol/l (Po = 0.72 +/- 0.06, n = 12). Adenosine 5'mono-, tri- and di-phosphate reduced Po to 29 +/- 14 (n = 5), 42 +/- 10 (n = 8) and 51 +/- 12 (n = 5) % of control Po, respectively, when they were added at a concentration of 10(-3) mol/l to the internal side. The channel was partially blocked by flufenamic acid (10(-4) mol/l) and 3',5'-dichlorodiphenylamine-2-carboxylic acid (DCDPC, 10(-5) mol/l). This type of channel, together with Ca(2+)-activated K+ channels, might participate in the control of membrane potential and modulate the motility of OHCs.

Charge displacement induced by rapid stretch in the basolateral membrane of the guinea-pig outer hair cell.

The properties of the basolateral membrane of cochlear outer hair cells were studied under whole-cell patch clamp to measure currents and capacitance changes associated with mechanical deformation. Stretching the membrane of outer hair cells along the cell axis generated a transient inward current, and subsequent relaxation of the membrane produced a similar transient outward current. These mechanically activated currents were velocity dependent with a mean sensitivity of 29 pA s mm-1. Unlike ionic currents, these currents did not reverse, but reached a peak magnitude at -33 mV. Stretching the cell also resulted in a measurable capacitance decrease of 0.3-1.1 pF microns-1. These results suggest that membrane stretch can induce a rapid charge movement resulting from the reversal of the electromechanical transduction process in outer hair cells.

ATP-induced cytoplasmic [Ca 2+]increases in isolated cochlear outer hair cells. Involved receptor and channel mechanisms

Outer hair cells (OHC) of the mammalian cochlea are thought to preprocess the sound signal by active movements, which can be induced by electrical or chemical stimulation, e.g. depolarization evoked by high [K +]or increased cytoplasmic [Ca 24]. Extracellular ATP has been found to induce cytoplasmic [Ca 2 +]increases in OHC but involved mechanisms have not been elucidated. Cytoplasmic [Ca 2+]was measured in non-enzymatically isolated single OHC using Fura-2 microspectrometry. Results, using ATP/derivatives and other P 2-purinergic receptor (P 2R) ligands, as well as Ca 2+-channel blockers and pertussis toxin, revealed several signal transduction pathways that increase cytoplasmic [Ca 2 +]in OHC: a P 2-purinergic receptor (P 2 R) -G-protein - effector (phospholipase C or an ion channel) system and a voltage-dependent Ca 2+ channel. Agonist potency studies denote a pattern analogous to that found in skeletal muscle, i.e. ATP- α-S > ATP = 2-methyl-S-ATP > ADP > α,β-methylene- but no activation by ADPβF or UTP, leaving a choice of P 2y or P 2zR subtypes. The latter possibility gained strength from calculations showing that up to 8% of ATP may have formed the P 2zR agonist ATP 4− in the experimental medium. Experiments in Ca 2+-free medium and with pertussis toxin revealed that the main Ca 2 + source was intracellular. Pertussis toxin did not affect [Ca 2 +]increase induced by carbachol. Acetylcholine, administered a few seconds before ATP. did not affect total cytoplasmic [Ca 2+]increases. Induced cytoplasmic [Ca 2 +]increases were high enough ( > 500 nM at 50 μM ATP/ derivatives) to hyperpolarize the OHC membrane by opening K +-channels and decreased little with time. Artifacts may have been caused by the sustained Ca 2+ levels, e.g. activation of proteases by the high cytoplasmic [Ca 2+]. Similar events in vivo may have pathological consequences.

Mapping the distribution of the outer hair cell motility voltage sensor by electrical amputation

The outer hair cell (OHC) possesses a nonlinear charge movement whose characteristics indicate that it represents the voltage sensor responsible for OHC mechanical activity. OHC mechanical activity is known to exist along a restricted extent of the cell's length. We have used a simultaneous partitioning microchamber and whole cell voltage clamp technique to electrically isolate sections of the OHC membrane and find that the nonlinear charge movement is also restricted along the cell's length. Apical and basal portions of the OHC are devoid of voltage sensors, corresponding to regions of the cell where the subsurface cisternae and/or the mechanical responses are absent. We conclude that the physical domain of the motility voltage sensor corresponds to that of the mechanical effector and speculate that sensor and effector reside within one intra membranous molecular species, perhaps an evolved nonconducting or poorly conducting voltage-dependent ion channel.

Structure, pharmacology and function of GABA-A receptors in cochlear outer hair cells.

There is evidence that the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is released from some efferent olivocochlear nerve endings terminating at outer hair cells (OHCs). Using monoclonal antibodies against postsynaptic GABAA receptor from bovine cerebral cortex we confirm the presence of GABA and benzodiazepine bindings sites of alpha- and beta-subunits of GABAA receptors at the basal pole of isolated OHCs. Whole-cell recording with viable OHCs revealed that the application of 10(-3)-10(-8) M GABA to the cell surface was followed by a concentration-dependent hyperpolarization of the outer cell membrane. Hyperpolarization was increased in the presence of 2.5 x 10(-5) M chlorazepate, a benzodiazepine derivative. Electrophysiological effects caused by GABA alone or in combination with chlorazepate were specifically inhibited by 10(-6) M of the GABA-receptor antagonist picrotoxin. Moreover, 10(-5)-10(-7) M GABA caused reversible slow elongation of the cylindrical hair cell body in OHCs examined. These neurotransmitter-induced motile responses were specifically blocked by 10(-4) M picrotoxin. The results suggest that a subpopulation of OHCs express alpha- and beta-subunits of GABAA receptors which both form a GABA/benzodiazepine-receptor complex at the basal pole of isolated OHCs. These receptors are thought to allow GABA which is released from efferent auditory nerve terminals to bind to the cell surface of OHCs, resulting in GABAA-receptor activation. This probably gates a GABAA-receptor-associated chloride channel in the postsynaptic OHC membrane, allowing hyperpolarization and elongation of the cell.

Hyposmotic activation hyperpolarizes outer hair cells of guinea pig cochlea

The electrophysiological responses of isolated guinea pig outer hair cells (OHCs) to hyposmotic activation were studied using the whole-cell patch-clamp technique. The cell swelling by hyposmotic activation hyperpolarized OHCs by 6.6 ± 2.3 mV from the resting membrane potential of −58.5 ± 5.9 mV ( n = 48). This hyperpolarization was associated with an outward current ( 97.7 ± 22.2, pA, n = 15). The hyperpolarization was inhibited by 300 μM quinine, 5 mN Ba 2+ and increasing the extracellular K + to 30 mM from 5 mM. In the absence of extracellular Ca 2+ (1 mM EGTA), the hyperpolarization during hyposmotic activation was also abolished while the following depolarization was preserved. 50 μM GdCl 3, which is known to block strecch-activated non-specific cation channels, inhibited the hyperpolarization reversibly. 50 μM GdCl 3 also inhibited [Ca 2+] i increase during hyposmotic activation as shown by the calcium-sensitive dye fura-2. Simultaneously, the [Ca 2+] i increase and the hyperpolarization during hyposmotic activation could be observed using the combined method of whole-cell patch clamp and fura-2 technique. It is concluded that the cell swelling by hyposmotic activation may activate the stretch-activated non-specific cation channels in the OHCs which allow a Ca 2+ influx. In turn, this [Ca 2+] i increase leads to an activation of the Ca 2+-activated K + channels at the basolateral membrane of OHCs which results finally in a reversible hyperpolarization of OHCs by K + efflux.

Acetylcholine increases intracellular Ca 2+ concentration and hyperpolarizes the guinea-pig outer hair cell

Extracellularly applied acetylcholine (ACh) induced outward currents in isolated outer hair cells of a guinea-pig cochlea. The ACh induced current was carried by K + ions. The current amplitude was ACh dose dependent with a K D of 12 μM. The ACh induced outward current was reversibly blocked by extracellularly applied atropine (1 μM), d-tubocurarine (d-TC, 1 μM), apamin (1 μM) and strychnine (0.1–10 μM). D-TC (10 μM) not only blocked the ACh induced outward current, but also reduced the amplitude of depolarization induced outward current. ACh induced a rise of intracellular Ca 2+ concentration ([Ca 2+]i). D-TC (10 μM) reduced but did not totally block the increase of [Ca 2+]i. In a low Ca 2+ (0.1 mM) extracellular medium, the amplitude of ACh induced current was reduced rapidly and was recovered gradually to the normal level after the extracellular Ca 2+ concentration was resumed. It is probable that ACh hyperpolarizes the guinea-pig outer hair cell membrane by activation of a Ca 2+-activated K − conductance.

Membrane potential measurement in isolated outer hair cells of the guinea pig cochlea using conventional microelectrodes

Membrane potential of the isolated outer hair cells (OHCs) from the guinea pig cochlea was measured using conventional microelectrodes filled with 200 mM KCl. The resting membrane potential during superfusion with the standard physiological saline solution containing 3.5 mM K + was −47.3 ± 1.4 mV ( N = 72), which was higher than those previously reported for isolated OHCs studied by using microelectrodes. Addition of ouabain (10 −5–10 −3 M), the specific Na +, K + ATPase inhibitor, depolarized the cell slowly and progressively, indicating the presence of low but definite Na +, K + ATPase activity in the plasma membrane of OHCs. The magnitude of membrane potential was mainly dependent on the extracellular K + concentration ([K +] o). A ten-fold increase of [K +] o depolarized the membrane potential by 49.6 ± 1.0 mV ( N = 58). A decrease of [Na +] o to one tenth of the control hyperpolarized the membrane potential by about 2 mV. Decreasing extracellular Cl − from 131.3 mM to 27.5 mM did not cause a significant change in the membrane potential. Using the Goldman-Hodgkin-Katz equation, assuming a negligible contribution of Cl − to the membrane potential and total monovalent cat ion concentration of the cytosol similar to the extracellular fluid, we calculated the permeability ratio of K + versus Na + to 131 ± 19 and intracellular K + concentration to 33.3 ± 1.9 mM.

C-type potassium channels in the lateral cell membrane of guinea-pig outer hair cells

The basolateral cell membrane of outer hair cells (OHC) from the mammalian cochlea is known to contain K +-channels. The prevailing type, a high-conductance K +-channel was further characterized in the present study in order to support its classification as C channel. OHC were isolated from the 3 rd and 4 th turn of the guinea-pig cochlea. Cell-attached and excised inside-out patches of the lateral cell membrane were investigated. The C-type channel had a selectivity for K + over Na + of 12:1 to 20:1 and displayed Goldman-type rectification and voltage-dependence of the open probability. The kinetics of both opening and closing could be described by time constants in the range of ms. The channel provides a calcium- and voltage-activated pathway through OHC lateral membranes for passive K + transport.

Carbohydrates in the cell surface of hair cells from the guinea pig cochlea.

The presence of cell surface carbohydrates was investigated in intact, non-fixed outer hair cells (OHCs) of guinea pigs using fluorescein isothiocyanate (FITC) and rhodamine (TRITC) lectins. By means of wheat germ agglutinin (WGA) N-acetyl-D-glucosamine was shown in the entire OHC membrane, including the stereocilia. Binding of WGA in OHCs to neuraminic acid was excluded by preincubation with neuraminidase. Moreover, FITC-Limulus polyphemus, a specific lectin for neuraminic acid, showed no fluorescence on OHCs. Neutral saccharides, like alpha-D-mannose and/or alpha-D-glucose, were mainly observed at the cuticular plate and at the basal cell pole with FITC-concanavalin A. A weaker fluorescence was seen at the lateral cell wall. Two branched oligosaccharides, composed of beta-galactose, N-acetyl-D-glucosamine and mannose, were demonstrated by TRITC-Phaseolus vulgaris (PHA-E/L) in the entire OHC membrane. A spot-like binding of soybean agglutinin to N-acetyl-D-galactosamine could be demonstrated in the region of the cuticular plate. However, using Helix pomatia, the subtype N-acetyl-alpha-D-galactosamine was not detectable. Moreover, there was no binding of Ulex europaeus or of Arachis hypogea (PNA) to OHCs, suggesting the absence of considerable amounts of L-fucose and of galactose-beta-3-N-acetylgalactosamine. The results indicate that N-acetyl-D-glucosamine, alpha-D-mannose and alpha-D-glucose can be considered the major components of the OHC glycocalix. We suggest that they have a function as an anchoring structure in interstereociliary links as well as in hair bundle connections to the tectorial membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

P-070 - Electrical property in outer hair cell membrane of guinea-pig cochlea

P-070 - Electrical property in outer hair cell membrane of guinea-pig cochlea

Funktionelle Morphologie der äußeren Haarzellen des Menschen: Neue Aspekte*

Serial sections through the outer hair cells of the human organ of Corti were investigated using high-resolution electron microscopy. Delicate monostratified tubular structures of differing lengths are located on the cytoplasmic side of the hair-cell membrane. They are connected to the cell membrane via short fibers (pillars) arranged in pairs. The tubular structures, which are also termed subsurface cisterns (SSC), constitute an extensive network. This network lines the entire inner surface of the outer hair-cell membrane, and in contrast to the mammals investigated so far, also lines the base of the cell. The postsynaptic cisterns of the efferent synapses are integrated into this system. At the surface of the inner lamina of the cisterns, pores 8.5 nm in diameter are to be found. These are surrounded by protein complexes with a stellate arrangement. Similar protein complexes are located on the inner lamina of the postsynaptic cisterns. Pores 8.5 nm in diameter are also present in the outer lamina of the SSC. These constitute the base for the pillar filaments. At high magnification, the pillars are seen to have a lumen about 6 nm in diameter. Pores about 8 nm in diameter are again to be found where the pillars are anchored in the outer cell membrane. The pillars, consisting of actin, are probably ionic channels, and the pores of the inner lamina of the SSC surrounded by protein complexes are thought to be acetylcholine receptors. Since the postsynaptic cisterns of the efferent innervation of the outer hair cells are part of the overall system of the SSC, and acetylcholine is regarded as the neurotransmitter of efferent innervation, it would seem possible to regulate the subsurface cisterns, as a wall-stabilizing, contractile system, via the efferent innervation. On the basis of serial sections, a three-dimensional, true-to-scale model of the contractile wall system of the human outer hair cell was reconstructed.

Cholinerge Innervation äußerer Haarzellen - Eine mögliche Bedeutung für den Diskriminationsverlust bei Perzeptionsschwerhörigkeiten

Cochlear outer hair cells (OHC) are capable of auditory perception. In addition, they influence actively the micromechanics of the cochlea. This concept is based on the observation of motile responses induced by electrical and chemical stimuli. OHC motility is probably controlled by the efferent olivo-cochlear system. By means of two monoclonal antibodies against intra- and extracellular epitopes of ACh-receptors a transmembraneous receptor molecule was visualized in the OHC membrane. Besides morphological studies functional investigations using acetylcholine and its analogues were carried out. We observed contractile responses of OHCs that were reversible and dependent on the applied transmitter concentration. We suggest that the high frequency selectivity and sensitivity of the cochlea is modulated by the efferent innervation and the observed ACh-induced OHC motility.

Actin in cochlear outer hair cells: A structural basis for cell shape and motility

Threshold sensitivity and frequency selectivity in the cochlea depend on intact outer hair cells and models to explain these phenomena suggest that active mechanical processes, residing in outer hair cells, alter micromechanical properties of the basilar membrane. Auditory sensory hair cells isolated from the inner ear change their shape in response to stimulation in vitro. Three areas of the cell have been suggested to be involved in this form of cell motility: (1) the infracuticular network of actin filaments that is seen as a central core; (2) the cortical layer of actin filaments along the lateral wall; and (3) actin in the outer hair cell cytoplasm. The actin filaments in the stereocilia, the cuticular plate, and the infracuticular network are not involved, since mechanically disrupted cells lacking these structures still shorten. Actin filaments along the lateral wall are not involved in shortening since outer hair cell membrane ghosts do not shorten, however, they retain the ability to constrict so...

Wheat germ agglutinin and helix pomatia agglutinin lectin binding on cochlear hair cells

The fluorescein labelled lectins FITC-WGA and FITC-HPA were used to identify specific carbohydrates in cochlear hair cells. Wheat germ agglutinin (WGA) bound with the cell coat of both inner and outer hair cells (IHC and OHC) suggesting the presence of either N- acetyl- d- glucosamine or sialic acid. In contrast, glycoconjugates with terminal N- acetyl- d- galactosamine residues that bind with Helix pomatiaagglutinin (HPA), were demonstrated inside the plasma membrane of outer hair cells. WGA and HPA lectin binding implies the presence of anionic glycoconjugates that furnish added negative charge on the membranes to which they are fixed. The presence of sialic acid or N- acetyl- d- glucosamine on the extracellular surface of cochlear hair cell plasma membrane is consistent with the normal distribution of these glycoconjugates in the cell coat. The presence of the membrane associated oligosaccharide N- acetyl- d- glucosamine within the outer hair cell is inconsistent with the distribution of glycoproteins in internal membrane systems of other cell types.

The response of hair cells in the basal turn of the guinea-pig cochlea to tones.

1. Intracellular recordings were made from inner and outer hair cells in the basal turn of the guinea-pig cochlea. The resting membrane potentials of the inner hair cells are more positive than -50 mV while those of outer hair cells are usually more negative than -70 mV. 2. At low frequencies the receptor potentials of inner hair cells are predominantly depolarizing while those from outer hair cells are hyperpolarizing at low and moderate sound pressure (e.g. less than 90 dB re 2 X 10(-5) Pa at 600 Hz). The potentials then become predominantly depolarizing at high sound pressure. 3. The asymmetry of the inner and outer hair cell receptor potentials are manifested instantaneously except at high stimulus levels when the depolarizing responses of outer hair cells take several cycles to develop. 4. At the offset of intense tones outer hair cell membrane potentials remain depolarized by 1-2 mV above their resting value and return to normal over a period depending on the level and duration of the tone. 5. In response to tones above about 2 kHz and at levels below about 90 dB the wave forms of outer hair cell receptor potentials are virtually symmetrical without measurable d.c. components. In response to tones close to their best frequencies (16-21 kHz), inner hair cells in the basal turn generate large depolarizing (d.c.) receptor potentials while outer hair cells from this region of the cochlea do not generate significant voltage responses. 6. Frequency tuning curves were derived for inner and outer hair cells from the amplitude-intensity relationships of their d.c. and phasic (a.c.) receptor potentials respectively. When the latter were compensated for the low-pass characteristics of the recording system and the hair cell time constant, the frequency selectivity of inner and outer hair cells are similar. 7. The response properties of inner and outer hair cells in the basal turn of the guinea-pig cochlea are discussed in relation to their proposed roles in mechano-electric transduction.

Uptake of horseradish peroxidase from perilymph by cochlear hair cells

The specific mechanisms involved in the uptake of an exogenous protein, horseradish peroxidase (HRP), by cochlear hair cells were studied morphologically in light and electron microscopy. HRP was internalized by coated vesicles which formed from the plasma membrane only in the basal portion of the hair cells. Inner hair cells demonstrated relatively greater uptake than adjacent outer hair cells. The lateral plasma membrane of outer hair cells was unique in that reaction product was never bound to this portion of the membrane. Subsequent to endocytosis, HRP was transported to the Golgi and its associated system of cndoplasmic reticulum and lysosomes. The exogenous protein was sequestered in a striking accumulation of secondary lysosomes and multivesicular bodies which were restricted to the infracuticular region of the hair cells and persisted for periods of at least 72 h after introduction of HRP. It is not clear whether the hair cells were slowly degrading the HRP or if the lysosomal enzymes necessary for its breakdown were lacking. The pathway demonstrated by HRP uptake and intracellular transport may represent one route by which macromolecules requisite for basic metabolic or nutritional requirements of the hair cells are supplied from the perilymph.

Ionic basis of membrane potential in outer hair cells of guinea pig cochlea

Mammalian hearing involves features not found in other species, for example, the separation of sound frequencies depends on an active control of the cochlear mechanics. The force-generating component in the cochlea is likely to be the outer hair cell (OHC), one of the two types of sensory cell through which current is gated by mechano-electrical transducer channels sited on the apical surface. Outer hair cells isolated in vitro have been shown to be motile and capable of generating forces at acoustic frequencies. The OHC membrane is not, however, electrically tuned, as found in lower vertebrates. Here we describe how the OHC resting potential is determined by a Ca2+-activated K+ conductance at the base of the cell. Two channel types with unitary sizes of 240 and 45 pS underlie this Ca2+-activated K+ conductance and we suggest that their activity is determined by a Ca2+ influx through the apical transducer channel, as demonstrated in other hair cells. This coupled system simultaneously explains the large OHC resting potentials observed in vivo and indicates how the current gated by the transducer may be maximized to generate the forces required in cochlear micromechanics.