Biomedical subjects
C J Kros
Publications and source records attributed to C J Kros.
Effects of trypsin on large-conductance Ca(2+)-activated K(+) channels of guinea-pig outer hair cells.
High-conductance Ca(2+)-activated K(+) (BK(Ca)) channels from isolated adult guinea-pig outer hair cells were studied in inside-out membrane patches. They had a 300 pS unitary conductance and were inhibited by tetraethyl ammonium (1 mM), iberiotoxin (33 nM) and charybdotoxin (50 nM). In symmetrical 144 mM KCl their K(+) permeability (P(K)) was 5.4 x 10(-13) cm(3)/s; this was reduced to around 4.5 x 10(-13) cm(3)/s with 160 mM Na(+) in place of K(+) on either internal or external membrane surface. BK(Ca) channels from trypsin-isolated hair cells had a high open probability, that depended on both membrane voltage (16 mV/e-fold change) and the concentration of calcium ions at their intracellular surface ([Ca(2+)](i)). The Hill coefficient was 3-4. About 50% of BK(Ca) channels from mechanically isolated outer hair cells had similar characteristics; the remainder had the same high conductance but a low open probability. Trypsin (<0.5 mg/ml) applied to the intracellular face of these 'inactive' channels markedly increased their open probability. It is possible that exposure to trypsin during cell isolation removes an inactivating beta subunit. This would account for the absence of 'inactive' BK(Ca) channels in trypsin-isolated cells.
A receptor-like inositol lipid phosphatase is required for the maturation of developing cochlear hair bundles.
A screen for protein tyrosine phosphatases (PTPs) expressed in the chick inner ear yielded a high proportion of clones encoding an avian ortholog of protein tyrosine phosphatase receptor Q (Ptprq), a receptor-like PTP. Ptprq was first identified as a transcript upregulated in rat kidney in response to glomerular nephritis and has recently been shown to be active against inositol phospholipids. An antibody to the intracellular domain of Ptprq, anti-Ptprq, stains hair bundles in mice and chicks. In the chick ear, the distribution of Ptprq is almost identical to that of the 275 kDa hair-cell antigen (HCA), a component of hair-bundle shaft connectors recognized by a monoclonal antibody (mAb) that stains inner-ear hair bundles and kidney glomeruli. Furthermore, anti-Ptprq immunoblots a 275 kDa polypeptide immunoprecipitated by the anti-HCA mAb from the avian inner ear, indicating that the HCA and Ptprq are likely to be the same molecule. In two transgenic mouse strains with different mutations in Ptprq, anti-Ptprq immunoreactivity cannot be detected in the ear. Shaft connectors are absent from mutant vestibular hair bundles, but the stereocilia forming the hair bundle are not splayed, indicating that shaft connectors are not necessary to hold the stereocilia together; however, the mice show rapid postnatal deterioration in cochlear hair-bundle structure, associated with smaller than normal transducer currents with otherwise normal adaptation properties, a progressive loss of basal-coil cochlear hair cells, and deafness. These results reveal that Ptprq is required for formation of the shaft connectors of the hair bundle, the normal maturation of cochlear hair bundles, and the long-term survival of high-frequency auditory hair cells.
Reduced climbing and increased slipping adaptation in cochlear hair cells of mice with Myo7a mutations.
Mutations in Myo7a cause hereditary deafness in mice and humans. We describe the effects of two mutations, Myo7a(6J) and Myo7a(4626SB), on mechano-electrical transduction in cochlear hair cells. Both mutations result in two major functional abnormalities that would interfere with sound transduction. The hair bundles need to be displaced beyond their physiological operating range for mechanotransducer channels to open. Transducer currents also adapt more strongly than normal to excitatory stimuli. We conclude that myosin VIIA participates in anchoring and holding membrane-bound elements to the actin core of the stereocilium. Myosin VIIA is therefore required for the normal gating of transducer channels.
FM1-43 dye behaves as a permeant blocker of the hair-cell mechanotransducer channel.
Hair cells in mouse cochlear cultures are selectively labeled by brief exposure to FM1-43, a styryl dye used to study endocytosis and exocytosis. Real-time confocal microscopy indicates that dye entry is rapid and via the apical surface. Cooling to 4 degrees C and high extracellular calcium both reduce dye loading. Pretreatment with EGTA, a condition that breaks tip links and prevents mechanotransducer channel gating, abolishes subsequent dye loading in the presence of calcium. Dye loading recovers after calcium chelation with a time course similar to that described for tip-link regeneration. Myo7a mutant hair cells, which can transduce but have all mechanotransducer channels normally closed at rest, do not label with FM1-43 unless the bundles are stimulated by large excitatory stimuli. Extracellular perfusion of FM1-43 reversibly blocks mechanotransduction with half-blocking concentrations in the low micromolar range. The block is reduced by high extracellular calcium and is voltage dependent, decreasing at extreme positive and negative potentials, indicating that FM1-43 behaves as a permeant blocker of the mechanotransducer channel. The time course for the relief of block after voltage steps to extreme potentials further suggests that FM1-43 competes with other cations for binding sites within the pore of the channel. FM1-43 does not block the transducer channel from the intracellular side at concentrations that would cause complete block when applied extracellularly. Calcium chelation and FM1-43 both reduce the ototoxic effects of the aminoglycoside antibiotic neomycin sulfate, suggesting that FM1-43 and aminoglycosides enter hair cells via the same pathway.
Hey presto! Electrophysiological characterisation of prestin, a motor protein from outer hair cells, transfected into kidney cells.
Explore the source record for details and available documents.
A genetic approach to understanding auditory function.
Little is known of the molecular basis of normal auditory function. In contrast to the visual or olfactory senses, in which reasonable amounts of sensory tissue can be gathered, the auditory system has proven difficult to access through biochemical routes, mainly because such small amounts of tissue are available for analysis. Key molecules, such as the transduction channel, may be present in only a few tens of copies per sensory hair cell, compounding the difficulty. Moreover, fundamental differences in the mechanism of stimulation and, most importantly, the speed of response of audition compared with other senses means that we have no well-understood models to provide good candidate molecules for investigation. For these reasons, a genetic approach is useful for identifying the key components of auditory transduction, as it makes no assumptions about the nature or expression level of molecules essential for hearing. We review here some of the major advances in our understanding of auditory function resulting from the recent rapid progress in identification of genes involved in deafness.
Gating energies and forces of the mammalian hair cell transducer channel and related hair bundle mechanics.
We quantified the molecular energies and forces involved in opening and closing of mechanoelectrical transducer channels in hair cells using a novel generally applicable method. It relies on a thermodynamic description of the free energy of an ion channel in terms of its open probability. The molecular gating force per channel as reflected in hair bundle mechanics is shown to equal kT/I(X) x dI(X)/dX, where I is the transducer current and X the deflection of the hair bundle. We applied the method to previously measured I(X) curves in mouse outer hair cells (OHCs) and vestibular hair cells (VHCs). Contrary to current models of transduction, gating of the transducer channel was found to involve only a finite range of free energy (< 10 kT), a consequence of our observation that the channel has a finite minimum open probability of ca. 1% for inhibitory bundle deflections. The maximum gating forces per channel of both cell types were found to be comparable (ca. 300-500 fN). Because of differences in passive restoring forces, gating forces result in very limited mechanical nonlinearity in OHC bundles compared to that in VHC bundles. A kinetic model of channel activation is proposed that accounts for the observed transducer currents and gating forces. It also predicts adaptation-like effects and spontaneous bundle movements ensuing from changes in state energy gaps possibly related to interactions of the channel with calcium ions.
A missense mutation in myosin VIIA prevents aminoglycoside accumulation in early postnatal cochlear hair cells.
Myosin VIIA is expressed by sensory hair cells in the inner ear and proximal tubule cells in the kidney, the two primary targets of aminoglycoside antibiotics. Using cochlear cultures prepared from early postnatal Myo7a6J mice with a missense mutation in the head region of the myosin VIIA molecule we show that this myosin is required for aminoglycoside accumulation in cochlear hair cells. Hair cells in homozygous mutant Myo7a6J cochlear cultures have disorganized hair bundles, but are otherwise morphologically normal and transduce. However, and in contrast to hair cells from heterozygous Myo7a6J cultures, the homozygous Myo7a6J hair cells do not accumulate [3H]gentamicin and do not exhibit an ototoxic response on exposure to aminoglycoside. Possible roles for myosin VIIA in the process of aminoglycoside accumulation are discussed.
Developmental expression of the potassium current IK,n contributes to maturation of mouse outer hair cells.
1. The expression of K+ currents in mouse outer hair cells (OHCs) was investigated as a function of developmental age between postnatal day (P) 0 and P26, using whole-cell patch clamp. 2. During the first postnatal week, a slow outward K+ current (IK,neo) was expressed by all OHCs from the apical coil of the cochlea. The amplitude of this current increased greatly between P0 and P6. Then, at the beginning of the second postnatal week, IK,neo decreased. At the same time, from P8 onwards, IK,n, a K+ current characteristic of mature OHCs, was rapidly expressed. 3. The expression of IK,n coincided with the onset of electromotility of the cell body of the OHCs, which could also be detected from P8 onwards and increased substantially in size thereafter. 4. IK,n was reversibly blocked by linopirdine, an inhibitor of members of the KCNQ family of K+ channels, with a KD of 0.7 microM. In the cochlea, KCNQ4 is only expressed in OHCs and is responsible for a form of non-syndromic autosomal dominant deafness. Linopirdine had no effect on other OHC K+ currents at concentrations up to 200 microM. We conclude that ion channels underlying IK,n contain the KCNQ4 subunit. 5. In current clamp, depolarizing current injections from the resting potential triggered action potentials in OHCs during the first postnatal week. Thereafter, more rapid and graded voltage responses occurred from more negative resting potentials. Thus, OHCs mature rapidly from P8 onwards, and IK,n contributes to this maturation.
Differentiation of mammalian vestibular hair cells from conditionally immortal, postnatal supporting cells.
We provide evidence from a newly established, conditionally immortal cell line (UB/UE-1) that vestibular supporting cells from the mammalian inner ear can differentiate postnatally into more than one variant of hair cell. A clonal supporting cell line was established from pure utricular sensory epithelia of H2k(b)tsA58 transgenic mice 2 d after birth. Cell proliferation was dependent on conditional expression of the immortalizing gene, the "T" antigen from the SV40 virus. Proliferating cells expressed cytokeratins, and patch-clamp recordings revealed that they all expressed small membrane currents with little time-dependence. They stopped dividing within 2 d of being transferred to differentiating conditions, and within a week they formed three defined populations expressing membrane currents characteristic of supporting cells and two kinds of neonatal hair cell. The cells expressed several characteristic features of normal hair cells, including the transcription factor Brn3.1, a functional acetylcholine receptor composed of alpha9 subunits, and the cytoskeletal proteins myosin VI, myosin VIIa, and fimbrin. Immunofluorescence labeling and electron microscopy showed that the cells formed complex cytoskeletal arrays on their upper surfaces with structural features resembling those at the apices of normal hair cells. The cell line UB/UE-1 provides a valuable in vitro preparation in which the expression of numerous structural and physiological components can be initiated or upregulated during early stages of mammalian hair cell commitment and differentiation.
Transient expression of an inwardly rectifying potassium conductance in developing inner and outer hair cells along the mouse cochlea.
Inwardly rectifying K+ currents in inner and outer hair cells (IHCs, OHCs) were studied during post-natal development of the mouse cochlea. Hyperpolarizing steps from a holding potential of -64 mV induced a rapidly activating current in both cell types. This current showed strong inward rectification around the K+ equilibrium potential and, at potentials negative to -130 mV, partial inactivation. The activation range varied with extracellular K+ concentration. External application of Ba2+ and Cs+ reversibly blocked the elicited current. The results are consistent with the presence of an IK1-type inwardly rectifying potassium conductance in these cells. The maximum current was 60% larger in IHCs than in OHCs. In OHCs, but not IHCs, the amplitude of IK1 varied significantly with the cells' position along the cochlea. IK1 was maximal in cells located in the most basal region of the cochlea and its amplitude decreased in the apical coil. IK1 disappeared upon functional maturation: in OHCs at the end of the first postnatal week, and in IHCs at the onset of auditory function 12 days after birth. The current is active at the resting potential of the cells and plays a role in regulating the spiking behaviour characteristic of developing hair cells.
Expression of a potassium current in inner hair cells during development of hearing in mice.
Excitable cells use ion channels to tailor their biophysical properties to the functional demands made upon them. During development, these demands may alter considerably, often associated with a change in the cells' complement of ion channels. Here we present evidence for such a change in inner hair cells, the primary sensory receptors in the mammalian cochlea. In mice, responses to sound can first be recorded from the auditory nerve and observed behaviourally from 10-12 days after birth; these responses mature rapidly over the next 4 days. Before this time, mouse inner hair cells have slow voltage responses and fire spontaneous and evoked action potentials. During development of auditory responsiveness a large, fast potassium conductance is expressed, greatly speeding up the membrane time constant and preventing action potentials. This change in potassium channel expression turns the inner hair cell from a regenerative, spiking pacemaker into a high-frequency signal transducer.
The paradox of hair cell adaptation.
Explore the source record for details and available documents.
Myosin VIIA is required for aminoglycoside accumulation in cochlear hair cells.
Myosin VIIA is expressed by sensory hair cells and has a primary structure predicting a role in membrane trafficking and turnover, processes that may underlie the susceptibility of hair cells to aminoglycoside antibiotics. [3H]Gentamicin accumulation and the effects of aminoglycosides were therefore examined in cochlear cultures of mice with different missense mutations in the myosin VIIA gene, Myo7a, to see whether myosin VIIA plays a role in aminoglycoside ototoxicity. Hair cells from homozygous mutant Myo7ash1 mice, with a mutation in a nonconserved region of the myosin VIIA head, respond rapidly to aminoglycoside treatment and accumulate high levels of gentamicin. Hair cells from homozygous mutant Myo7a6J mice, with a mutation at a highly conserved residue close to the ATP binding site of the myosin VIIA head, do not accumulate [3H]gentamicin and are protected from aminoglycoside ototoxicity. Hair cells from heterozygotes of both alleles accumulate [3H]gentamicin and respond to aminoglycosides. Although aminoglycoside uptake is thought to be via apical surface-associated endocytosis, coated pit numbers on the apical membrane of heterozygous and homozygous Myo7a6J hair cells are similar. Pulse-chase experiments with cationic ferritin confirm that the apical endocytotic pathway is functional in homozygous Myo7a6J hair cells. Transduction currents can be recorded from both heterozygous and homozygous Myo7a6J hair cells, suggesting it is unlikely that the drug enters via diffusion through the mechanotransducer channel. The results show that myosin VIIA is required for aminoglycoside accumulation in hair cells. Myosin VIIA may transport a putative aminoglycoside receptor to the hair cell surface, indirectly translocate it to sites of membrane retrieval, or retain it in the endocytotic pathway.
A quantitative comparison of mechanoelectrical transduction in vestibular and auditory hair cells of neonatal mice.
Vestibular hair cells (VHCs) and cochlear outer hair cells (OHCs) of neonatal mice were stimulated by a fluid jet directed at their stereociliary bundles. Relations between the force exerted by the jet, bundle displacement, and the resulting transducer current were studied. The mean maximum transducer conductance in VHCs (2.6 nS) was about half that of the OHCs (5.5 nS), with the largest recorded values being 4.1 nS and 9.2 nS, respectively. In some OHCs activity of a single, 112 pS transducer channel was observed, allowing an estimate of the maximum number of channels: up to 36 in VHCs and 82 in OHCs, corresponding to about one transducer channel per tip link. The VHC bundles required about 330 nm of tip displacement to activate 90% of the maximum transducer conductance, compared to 150 nm for the OHC bundles. This corresponded to 2 deg of rotation about their pivots for both, due to the greater length of the VHC bundles. The VHC bundles' translational stiffness was one-seventh of that of the OHCs. Conversion to rotational stiffness almost abolished this difference. Rotation of the hair bundle rather than translation determines the gating of the transducer channels, independent of bundle height or origin of the cells.
Displacement sensitivity of mammalian vestibular transducers.
Explore the source record for details and available documents.
Patch clamped responses from outer hair cells in the intact adult organ of Corti.
Outer hair cells (OHCs) from the mammalian cochlea act as both sensory cells and motor cells. We report here whole-cell tight seal recordings of OHC activity in their natural embedding tissue, the intact organ of Corti, using a temporal bone preparation. The mean cell resting potential, -76 +/- 4 mV (n = 19) and input conductance (10 +/- 3 nS at -70 mV) of third turn hair cells were significantly lower than have been found in isolated cells. Two main K+ currents in the cell were identified. One current, activated positive to -100 mV, was reduced by 5 mM BaCl2. The other current, activated above -40 mV, was reduced by 100 microM 4-aminopyridine (4-AP) and by 30 mM tetraethylammonium (TEA). Both of these currents have been also identified in recordings reported from isolated cells. On stepping to different membrane potentials, cells imaged in the organ of Corti changed length by an amount large enough to cause visible distortions in neighbouring cells. By quantifying such distortions we estimate that the forces generated by OHCs can account for the enhanced response to sound required by the cochlear amplifier.