Effect of salicylate on KCNQ4 of the guinea pig outer hair cell.

Salicylate causes a moderate hearing loss and tinnitus in humans at high-dose levels. Salicylate-induced hearing loss has been attributed to impaired sound amplification by outer hair cells (OHCs) through its direct action on the OHC motility sensor and/or motor. However, there is a disparity of salicylate concentrations between the clinical and animal studies, i.e., extremely high extracellular concentrations of salicylate (from 1 to 10 mM) is required to produce a significant reduction of electromotility in animal studies. Such concentrations are above the clinical/physiological range for humans. Here, we showed that clinical/physiological concentration range of salicylate caused concentration-dependent and reversible reductions in I(K,n) (KCNQ4) and subsequent depolarization of OHCs. Salicylate reduced the maximal tail current of the activation curve of I(K,n) without altering the voltage-sensitivity (V(half)). The salicylate-induced reduction of I(K,n) was almost completely blocked by linopirdine (0.1 mM) and BaCl2 (10 mM). Consistent with the finding in OHCs, salicylate significantly reduced KCNQ4-mediated current expressed in Chinese hamster ovarian (CHO) cells by comparable amplitude to OHCs without significantly shifting V(half). Nonstationary fluctuation analysis shows that salicylate significantly reduced the estimated single-channel current amplitude and numbers. Intracellular Ca²+ elevation resulting from cytoplasmic acidosis also contributes to the current reduction of I(K,n) (KCNQ4) of OHCs. These results indicate a different model for the salicylate-induced hearing loss through the reduction of KCNQ4 and subsequent depolarization of OHCs, which reduces the driving force for transduction current and electromotility. The major mechanism underlying the reduction of I(K,n) (KCNQ4) is the direct blocking action of salicylate on KCNQ4.

Otoconin-90 deletion leads to imbalance but normal hearing: a comparison with other otoconia mutants.

Our sense of gravitation and linear acceleration is mediated by stimulation of vestibular hair cells through displacement of otoconia in the utricle and saccule (the gravity receptor organ). We recently showed that otoconin-90 (Oc90) deletion led to formation of giant otoconia. In the present study, we determined the extent to which the giant otoconia affected balance and gravity receptor sensory input and compared the findings with other otoconia mutants. We employed a wide spectrum of balance behavioral tests, including reaching and air-righting reflexes, gait, swimming, beam-crossing, rotorod latencies, and a direct measure of gravity receptor input, vestibular evoked potentials (VsEPs). All tests on homozygous adult mutants consistently ranked the order of imbalance as (from worst to best) Nox3(het)

Hair cells--beyond the transducer.

OVERVIEW: This review considers the "tween twixt and twain" of hair cell physiology, specifically the signaling elements and membrane conductances which underpin forward and reverse transduction at the input stage of hair cell function and neurotransmitter release at the output stage. Other sections of this review series outline the advances which have been made in understanding the molecular physiology of mechanoelectrical transduction and outer hair cell electromotility. Here we outline the contributions of a considerable array of ion channels and receptor signaling pathways that define the biophysical status of the sensory hair cells, contributing to hair cell development and subsequently defining the operational condition of the hair cells across the broad dynamic range of physiological function.

Multisensory plasticity in congenitally deaf mice: how are cortical areas functionally specified?

The neocortex of congenitally deaf mice was examined using electrophysiological recording techniques combined with cortical myeloarchitecture. Our results indicate that relative activity patterns across sensory systems during development contribute to modality assignment of cortical fields as well as the size of cortical fields. In congenitally deaf mice, "auditory cortex" contained neurons that responded to somatosensory, visual, or both somatosensory and visual stimulation; the primary visual area contained a larger proportion of neurons that responded to somatosensory stimulation than in normal animals, and the primary visual area had significantly increased in size. Thus, cortical architecture and functional specification were de-correlated. When results are considered in the light of molecular studies and studies in which peripheral activity is altered in development, it becomes clear that similar types of changes to the neocortex, such as alterations in cortical field size, can be achieved in more than one way in the developing and evolving neocortex.

Direct measurement of single-channel Ca(2+) currents in bullfrog hair cells reveals two distinct channel subtypes.

1. To confer their acute sensitivity to mechanical stimuli, hair cells employ Ca(2+) ions to mediate sharp electrical tuning and neurotransmitter release. We examined the diversity and properties of voltage-gated Ca(2+) channels in bullfrog saccular hair cells by means of perforated and cell-attached patch-clamp techniques. Whole-cell Ca(2+) current records provided hints that hair cells express L-type as well as dihydropyridine-insensitive Ca(2+) currents. 2. Single Ca(2+) channel records confirmed the presence of L-type channels, and a distinct Ca(2+) channel, which has sensitivity towards omega-conotoxin GVIA. Despite its sensitivity towards omega-conotoxin GVIA, the non-L-type channel cannot necessarily be considered as an N-type channel because of its distinct voltage-dependent gating properties. 3. Using 65 mM Ca(2+) as the charge carrier, the L-type channels were recruited at about -40 mV and showed a single-channel conductance of 13 pS. Under similar recording conditions, the non-L-type channels were activated at approximately -60 mV and had a single-channel conductance of approximately 16 pS. 4. The non-L-type channel exhibited at least two fast open time constants (tau(o) = 0.2 and 5 ms). In contrast, the L-type channels showed long openings (tau(o) = approximately 23 ms) that were enhanced by Bay K 8644, in addition to the brief openings (tau(o) = 0.3 and 10 ms). 5. The number of functional channels observed in patches of similar sizes suggests that Ca(2+) channels are expressed singly, in low-density clusters (2-15 channels) and in high-density clusters (20-80 channels). Co-localization of the two channel subtypes was observed in patches containing low-density clusters, but was rare in patches containing high-density clusters. 6. Finally, we confirmed the existence of two distinct Ca(2+) channel subtypes by using immunoblot and immunohistochemical techniques.

Mice lacking the basolateral Na-K-2Cl cotransporter have impaired epithelial chloride secretion and are profoundly deaf.

In chloride-secretory epithelia, the basolateral Na-K-2Cl cotransporter (NKCC1) is thought to play a major role in transepithelial Cl(-) and fluid transport. Similarly, in marginal cells of the inner ear, NKCC1 has been proposed as a component of the entry pathway for K(+) that is secreted into the endolymph, thus playing a critical role in hearing. To test these hypotheses, we generated and analyzed an NKCC1-deficient mouse. Homozygous mutant (Nkcc1(-/-)) mice exhibited growth retardation, a 28% incidence of death around the time of weaning, and mild difficulties in maintaining their balance. Mean arterial blood pressure was significantly reduced in both heterozygous and homozygous mutants, indicating an important function for NKCC1 in the maintenance of blood pressure. cAMP-induced short circuit currents, which are dependent on the CFTR Cl(-) channel, were reduced in jejunum, cecum, and trachea of Nkcc1(-/-) mice, indicating that NKCC1 contributes to cAMP-induced Cl(-) secretion. In contrast, secretion of gastric acid in adult Nkcc1(-/-) stomachs and enterotoxin-stimulated fluid secretion in the intestine of suckling Nkcc1(-/-) mice were normal. Finally, homozygous mutants were deaf, and histological analysis of the inner ear revealed a collapse of the membranous labyrinth, consistent with a critical role for NKCC1 in transepithelial K(+) movements involved in generation of the K(+)-rich endolymph and the endocochlear potential.

Expression of different types of inward rectifier currents confers specificity of light and dark responses in type A and B photoreceptors of Hermissenda.

Each eye of the mollusc Hermissenda consists of five photoreceptors, two type A and three type B cells. Type A cells are quiescent, whereas B cells are spontaneously active in the dark. Differences in the intrinsic membrane properties of type A and B photoreceptors were studied using voltage- and current-clamp techniques. The current density of a Ni2+-sensitive, low-voltage activated Ca2+ current was similar in the two cell types. However, type B cells express an inward rectifier current (Ih) that has different permeation and pharmacological properties from the inward rectifier current in type A cells. The current in the B cells was time-dependent and was blocked by Cs+. Na+ and K+ were the charge carriers for Ih. The inward rectifier current in A cells (IK1) was time-independent, was selectively permeable to K+, and was blocked by Ba2+. Ni2+ reduced the spontaneous spike activities of type A and B cells, whereas Cs+ produced membrane hyperpolarization and reduced the spike activities of dark-adapted B cells. The application of both Cs+ and Ni2+ completely blocked dark-adapted spontaneous activities of B cells. Moreover, Ba2+ increased the excitability of type A cells but not B cells. Hence, differential expression of the two distinct inward rectifiers found in type A and B cells contributes to differences in their intrinsic membrane properties. Because changes in the excitability of the two cell types are correlates of conditioning in Hermissenda, modulation of these underlying currents may play a major role during conditioning-induced plasticity.

Balance and hearing deficits in mice with a null mutation in the gene encoding plasma membrane Ca2+-ATPase isoform 2.

Plasma membrane Ca2+-ATPase isoform 2 (PMCA2) exhibits a highly restricted tissue distribution, suggesting that it serves more specialized physiological functions than some of the other isoforms. A unique role in hearing is indicated by the high levels of PMCA2 expression in cochlear outer hair cells and spiral ganglion cells. To analyze the physiological role of PMCA2 we used gene targeting to produce PMCA2-deficient mice. Breeding of heterozygous mice yielded live homozygous mutant offspring. PMCA2-null mice grow more slowly than heterozygous and wild-type mice and exhibit an unsteady gait and difficulties in maintaining balance. Histological analysis of the cerebellum and inner ear of mutant and wild-type mice revealed that null mutants had slightly increased numbers of Purkinje neurons (in which PMCA2 is highly expressed), a decreased thickness of the molecular layer, an absence of otoconia in the vestibular system, and a range of abnormalities of the organ of Corti. Analysis of auditory evoked brainstem responses revealed that homozygous mutants were deaf and that heterozygous mice had a significant hearing loss. These data demonstrate that PMCA2 is required for both balance and hearing and suggest that it may be a major source of the calcium used in the formation and maintenance of otoconia.

Plasma membrane Ca2+-ATPase extrudes Ca2+ from hair cell stereocilia.

Mechanically sensitive hair cells of the auditory and vestibular systems use Ca2+ to control adaptation of mechanical transduction, to effect frequency tuning, to trigger neurotransmitter release, and to mediate efferent synaptic signaling. To determine the role that pumps play in regulation of Ca2+ in the hair bundle, the organelle responsible for mechanoelectrical transduction, we localized and quantified the plasma membrane Ca2+-ATPase (PMCA) of the bundle. We found that each hair bundle contains approximately 10(6) PMCA molecules or approximately 2000 per square micrometer of bundle membrane and that PMCA is the principal calmodulin binding protein of the bundle. Consistent with biochemical estimates of PMCA density, we measured with extracellular Ca2+-selective electrodes a substantial Ca2+ efflux from bundles. The number of bundle Ca2+ pumps and magnitude of resting Ca2+ efflux suggested that PMCA should generate a substantial membrane current as bundles expel Ca2+. Measurement of whole-cell currents revealed a transduction-dependent outward current that was consistent with the activity of PMCA. Finally, dialysis of hair cells with PMCA inhibitors led to a large increase in the concentration of Ca2+ in bundles, which suggests that PMCA plays a major role in regulating bundle Ca2+ concentration. Our data further indicate that PMCA could elevate the extracellular Ca2+ concentration close to hair bundles above the low level found in bulk endolymph.

Potassium currents in presynaptic hair cells of Hermissenda.

Apart from their primary function as balance sensors, Hermissenda hair cells are presynaptic neurons involved in the Ca(2+)-dependent neuronal plasticity in postsynaptic B photoreceptors that accompanies classical conditioning. With a view to beginning to understand presynaptic mechanisms of plasticity in the vestibulo-visual system, a locus for conditioning-induced neuronal plasticity, outward currents that may govern the excitability of hair cells were recorded by means of a whole-cell patch-clamp technique. Three K+ currents were characterized: a 4-aminopyridine-sensitive transient outward K+ current (IA), a tetraethyl ammonium-sensitive delayed rectifier K+ current (IK,V), and a Ca(2+)-activated K+ current (IK,Ca). IA activates and decays rapidly; the steady-state activation and inactivation curves of the current reveal a window current close to the apparent resting voltage of the hair cells, suggesting that the current is partially activated at rest. By modulating firing frequency and perhaps damping membrane oscillations, IA may regulate synaptic release at baseline. In contrast, IK,V and IK,Ca have slow onset and exhibit little or no inactivation. These two K+ currents may determine the duration of the repolarization phase of hair-cell action potentials and hence synaptic release via Ca2+ influx through voltage-gated Ca2+ channels. In addition, IK,Ca may be responsible for the afterhyperpolarization of hair cell membrane voltage following prolonged stimulation.

Regeneration of broken tip links and restoration of mechanical transduction in hair cells.

A hair cell's tip links are thought to gate mechanoelectrical transduction channels. The susceptibility of tip links to acoustic trauma raises questions as to whether these fragile structures can be regenerated. We broke tip links with the calcium chelator 1,2-bis(O-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid and found that they can regenerate, albeit imperfectly, over several hours. The time course of tip-link regeneration suggests that this process may underlie recovery from temporary threshold shifts induced by noise exposure. Cycloheximide does not block tip-link regeneration, indicating that new protein synthesis is not required. The calcium ionophore ionomycin prevents regeneration, suggesting regeneration normally may be stimulated by the reduction in stereociliary Ca2+ when gating springs rupture and transduction channels close. Supporting the equivalence of tip links with gating springs, mechanoelectrical transduction returns over the same time period as tip links; strikingly, adaptation is substantially reduced, even 24 hr after breaking tip links.

Phosphate analogs block adaptation in hair cells by inhibiting adaptation-motor force production.

To ensure optimal sensitivity for mechanoelectrical transduction, hair cells adapt to prolonged stimuli using active motors. Adaptation motors are thought to employ myosin molecules as their force-producing components. We find that beryllium fluoride, vanadate, and sulfate, phosphate analogs that inhibit the ATPase activity of myosin, inhibit adaptation by abolishing motor force production. Phosphate analogs interact with a 120-kDa bundle protein, most likely myosin 1 beta, in a manner that coincides with their effects on adaptation. Features of transduction following inhibition of motor force production suggest that the gating and extent springs of the hair cell orient in parallel at rest and that the negative limit of adaptation arises when force in the stretched extent spring matches the force output of the adaptation motor.

Protein kinase and G-protein regulation of Ca2+ currents in Hermissenda photoreceptors by 5-HT and GABA.

The effects of serotonin (5-HT) and GABA on two Ca2+ currents, a transient low-voltage-activated current (tLVA) and a sustained high-voltage-activated current (sHVA) were examined in isolated photoreceptors of Hermissenda. The sHVA current was blocked by 5-HT and reduced by activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate. The effects of 5-HT were transiently reversed by staurosporine and partially blocked by the PKC inhibitor peptide [PKC(19-36)]. GABA enhanced both the tLVA and sHVA currents at low concentrations (5 nM to 5 microM) and reduced the sHVA current at high concentrations (>10 microM). The GABA-mediated enhancement of the Ca2+ current at low concentrations was sensitive to block by picrotoxin. The protein kinase A (PKA) inhibitor peptide [PKI(6-22)amide] blocked enhancement of both Ca2+ currents produced by cAMP analogs and GABA, suggesting that the effects at low concentrations may be PKA mediated. Caged GTP-gamma-S released by flash photolysis reduced the sHVA current, and pretreatment of the photoreceptors with pertussis toxin blocked the effects of higher concentrations of GABA, indicating that at higher concentrations, the effects may be G-protein mediated.

Evidence for a contribution of ICa to serotonergic modulation of IK,Ca in Hermissenda photoreceptors.

1. The Ca(2+)-dependent K+ current (IK,Ca) contributes to both the plateau phase of light-elicited generator potentials and enhanced excitability of identified type B photoreceptors of Hermissenda detected after classical conditioning. Serotonergic modulation of membrane conductances mimics some of the effects of conditioning. Serotonin (5-HT) reduces the magnitude of IK,Ca and decreases the sustained voltage-activated Ca2+ current (ICa) in type B photoreceptors. We have examined the modulatory role of 5-HT in regulation of IK,Ca by ICa using a Ca2+ ionophore in conjunction with the whole cell patch-clamp technique in isolated photoreceptors. 2. The 40-50% reduction of IK,Ca by 5-HT was voltage independent. Cd2+ blocked ICa and reduced IK,Ca by 70-80%. The remaining 20-30% of IK,Ca may result from Ca2+ release from intracellular stores, because IK,Ca was further reduced to 5-10% as the pipette ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) was raised from 0.5 to 5 mM. The application of the Ca2+ ionophore A23187, which was designed to produce Ca2+ influx independent of the voltage-activated Ca2+ channels, restored IK,Ca. 3. The application of A23187 reversed the effects of 5-HT and Cd2+ on IK,Ca for experiments lasting 15-20 min. However, for longer time periods (> 25 min), complete restoration of IK,Ca by A23187 was obtained in the presence of Cd2+ but not 5-HT. These results suggest that for 15 to 20 min exposures the reduction of IK,Ca by 5-HT is a consequence of modulation of ICa by 5-HT and not a direct effect of 5-HT on IK,Ca.

Calcium current and inactivation in identified neurons in Hermissenda crassicornis.

1. N-type (omega-conotoxin sensitive) calcium currents (ICa) were recorded in identified neurons in Hermissenda crassicornis using low-resistance patch electrodes (0.7 +/- 0.3 M omega; n = 101) under conditions that eliminated inward Na+ currents (choline ions substitution) and suppressed outward K+ currents (Cs+, tetraethylammonium, and 4-AP). Step depolarization from a holding potential of -60 mV to potentials above -30 mV elicited ICa, which peaked approximately 20 mV and declined with increasing depolarizations. 2. Evidence for a low-threshold current was present. Step depolarization from a more hyperpolarizing potentials (e.g., -90 mV) revealed a small shoulder (< 100 pA) at -60 to -40 mV that was sensitive to Co2+ and Ni2+. However, under the conditions examined here (holding potential of -60 mV), the high-voltage-activated current predominated. 3. Barium (Ba2+) and strontium (Sr2+) permeate the Ca2+ channel with similar activation kinetics (ease of permeation; Ba2+ > Ca2+ > Sr2+). Steady-state activation of permeability versus membrane potentials for Ca2+, Ba2+, and Sr2+ as charge carriers could be fitted with the Boltzmann equation, with half-activation voltage and slope factor of 2.9 and 7.7 mV for ICa, -13.1 mV and 7.8 for Ba2+ current (IBa) and -2.3 mV and 7.8 for Sr2+ current (ISr). The time course of activation was monotonic with time constant (tau) for ICa ranging from 2 to 8 ms. 4. The inactivation profile was complex. At negative step potentials (e.g., -20 mV), inactivation of the current was slow. Depolarization steps to relatively positive voltages (e.g., 10 mV) showed more rapid inactivation than those at more positive potentials (e.g., 40 mV). When extracellular Ca2+ was raised from 5 to 10 mM, a biphasic decay (tau fast of 25 +/- 4 ms; and tau slow of 473 +/- 64 ms; mean +/- SD, n = 9) was seen. Such an observation suggested a current-mediated inactivation. 5. With a pulse duration of approximately 350 ms, ISr showed inactivation whereas Ba2+ virtually removed the decay. However, IBa turned off with more prolonged depolarization. 6. A twin-pulse protocol was used to assess the voltage dependence of inactivation: an incomplete U-shaped inactivation curve was observed for ICa, IBa, and ISr. Channels available for inactivation were increased in the presence of Ca2+ ions. 7. Inactivation was further studied with the Ca2+ chelators, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and bis(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA). With 10 mM of BAPTA, in the pipette, inactivation was reduced but not removed.(ABSTRACT TRUNCATED AT 250 WORDS)

Two components of calcium currents in the soma of photoreceptors of Hermissenda.

1. The proposed mechanism of cellular plasticity underlying classical conditioning of Hermissenda involves Ca2+ influx through voltage-activated channels. This influx triggers several molecular cascades and leads to the phosphorylation of K+ channels in identified photoreceptors. We studied Ca2+ currents from isolated photoreceptors of Hermissenda with the whole cell patch-clamp technique. Two distinct Ca2+ currents were identified in isolated photoreceptors on the basis of differences in their voltage dependence, kinetics, and pharmacology. 2. One Ca2+ current was transient (ICa(t)), with a fast onset (approximately 5 ms), activated at -50 mV from a holding potential of -90 mV, and peaked at 0 mV. The second Ca2+ current, designated as sustained (ICa(s)), exhibited a delayed time-to-peak, activated at -30 mV, and reached maximum at 30 mV. 3. Steady-state activation curves for both currents were generated from normalized currents and fitted with the Boltzmann function; estimates of half-activation voltages for ICa(t) were -38.8 +/- 6.7 mV (mean +/- SD; n = 9) and 3.2 +/- 8.2 mV for ICa(s) (n = 11) with maximum slopes of 8.9 +/- 1.6 mV (n = 9) and 11.0 +/- 2.4 mV (n = 11). 4. The inactivation of ICa(s) was slow (time constants > 3 s) whereas ICa(t) inactivated rapidly (time constant of inactivation at various voltages; 75-600 ms). 5. Ni2+ (0.8 mM), Gd3+ (0.5 mM), and amiloride (10 microM) produced a reversible block of ICa(t) without affecting ICa(s). omega-Conotoxin GVIA (10 nM) irreversibly blocked ICa(s) whereas nitrendipine (20 microM) produced a reversible block. 6. ICa(t) may be responsible for steady-state membrane potential oscillations. ICa(s) may contribute to the maintenance of the amplitude of the plateau phase of the generator potential.

Outgrowths from Hermissenda photoreceptor somata are associated with activation of protein kinase C.

We have found changes in the morphology of photoreceptor somata from the mollusc Hermissenda that are produced by application of 12,13-phorbol dibutyrate (PDBU), an activator of PKC, in combination with elevated intracellular Ca2+ levels. The changes in morphology were expressed as rapid and repetitive outgrowths and additionally as more general changes in shape of the soma. Application of 4 alpha-PMA, a phorbol ester which does not activate PKC, did not produce these changes. The functional integrity of the photoreceptors in these dissociated eye preparations was maintained throughout the period of incubation with PDBU according to standard electrophysiological criteria. It has previously been shown that classical conditioning produced a reduction of dendritic volume in the type B photoreceptor of Hermissenda, a specific locus for associative memory storage. These changes in dendritic morphology were correlated with increased resistance across the cell membrane caused by learning-induced reductions of outward somatic K+ currents. Such conditioning-specific reductions of somatic K+ currents appear to depend on the phosphorylation of a 20-kDa G-protein (CP20) mediated by the Ca2+ and phospholipid-dependent kinase, protein kinase C (PKC). Thus PKC activity may be important in structural changes of the synaptic region of specific neurons involved in associative memory. The results of the present study suggest that the effects of PKC activation may also include structural changes in the soma of these same neurons.