Tuning Of Hair Cells Research Articles

Probing electrical tuning of hair cells with a Zap current method in the intact amphibian papilla of bullfrogs.

Most, if not all, modern vertebrate species have evolved exquisite inner ears to discriminate acoustic signals of different frequencies, through a process called frequency tuning. For non-mammalian species, at least part of frequency tuning has been attributed to intrinsic electrical properties of hair cells, i.e. electrical tuning. Since it was first discovered, the traditional method to assess electrical tuning has been to inject step current into hair cells and examine dampened membrane voltage oscillation. However, this method is not applicable for hair cells that do not oscillate. In this study, we developed a Zap current method that can be unbiasedly applied to all hair cells regardless of their oscillating behavior. Similar to a chirp sound in acoustic stimulation, a Zap current is a sinusoidal current with the frequency increased linearly with time. We first validated this new method with the traditional step current method on hair cells with dampened membrane voltage oscillation, and then applied it to all hair cells in the intact amphibian papilla of bullfrogs. We found that while hair cells with dampened membrane voltage oscillation are sharply tuned, non-oscillating hair cells are broadly tuned. In addition, we found a third type of hair cells, which oscillate continuously and are extremely sharply tuned, with multiple peaks that are reminiscent of harmonics in the mammalian cochlea. In conclusion, the new Zap current method provides an unbiased way to assess electrical tuning, and it reveals an underappreciated heterogeneity of electrical tuning in the bullfrog amphibian papilla.

The Contribution of RCK Domains to Human BK Channel Allosteric Activation

Large conductance voltage- and Ca(2+)-activated K(+) (BK) channels are potent regulators of cellular processes including neuronal firing, synaptic transmission, cochlear hair cell tuning, insulin release, and smooth muscle tone. Their unique activation pathway relies on structurally distinct regulatory domains including one transmembrane voltage-sensing domain (VSD) and two intracellular high affinity Ca(2+)-sensing sites per subunit (located in the RCK1 and RCK2 domains). Four pairs of RCK1 and RCK2 domains form a Ca(2+)-sensing apparatus known as the "gating ring." The allosteric interplay between voltage- and Ca(2+)-sensing apparati is a fundamental mechanism of BK channel function. Using voltage-clamp fluorometry and UV photolysis of intracellular caged Ca(2+), we optically resolved VSD activation prompted by Ca(2+) binding to the gating ring. The sudden increase of intracellular Ca(2+) concentration ([Ca(2+)](i)) induced a hyperpolarizing shift in the voltage dependence of both channel opening and VSD activation, reported by a fluorophore labeling position 202, located in the upper side of the S4 transmembrane segment. The neutralization of the Ca(2+) sensor located in the RCK2 domain abolished the effect of [Ca(2+)](i) increase on the VSD rearrangements. On the other hand, the mutation of RCK1 residues involved in Ca(2+) sensing did not prevent the effect of Ca(2+) release on the VSD, revealing a functionally distinct interaction between RCK1 and RCK2 and the VSD. A statistical-mechanical model quantifies the complex thermodynamics interplay between Ca(2+) association in two distinct sites, voltage sensor activation, and BK channel opening.

Open structure of the Ca2+ gating ring in the high-conductance Ca2+-activated K+ channel.

High-conductance voltage- and Ca(2+)-activated K(+) channels function in many physiological processes that link cell membrane voltage and intracellular Ca(2+) concentration, including neuronal electrical activity, skeletal and smooth muscle contraction, and hair cell tuning. Like other voltage-dependent K(+) channels, Ca(2+)-activated K(+) channels open when the cell membrane depolarizes, but in contrast to other voltage-dependent K(+) channels, they also open when intracellular Ca(2+) concentrations rise. Channel opening by Ca(2+) is made possible by a structure called the gating ring, which is located in the cytoplasm. Recent structural studies have defined the Ca(2+)-free, closed, conformation of the gating ring, but the Ca(2+)-bound, open, conformation is not yet known. Here we present the Ca(2+)-bound conformation of the gating ring. This structure shows how one layer of the gating ring, in response to the binding of Ca(2+), opens like the petals of a flower. The degree to which it opens explains how Ca(2+) binding can open the transmembrane pore. These findings present a molecular basis for Ca(2+) activation of K(+) channels and suggest new possibilities for targeting the gating ring to treat conditions such as asthma and hypertension.

Metal-driven Operation of the Human Large-conductance Voltage- and Ca2+-dependent Potassium Channel (BK) Gating Ring Apparatus

Large-conductance voltage- and Ca(2+)-dependent K(+) (BK, also known as MaxiK) channels are homo-tetrameric proteins with a broad expression pattern that potently regulate cellular excitability and Ca(2+) homeostasis. Their activation results from the complex synergy between the transmembrane voltage sensors and a large (>300 kDa) C-terminal, cytoplasmic complex (the "gating ring"), which confers sensitivity to intracellular Ca(2+) and other ligands. However, the molecular and biophysical operation of the gating ring remains unclear. We have used spectroscopic and particle-scale optical approaches to probe the metal-sensing properties of the human BK gating ring under physiologically relevant conditions. This functional molecular sensor undergoes Ca(2+)- and Mg(2+)-dependent conformational changes at physiologically relevant concentrations, detected by time-resolved and steady-state fluorescence spectroscopy. The lack of detectable Ba(2+)-evoked structural changes defined the metal selectivity of the gating ring. Neutralization of a high-affinity Ca(2+)-binding site (the "calcium bowl") reduced the Ca(2+) and abolished the Mg(2+) dependence of structural rearrangements. In congruence with electrophysiological investigations, these findings provide biochemical evidence that the gating ring possesses an additional high-affinity Ca(2+)-binding site and that Mg(2+) can bind to the calcium bowl with less affinity than Ca(2+). Dynamic light scattering analysis revealed a reversible Ca(2+)-dependent decrease of the hydrodynamic radius of the gating ring, consistent with a more compact overall shape. These structural changes, resolved under physiologically relevant conditions, likely represent the molecular transitions that initiate the ligand-induced activation of the human BK channel.

A search for factors specifying tonotopy implicates DNER in hair-cell development in the chick's cochlea

The accurate perception of sound frequency by vertebrates relies upon the tuning of hair cells, which are arranged along auditory organs according to frequency. This arrangement, which is termed a tonotopic gradient, results from the coordination of many cellular and extracellular features. Seeking the mechanisms that orchestrate those features and govern the tonotopic gradient, we used expression microarrays to identify genes differentially expressed between the high- and low-frequency cochlear regions of the chick ( Gallus gallus). Of the three signaling systems that were represented extensively in the results, we focused on the notch pathway and particularly on DNER, a putative notch ligand, and PTPζ, a receptor phosphatase that controls DNER trafficking. Immunohistochemistry confirmed that both proteins are expressed more strongly in hair cells at the cochlear apex than in those at the base. At the apical surface of each hair cell, the proteins display polarized, mutually exclusive localization patterns. Using morpholinos to decrease the expression of DNER or PTPζ as well as a retroviral vector to overexpress DNER, we observed disturbances of hair-bundle morphology and orientation. Our results suggest a role for DNER and PTPζ in hair-cell development and possibly in the specification of tonotopy.

Mechanics of the frog ear

The frog inner ear contains three regions that are sensitive to airborne sound and which are functionally distinct. (1) The responses of nerve fibres innervating the low-frequency, rostral part of the amphibian papilla (AP) are complex. Electrical tuning of hair cells presumably contributes to the frequency selectivity of these responses. (2) The caudal part of the AP covers the mid-frequency portion of the frog’s auditory range. It shares the ability to generate both evoked and spontaneous otoacoustic emissions with the mammalian cochlea and other vertebrate ears. (3) The basilar papilla functions mainly as a single auditory filter. Its simple anatomy and function provide a model system for testing hypotheses concerning emission generation. Group delays of stimulus-frequency otoacoustic emissions (SFOAEs) from the basilar papilla are accounted for by assuming that they result from forward and reverse transmission through the middle ear, a mechanical delay due to tectorial membrane filtering and a rapid forward and reverse propagation through the inner ear fluids, with negligible delay.

Gerbils can tune in

Gerbils can tune in

Beta1 subunits facilitate gating of BK channels by acting through the Ca2+, but not the Mg2+, activating mechanisms.

The beta1 subunit of BK (large conductance Ca2+ and voltage-activated K+) channels is essential for many key physiological processes, such as controlling the contraction of smooth muscle and the tuning of hair cells in the cochlea. Although it is known that the beta1 subunit greatly increases the open probability of BK channels, little is known about its mechanism of action. We now explore this mechanism by using channels in which the Ca2+- and Mg2+-dependent activating mechanisms have been disrupted by mutating three sites to remove the Ca2+ and Mg2+ sensitivity. We find that the presence of the beta1 subunit partially restores Ca2+ sensitivity to the triply mutated channels, but not the Mg2+ sensitivity. We also find that the beta1 subunit has no effect on the Mg2+ sensitivity of WT BK channels, in contrast to its pronounced effect of increasing the apparent Ca2+ sensitivity. These observations suggest that the beta1 subunit increases open probability by working through the Ca2+-dependent, rather than Mg2+-dependent, activating mechanisms, and that the action of the beta1 subunit is not directly on the Ca2+ binding sites, but on the allosteric machinery coupling the sites to the gate. The differential effects of the beta1 subunit on the Ca2+ and Mg2+ activation of the channel suggest that these processes act separately. Finally, we show that Mgi2+ inhibits, rather than activates, BK channels in the presence of the beta1 subunit for intermediate levels of Cai2+. This Mg2+ inhibition in the presence of the beta1 subunit provides an additional regulatory mechanism of BK channel activity.

Modeling Hair Cell Tuning by Expression Gradients of Potassium Channel β Subunits

The receptor potential of sensory hair cells arises from the gating of mechanosensitive cation channels, but its amplitude and time course also depend on the number and kinetics of voltage-gated ion channels in each cell. Prominent among these are “BK” potassium channels encoded by the slo gene that support electrical tuning in some hair cells. Hair cells tuned to low frequencies have slowly gating BK channels, whereas those of higher-frequency hair cells gate more rapidly. Alternative splicing of the slo gene mRNA that encodes the pore-forming α subunit can alter BK channel kinetics, and gating is dramatically slowed by coexpression with modulatory β subunits. The effect of the β subunit is consistent with low-frequency tuning, and β mRNA is expressed at highest levels in the low frequency apex of the bird’s auditory epithelium. How might an expression gradient of β subunits contribute to hair cell tuning? The present work uses a computational model of hair cell-tuning based on the functional properties of BK channels expressed from hair cell α and β slo cDNA. The model reveals that a limited tonotopic gradient could be achieved simply by altering the fraction of BK channels in each hair cell that are combined with β subunits. However, complete coverage of the tuning spectrum requires kinetic variants in addition to those modeled here.

Mechanisms of hair cell tuning.

Mechanosensory hair cells of the vertebrate inner ear contribute to acoustic tuning through feedback processes involving voltage-gated channels in the basolateral membrane and mechanotransduction channels in the apical hair bundle. The specific number and kinetics of calcium-activated (BK) potassium channels determine the resonant frequency of electrically tuned hair cells. Kinetic variation among BK channels may arise through alternative splicing of slo gene mRNA and combination with modulatory beta subunits. The number of transduction channels and their rate of adaptation rise with hair cell response frequency along the cochlea's tonotopic axis. Calcium-dependent feedback onto transduction channels may underlie active hair bundle mechanics. The relative contributions of electrical and mechanical feedback to active tuning of hair cells may vary as a function of sound frequency.

Effects of streptomycin and amiloride on the function of the pigeon inner ear

In vitro, streptomycin and amiloride block the mechano-electrical transduction channels in isolated auditory hair cells of mammals and birds. The present in vivo experiments investigate their action in the intact pigeon inner ear. Streptomycin dissolved in artificial endolymph at concentrations between 1.0 and 6.2 mmol/l was applied into the scala media. After a bolus injection (0.09 to 0.36 μl) the endocochlear potential (EP) was elevated from 6.8 to 7.9 mV. Similarly applied boli of 0.09 to 0.55 μl of amiloride in concentrations of 1–2 mmol/l did not significantly elevate EP (8.7 versus 9.4 mV). Compound action potential (CAP) frequency threshold curves (FTCs) were elevated by both streptomycin and amiloride. The threshold elevation increased with frequency. With streptomycin injection, a complete loss of CAPs was found at frequencies above 1.2 kHz. With amiloride, the loss in sensitivity was 40 dB for the same frequency range. Single fibre recordings showed elevated thresholds and decreased sharpness of tuning with both drugs. With streptomycin, the evoked activity was affected regard-less of stimulation frequencies (above or below the fibre's characteristic frequency). Amiloride, however, preferentially elevated thresholds at frequencies above characteristic frequency. Thus amiloride seems to interfere with hair cell tuning mechanisms. Spontaneous activity decreased by about 30%, following the application of streptomycin. In contrast, injections of amiloride into the scala media were followed by an increase in spontaneous discharge rates.

Ionic conductances and hair cell tuning in the turtle cochlea.

Ionic conductances and hair cell tuning in the turtle cochlea.

A kinetic description of the calcium-activated potassium channel and its application to electrical tuning of hair cells.

A kinetic description of the calcium-activated potassium channel and its application to electrical tuning of hair cells.

The effects of low calcium on the voltage-dependent conductances involved in tuning of turtle hair cells.

1. The voltage-dependent conductances of turtle cochlear hair cells of known resonant frequency were characterized by tight-seal, whole-cell recording during superfusion with solutions containing normal (2.8 mM) and reduced (0.1-10 microM) Ca2+. 2. In 1 microM Ca2+, the current flowing through the voltage-dependent Ca2+ channels was increased roughly fivefold and had a reversal potential near 0 mV. This observation may be explained by the Ca2+ channels becoming non-selectively permeable to monovalent cations in low-Ca2+ solutions. Lowering the Ca2+ further to 0.1 microM produced little increase in the current. 3. The size of the non-selective current increased systematically with the resonant frequency of the hair cell over the range from 10 to 320 Hz. This suggests that hair cells tuned to higher frequencies contain more voltage-dependent Ca2+ channels. 4. There was a good correlation between the amplitudes of the non-selective current and the K+ current which underlies electrical tuning of these hair cells. The amplitude of the K+ current also increased systematically with resonant frequency. 5. In cells with resonant frequencies between 120 and 320 Hz, the K+ current was completely abolished in 1 microM Ca2+, consistent with prior evidence that this current flows through Ca2+ activated K+ channels. In a majority of cells tuned between 50 and 120 Hz, the K+ current was incompletely blocked in 1 microM Ca2+, but was eliminated in 0.1 microM Ca2+. In all hair cells the K+ current was abolished by 25 mM tetraethylammonium chloride. 6. In cells tuned to 10-20 Hz, the K+ current was not substantially diminished even in 0.1 microM Ca2+, which argues that it may not be Ca2+ activated. 7. In cells tuned to frequencies above 100 Hz, the K+ current could still be evoked by depolarization during superfusion with 10 microM Ca2+. However, its half-activation voltage was shifted to more depolarized levels and its maximum amplitude was systematically reduced with increasing resonant frequency. 8. These observations are consistent with the notion that in cells tuned to more than 50 Hz, there is a fixed ratio of the number of voltage-dependent Ca2+ channels to Ca(2+)-activated K+ channels, the numbers of each increasing in proportion to resonant frequency. The results also provide indirect evidence that the Ca(2+)-activated K+ channels in cells tuned to higher frequencies may be less sensitive to intracellular Ca2+.

Electrical tuning in hair cells isolated from the chick cochlea

Tall (inner) hair cells were isolated from specific locations in the chick cochlea. The electrical membrane properties of these cells were recorded using the tight-seal whole-cell technique. Depolarizing current steps elicited damped voltage oscillations that ranged in frequency from 100 to 250 Hz among cells from the middle third of the cochlea (basal cells). The current-voltage relation obtained under voltage clamp was dominated by calcium-activated potassium current in the voltage range over which these oscillations occurred. Tall hair cells isolated from the apical tip of the cochlea (apical cells) exhibited action potentials and lower frequency voltage oscillations (5-14 Hz) during depolarizing current steps. Outward currents in these cells were 20-fold slower than those found in the basal cells. These results suggest that electrical tuning of hair cells may play a role in determining the frequency selectivity of the chick cochlea.

Single unit clues to cochlear mechanisms.

In recent years studies on isolated hair cells have suggested that there is an inherent tuning of hair cells determined by their mechanical and electrical properties. However, tuning for mammalian cochleas appears to be much more complicated since there are typically two types of receptor cells (inner and outer hair cells) imbedded in a highly organized framework of supporting cells, membranes and fluids. The major neural output of the cochlea can be monitored by recording the activity of myelinated axons of spiral ganglion cells, not only under normal conditions, but also when the discharge patterns are altered by ototoxic drugs, acoustic trauma or olivocochlear bundle stimulation. A model system with two excitatory influences, one sharply tuned and highly sensitive, and a second, broadly tuned and relatively insensitive, can account for much of the existing data. Results from single-neuron marking studies support the notion that these two influences probably involve interactions between inner and outer hair cells. More global influences such as the endocochlear potential also can act on auditory-nerve fibers through the hair-cell systems. Thus, the inherent frequency selectivity of the receptor cell is only one of many factors that determine the tuning of mammalian auditory-nerve fibers.

Models for electrical tuning in hair cells

We analyse several models for the electrical properties of vertebrate hair cell membranes to assess whether they can account for the electrical resonant tuning that these cells possess. The presence of either a voltage-gated potassium current or a calcium-gated potassium current in the cell membrane is shown, with suitable assumptions, to make the cell behave as a simple resistance-inductance-capacitance circuit showing resonant behaviour. With plausible values for the model parameters however, the presence of a voltage-gated current alone cannot account for the high Q values of the resonance behaviour seen in hair cells. A calcium-gated current could account for the high Q values. Mechanisms that allow variation of optimal frequency between different hair cells are discussed. It is concluded that the variation may be produced by systematic changes in the number of calcium channels and calcium pumps in the cell membrane.

Efferent modulation of hair cell tuning in the cochlea of the turtle.

Intracellular recordings were made from turtle cochlear hair cells in order to study the changes in their tuning properties resulting from electrical stimulation of the efferent axons. Efferent stimulation caused a reduction in the amplitude of the receptor potential at the hair cell's most sensitive or characteristic frequency, an increased amplitude at frequencies more than an octave below the characteristic frequency, and no change at very high frequencies. These differential effects resulted in a broadening of each cell's tuning curve, which, during maximal efferent stimulation degenerated from a sharply tuned resonance to a critically damped low-pass filter. Efferent alterations in tuning were also inferred from the oscillations in membrane potential produced by acoustic clicks or extrinsic currents. The quality factor (Q) of tuning, derived from the decay of the oscillations, was progressively reduced with synaptic hyperpolarizations up to about 5 mV in amplitude. A consequence of efferent action was that the wave forms of transient pressure changes were more faithfully encoded as changes in hair cell membrane potential. Hyperpolarization of a hair cell by steady current injection resulted in a lowering of its characteristic frequency and quality factor, and an increase in steady-state resistance. By comparison, for a given reduction in quality factor, efferent stimulation was associated with a smaller change in characteristic frequency. This difference is expected if the resonance is also damped by the shunting action of the synaptic conductance. Perfusion with perilymphs containing 0.5-15 mM of the potassium channel blocker, tetraethylammonium bromide (TEA) reduced the hair cell's frequency selectivity, whether assayed acoustically or with extrinsic currents. Lower TEA concentrations abolished the efferent inhibitory post-synaptic potential with only a minor change in tuning. TEA produced other effects different from efferent stimulation including (i) a lowering of the characteristic frequency, and (ii) a highly asymmetric receptor potential. These observations suggest that the efferents do not simply block membrane conductances associated with tuning. We conclude that the efferent modification of the shape of the tuning curve may be a composite result of the synaptic conductance and the hyperpolarization of the hair cell membrane.

Morphology of inner hair cell stereocilia in C57BL/6J mice as studied by scanning electron microscopy.

The observation that hair cell tuning curves exhibit frequency selectivity as sharply tuned as that seen in auditory nerve fibers has prompted closer examination of the sensory hairs or stereocilia. The present study was designed to examine the morphologic organization of inner hair cell stereocilia in a mammalian species, the neonatal C57BL/6J mouse. The cochleae of mice were fixed in OSO4, dehydrated, dissected, and prepared for scanning electron microscopy. An examination of the number of stereocilia per inner hair cell revealed an orderly decrease from base to apex. Conversely, there was a 300% increase in the height of the tallest stereocilia, a 100% increase in the height of the middle row stereocilia, and a 30% increase in shortest stereocilia from base to apex. The total surface area of the stereocilia, per hair cell, was shown to increase by approximately 250% from the base to the apex of the cochlea.

Basilar membrane tuning properties in the specialised cochlea of the CF-bat, Rhinolophus ferrumequinum.

The greater horseshoe bat has greatly expanded frequency mapping, and morphological specialisations, in the first half turn of its cochlea and a sudden transition to normal mapping. Amplitude and phase of vibration have been measured on various structures in the expanded and normal regions and have not revealed any sharply tuned responses. Amplitudes are much lower than those found in other species and frequently show a deep notch in the 77-84 kHz region. The high-frequency cut-off frequencies are tonotopically organised but deviate from the Bruns map, so that hair-cell tuning appears to occur at a frequency at which basilar membrane vibration is small. In the basal region, phase differences were frequently found between the inner and outer parts of the basilar membrane. These appear to be due to interaction between two components of motion and are probably not indicative of a further filtering mechanism. There is no evidence for reflection of the travelling wave at the transition.