Compliance of the hair bundle associated with gating of mechanoelectrical transduction channels in the bullfrog's saccular hair cell

Mechanical stimuli are thought to open the transduction channels of a hair cell by tensing elastic components, the gating springs, that pull directly on the channels. To test this model, we measured the stiffness of hair bundles during mechanical stimulation. A bundle's compliance increased by about 40% at the position where half of the channels opened. This we attribute to conformational changes of transduction channels as they open and close. The magnitude and displacement dependence of the gating compliance provide quantitative information about the molecular basis of mechanoelectrical transduction: the force required to open each channel, the number of transduction channels per hair cell, the stiffness of a gating spring, and the swing of a channel's gate as it opens.     

A molecular level prototype for mechanoelectrical transducer in mammalian hair cells

The mechanoelectrical transducer (MET) is a crucial component of mammalian auditory system. The gating mechanism of the MET channel remains a puzzling issue, though there are many speculations, due to the lack of essential molecular building blocks. To understand the working principle of mammalian MET, we propose a molecular level prototype which constitutes a charged blocker, a realistic ion channel and its surrounding membrane. To validate the proposed prototype, we make use of a well-established ion channel theory, the Poisson–Nernst–Planck equations, for three-dimensional (3D) numerical simulations. A wide variety of model parameters, including bulk ion concentration, applied external voltage, blocker charge and blocker displacement, are explored to understand the basic function of the proposed MET prototype. We show that our prototype prediction of channel open probability in response to blocker relative displacement is in remarkable accordance with experimental observation of rat cochlea outer hair cells. Our results appear to suggest that tip links which connect hair bundles gate MET channels.     

How the ear's works work: mechanoelectrical transduction and amplification by hair cells

The sensitivity of our hearing is enhanced by an active process that both amplifies and tunes the movements of the ear's sensory receptors, the hair cells. In a quiet environment, the active process can even evoke spontaneous emission of sounds from an ear. Recent research indicates that, at least in non-mammalian tetrapods, the active process results from the interaction of negative stiffness in the mechanosensitive hair bundles with two motor processes, one due to myosin-based adaptation and the other to Ca2+ -dependent reclosure of transduction channels. These three processes together explain many of the complex phenomena characteristic of the hearing process.     

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.     

Developmental expression of BK channels in chick cochlear hair cells

Background  

Cochlear hair cells are high-frequency sensory receptors. At the onset of hearing, hair cells acquire fast, calcium-activated potassium (BK) currents, turning immature spiking cells into functional receptors. In non-mammalian vertebrates, the number and kinetics of BK channels are varied systematically along the frequency-axis of the cochlea giving rise to an intrinsic electrical tuning mechanism. The processes that control the appearance and heterogeneity of hair cell BK currents remain unclear.     

The actions of calcium on hair bundle mechanics in mammalian cochlear hair cells

Sound stimuli excite cochlear hair cells by vibration of each hair bundle, which opens mechanotransducer (MT) channels. We have measured hair-bundle mechanics in isolated rat cochleas by stimulation with flexible glass fibers and simultaneous recording of the MT current. Both inner and outer hair-cell bundles exhibited force-displacement relationships with a nonlinearity that reflects a time-dependent reduction in stiffness. The nonlinearity was abolished, and hair-bundle stiffness increased, by maneuvers that diminished calcium influx through the MT channels: lowering extracellular calcium, blocking the MT current with dihydrostreptomycin, or depolarizing to positive potentials. To simulate the effects of Ca²⁺, we constructed a finite-element model of the outer hair cell bundle that incorporates the gating-spring hypothesis for MT channel activation. Four calcium ions were assumed to bind to the MT channel, making it harder to open, and, in addition, Ca²⁺ was posited to cause either a channel release or a decrease in the gating-spring stiffness. Both mechanisms produced Ca²⁺ effects on adaptation and bundle mechanics comparable to those measured experimentally. We suggest that fast adaptation and force generation by the hair bundle may stem from the action of Ca²⁺ on the channel complex and do not necessarily require the direct involvement of a myosin motor. The significance of these results for cochlear transduction and amplification are discussed.     

哺乳动物耳蜗外毛细胞的马达蛋白:Prestin

近几年的研究发现,在耳蜗基底膜的外毛细胞膜上有一种新奇的蛋白质:prestin(马达蛋白),它能感受细胞膜电位的变化,进而发生构象改变,引发外毛细胞的形状和表面积的改变.Prestin作为一种独特的马达蛋白,能驱动耳蜗外毛细胞的电能动性(electromotility),产生耳蜗的放大器作用,因而使哺乳动物的听觉具有高度的敏感性,广阔的听觉域,敏锐的频率选择性.这种蛋白质的缺失或基因的突变会导致听觉功能严重受损,对于prestin的深入细致的研究,也许可以使人们进一步认识和理解哺乳动物的听觉调谐机制,通过对这种蛋白质基因的表达的调控,是否能够防治一些与之相关的疾病?这或许将是今后听觉研究领域的一个重要课题.     

Structural features of the lateral walls in mammalian cochlear outer hair cells

Summary Freeze-fracture, freeze-etching and thin sections have been used to determine features of the structural organisation of the lateral walls in cochlear outer hair cells. The presence of an organised meshwork of filaments in the lateral cortex of the cell is confirmed in intact unfixed cells. This meshwork showed morphological features similar to the cytoskeletal lattice. The lateral plasma membrane is shown to be protein-rich and to contain cholesterol. The membranes of the subplasmalemmal lateral cisternae contain much less protein, and little cholesterol as judged by their responses to filipin and tomatin. These findings indicate differences in the physical properties of the two membrane systems. On the fracture faces of the plasma membrane there is a high density of intramembrane particles and this particle population is heterogeneous. Some particles show morphological features consistent with those of transmembrane channels. Regularly spaced pillars crossing the space between the plasma and cisternal membranes were identified both in thin sections and in freezeetched preparations, but neither the plasma nor cisternal membrane fracture faces showed any feature corresponding directly to the pillar. This suggests the pillars do not insert directly into either membrane. Freeze-fracture and freeze-etching of unfixed cells indicated that the pillar is indirectly associated with the cytoplasmic surface of the plasma membrane, and, at its inner end, linked to the cortical cytoskeletal lattice on the outer surface of the cisternal membrane.     

DEG/ENaC but not TRP channels are the major mechanoelectrical transduction channels in a C. elegans nociceptor

Many nociceptors detect mechanical cues, but the ion channels responsible for mechanotransduction in these sensory neurons remain obscure. Using in?vivo recordings and genetic dissection, we identified the DEG/ENaC protein, DEG-1, as the major mechanotransduction channel in ASH, a polymodal nociceptor in Caenorhabditis elegans. But DEG-1 is not the only mechanotransduction channel in ASH: loss of deg-1 revealed a minor current whose properties differ from those expected of DEG/ENaC channels. This current was independent of two TRPV channels expressed in ASH. Although loss of these TRPV channels inhibits behavioral responses to noxious stimuli, we found that both mechanoreceptor currents and potentials were essentially wild-type in TRPV mutants. We propose that ASH nociceptors rely on two genetically distinct mechanotransduction channels and that TRPV channels contribute to encoding and transmitting information. Because mammalian and insect nociceptors also coexpress DEG/ENaCs and TRPVs, the cellular functions elaborated here for these ion channels may be conserved.     

Effect of cytoskeletal elastic properties on the mechanoelectrical transduction in excitable cells

This paper addresses the possible mechanism of stretch on cell electrochemical potential change, based on the physicochemical properties of cytoskeletal network. Synthetic polyelectrolyte gel was used as an experimental model of the cytoskeleton. Gel samples with different density of network cross linking were studied. Triangular axial deformations of samples were applied. Simultaneously, the electrochemical (Donnan) potential of the gel was measured between a micropipette electrode pinned into the swollen gel, and a reference electrode in the outer solution. We found that axial deformation shifts the gel potential toward depolarization. The extent of gel depolarization showed a close negative correlation with the Young modulus of the gel. We suggest that the underlying mechanism is likely to be a universal process of counterion adsorption on charged polymer filaments due to the decrease of distance between polymer filaments owing to gel elongation.     

BK channels mediate cholinergic inhibition of high frequency cochlear hair cells

Background

Outer hair cells are the specialized sensory cells that empower the mammalian hearing organ, the cochlea, with its remarkable sensitivity and frequency selectivity. Sound-evoked receptor potentials in outer hair cells are shaped by both voltage-gated K⁺ channels that control the membrane potential and also ligand-gated K⁺ channels involved in the cholinergic efferent modulation of the membrane potential. The objectives of this study were to investigate the tonotopic contribution of BK channels to voltage- and ligand-gated currents in mature outer hair cells from the rat cochlea.

Methodology/Principal

Findings In this work we used patch clamp electrophysiology and immunofluorescence in tonotopically defined segments of the rat cochlea to determine the contribution of BK channels to voltage- and ligand-gated currents in outer hair cells. Although voltage and ligand-gated currents have been investigated previously in hair cells from the rat cochlea, little is known about their tonotopic distribution or potential contribution to efferent inhibition. We found that apical (low frequency) outer hair cells had no BK channel immunoreactivity and little or no BK current. In marked contrast, basal (high frequency) outer hair cells had abundant BK channel immunoreactivity and BK currents contributed significantly to both voltage-gated and ACh-evoked K⁺ currents.

Conclusions/Significance

Our findings suggest that basal (high frequency) outer hair cells may employ an alternative mechanism of efferent inhibition mediated by BK channels instead of SK2 channels. Thus, efferent synapses may use different mechanisms of action both developmentally and tonotopically to support high frequency audition. High frequency audition has required various functional specializations of the mammalian cochlea, and as shown in our work, may include the utilization of BK channels at efferent synapses. This mechanism of efferent inhibition may be related to the unique acetylcholine receptors that have evolved in mammalian hair cells compared to those of other vertebrates.     

Role of myosin VI in the differentiation of cochlear hair cells.

The mouse mutant Snell's waltzer (sv) has an intragenic deletion of the Myo6 gene, which encodes the unconventional myosin molecule myosin VI (K. B. Avraham et al., 1995, Nat. Genet. 11, 369-375). Snell's waltzer mutants exhibit behavioural abnormalities suggestive of an inner ear defect, including lack of responsiveness to sound, hyperactivity, head tossing, and circling. We have investigated the effects of a lack of myosin VI on the development of the sensory hair cells of the cochlea in these mutants. In normal mice, the hair cells sprout microvilli on their upper surface, and some of these grow to form a crescent or V-shaped array of modified microvilli, the stereocilia. In the mutants, early stages of stereocilia development appear to proceed normally because at birth many stereocilia bundles have a normal appearance, but in places there are signs of disorganisation of the bundles. Over the next few days, the stereocilia become progressively more disorganised and fuse together. Practically all hair cells show fused stereocilia by 3 days after birth, and there is extensive stereocilia fusion by 7 days. By 20 days, giant stereocilia are observed on top of the hair cells. At 1 and 3 days after birth, hair cells of mutants and controls take up the membrane dye FM1-43, suggesting that endocytosis occurs in mutant hair cells. One possible model for the fusion is that myosin VI may be involved in anchoring the apical hair cell membrane to the underlying actin-rich cuticular plate, and in the absence of normal myosin VI this apical membrane will tend to pull up between stereocilia, leading to fusion.     

Reversible mechanosensitive ion pumping as a part of mechanoelectrical transduction.

To explain the ability of some mechanosensitive cells to reverse the process of mechanotransduction and to generate mechanical oscillations and emit sound, a piezo-conformational coupling model (PCC model) is proposed. The model includes a transport protein which changes either its volume (PV-coupling) or its area in the membrane (gamma A-coupling) when undergoing conformational transitions. Such a protein can interact with an oscillating pressure to pump ions and create a transmembrane gradient if the affinities of the protein for ions are different at the two sides of membrane. The frequency and concentration windows for mechanical energy transduction were determined. Under optimal conditions, the efficiency of energy transduction can approach the theoretical maximum of 100%. If the concentration gradient exceeds the static head value (quasi-equilibrium which can be built up and maintained by this transport system), the energy transduction reverses and the transporter becomes a generator of mechanical oscillations at the expense of a concentration gradient. Estimation of thermodynamic parameters of the pump shows that the PV-coupling model would require large pressure oscillations to work while the gamma A-coupling model could work in physiological conditions. The gamma A-coupling mechanism may be used by cells for two purposes. In the reverse mode, it can be a force generator for various applications. In the direct mode, it may serve bioenergetic purposes by harvesting the energy of mechanical oscillations and storing it in the form of a concentration gradient.(ABSTRACT TRUNCATED AT 250 WORDS)     

A cyclic-AMP-gated conductance in cochlear hair cells

The patch clamp technique was used to record cAMP-dependent currents of the guinea pig cochlear hair cell plasma membrane. Data obtained indicate that the channels passing this current are moderately selective for monovalent cations and are effectively blocked by L-cis-diltiazem and reversibly blocked by 1 mM Mg²⁺ or Ca²⁺. The single-channel unit conductance estimated in the absence of divalent cations is about 16 pS. The results demonstrate that cyclic nucleotide-dependent channels of cochlear hair cells are virtually identical to the photoreceptor and olfactory ones.     

Tip-link integrity and mechanical transduction in vertebrate hair cells.

An attractive hypothesis for hair-cell transduction is that fine, filamentous "tip links" pull directly on mechanically sensitive ion channels located at the tips of the stereocilia. We tested the involvement of tip links in the transduction process by treating bundles with a BAPTA-buffered, low-Ca2+ saline (10(-9) M). BAPTA abolished the transduction current in a few hundred milliseconds. BAPTA treatment for a few seconds eliminated the tip links observed by either scanning or transmission electron microscopy. BAPTA also eliminated the voltage-dependent movement and caused a positive bundle displacement of 133 nm, in quantitative agreement with a model for regulation of tension. We conclude that tip links convey tension to the transduction channels of hair cells.     

Kinetic analysis of barium currents in chick cochlear hair cells.

Inward barium current (IBa) through voltage-gated calcium channels was recorded from chick cochlear hair cells using the whole-cell clamp technique. IBa was sensitive to dihydropyridines and insensitive to the peptide toxins omega-agatoxin IVa, omega-conotoxin GVIa, and omega-conotoxin MVIIC. Changing the holding potential over a -40 to -80 mV range had no effect on the time course or magnitude of IBa nor did it reveal any inactivating inward currents. The activation of IBa was modeled with Hodgkin-Huxley m2 kinetics. The time constant of activation, tau m, was 550 microseconds at -30 mV and gradually decreased to 100 microseconds at +50 mV. A Boltzmann fit to the activation curve, m infinity, yielded a half activation voltage of -15 mV and a steepness factor of 7.8 mV. Opening and closing rate constants, alpha m and beta m, were calculated from tau m and m infinity, then fit with modified exponential functions. The H-H model derived by evaluating the exponential functions for alpha m and beta m not only provided an excellent fit to the time course of IBa activation, but was predictive of the time course and magnitude of the IBa tail current. No differences in kinetics or voltage dependence of activation of IBa were found between tall and short hair cells. We conclude that both tall and short hair cells of the chick cochlea predominantly, if not exclusively, express noninactivating L-type calcium channels. These channels are therefore responsible for processes requiring voltage-dependent calcium entry through the basolateral cell membrane, such as transmitter release and activation of Ca(2+)-dependent K+ channels.     

Signal transduction and ion channels in guard cells.

Our understanding of the signalling mechanisms involved in the process of stomatal closure is reviewed. Work has concentrated on the mechanisms by which abscisic acid (ABA) induces changes in specific ion channels at both the plasmalemma and the tonoplast, leading to efflux of both K+ and anions at both membranes, requiring four essential changes. For each we need to identify the specific channels concerned, and the detailed signalling chains by which each is linked through signalling intermediates to ABA. There are two global changes that are identified following ABA treatment: an increase in cytoplasmic pH and an increase in cytoplasmic Ca2+, although stomata can close without any measurable global increase in cytoplasmic Ca2+. There is also evidence for the importance of several protein phosphatases and protein kinases in the regulation of channel activity. At the plasmalemma, loss of K+ requires depolarization of the membrane potential into the range at which the outward K+ channel is open. ABA-induced activation of a non-specific cation channel, permeable to Ca2+, may contribute to the necessary depolarization, together with ABA-induced activation of S-type anion channels in the plasmalemma, which are then responsible for the necessary anion efflux. The anion channels are activated by Ca2+ and by phosphorylation, but the precise mechanism of their activation by ABA is not yet clear. ABA also up-regulates the outward K+ current at any given membrane potential; this activation is Ca(2+)-independent and is attributed to the increase in cytoplasmic pH, perhaps through the marked pH-sensitivity of protein phosphatase type 2C. Our understanding of mechanisms at the tonoplast is much less complete. A total of two channels, both Ca(2+)-activated, have been identified which are capable of K+ efflux; these are the voltage-independent VK channel specific to K+, and the slow vacuolar (SV) channel which opens only at non-physiological tonoplast potentials (cytoplasm positive). The SV channel is permeable to K+ and Ca2+, and although it has been argued that it could be responsible for Ca(2+)-induced Ca2+ release, it now seems likely that it opens only under conditions where Ca2+ will flow from cytoplasm to vacuole. Although tracer measurements show unequivocally that ABA does activate efflux of Cl- from vacuole to cytoplasm, no vacuolar anion channel has yet been identified. There is clear evidence that ABA activates release of Ca2+ from internal stores, but the source and trigger for ABA-induced increase in cytoplasmic Ca2+ are uncertain. The tonoplast and another membrane, probably ER, have IP3-sensitive Ca2+ release channels, and the tonoplast has also cADPR-activated Ca2+ channels. Their relative contributions to ABA-induced release of Ca2+ from internal stores remain to be established. There is some evidence for activation of phospholipase C by ABA, by an unknown mechanism; plant phospholipase C may be activated by Ca2+ rather than by the G-proteins used in many animal cell signalling systems. A further ABA-induced channel modulation is the inhibition of the inward K+ channel, which is not essential for closing but will prevent opening. It is suggested that this is mediated through the Ca(2+)-activated protein phosphatase, calcineurin. The question of Ca(2+)-independent stomatal closure remains controversial. At the plasmalemma the stimulation of K+ efflux is Ca(2+)-independent and, at least in Arabidopsis, activation of anion efflux by ABA may also be Ca(2+)-independent. But there are no indications of Ca(2+)-independent mechanisms for K+ efflux at the tonoplast, and the appropriate anion channel at the tonoplast is still to be found. There is also evidence that ABA interferes with a control system in the guard cell, resetting its set-point to lower contents, suggesting that stretch-activated channels also feature in the regulation of guard cell ion channels, perhaps through interactions with cytoskeletal proteins. (ABSTRACT TRUN