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The Auditory Periphery

2 – Hair Cell Structure and Transduction

Dr. Elisabeth Glowatzki955-3877

eglowatz@bme.jhu.edu521 Traylor Building

Websites:Promenade ‘round the cochlea

(http://www.iurc.montp.inserm.fr/cric/audition/english/start.htm)Auditory Animations, Univ. of Wisconsin(http://www.neurophys.wisc.edu/animations/)

Texts (at Welch or Eisenhower):From Sound to Synapse, C. D. Geisler, New York: Oxford Univ. Press, 1998An Introduction to the Physiology of Hearing, J. O. Pickles, New York:Academic Press, 1982Fundamentals of Hearing: An Introduction (3rd ed.), W. A. Yost, San Diego:Academic Press, 1994Hackney CM, Furness DN (1995) Mechanotransduction in vertebrate haircells: structure and function of the stereociliary bundle. Am. J. Physiol.268:C1-C13.

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The Organ of Corti

Stephan Blatrix

Overview over the organ of Corti

One row of inner hair cells (IHCs) and three rows of outer hair cells (OHCs),both with stereocilia bundles. The IHCs are flask-shaped, the OHCs are rod-

shaped.Both have stereocilia bundles at the apex and synapses at the base.The OHC stereocilia bundles contact the tectorial membrane, the IHC

stereocilia bundles seem not to contact the tectorial membrane.

Innervation:1. IHCs make 95% of afferent glutaminergic synapses (blue).2. OHCs make 5 % of afferent synapses; their function is unknown (green).3. OHCs make efferent cholinergic (ACh-activated synapses (red).4. During development IHCs make have cholinergic synapses (not shown).

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Cross sections ofOrgan of Corti ofguinea pig. Upperfrom apex, lowerfrom base ofcochlear spiral. R.

Pujol.

Two histological sections of the organ of Corti, one apical, one basal. One rowof IHCs, three rows OHCs, supporting cells around the IHC and under theOHCs. The tectorial membrane always lifts up from the stereocilia inhistological sections (due to the change in ionic environment?).

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Deflection of the Stereocilia Bundles

Transduction process:

Stereocilia of IHCs and OHCs are deflected against the tectorial membrane,when the basilar membrane is set in motion.

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Stephan Blatrix

Transduction and Synaptic Transmission at theInner Hair Cell

How is the transduction signal transmitted to the brain?Sound sets the basilar membrane in motion.The stereocilia bundles are deflected against the tectorial membrane.The hair cell is depolarized by K+ influx at the apex of the hair cell throughtransduction channels.The transduction current generates a receptor potential.Depolarization of the hair cell opens voltage gated Ca2+-channels at the baseof the hair cell and induces transmitter release.Vesicles filled with glutamate fuse with the synaptic membrane in a Ca 2+ -dependent manner.Glutamate in the synaptic cleft activates glutamate receptors on the afferentfiber terminal and induces excitatory postsynaptic potentials (EPSPs).The EPSPs activate action potentials that travel down the auditory nerve.Deflection of the stereocilia bundles towards the biggest stereocilium inducean increase in the firing rate in auditory nerve fibers. Deflection in the oppositedirection induce a reduction in firing rate.At rest (when no signal is applied to the hair cells), there is still some influx ofK+ into the hair cell (about 10 % of the maximal current), some transmitter isreleased causing ‘spontaneous activity’ in the auditory nerve fibers.

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Conductances in the Lateral Wall of the Hair Cells Shape the Receptor Potential

g transductiong (ATP)

The receptor potential is shaped by the transduction current and a number ofbasolateral conductances, some of which are illustrated here in this figure. Forexample voltage-gated potassium conductances g Kv, Ca2+-dependentpotassium conductances, ligand-gated conductances (ATP, Acetylcholine) etc.can impact the shape of the receptor potential.

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Hair Cell & Hair Bundle Examples

Turtle VHC

Turtle VHC Bullfrog VHC

IHC OHC

Hair cell and hair bundle examples in electron-microscopic images.Note: in vivo, the stereocilia and kinocilium (the tallest stereocilium in vestibular hair cells) arerigid and upright (not curved, as shown particularly in the turtle vestibular hair cell bundles dueto fixation).Observations:

• Pipe-organ arrangement in vestibular hair cell hair bundles (mammalian and non-mammalian) and cochlear hair bundles of non-mammalian vertebrates.• Staircase arrangement in mammalian cochlear hair bundles (3-4 rows).• Axis of bilateral symmetry• Tapered base of stereocilia• Tilted inward• Number of stereocilia per bundle varies widely

• Chick cochlear hair bundles: 50-300• Across species, number of stereocilia decreases from base (HF) to apex(LF).• Stereocilium length and cell size increases from base to apex.• Kinocilia in VHC end as a bulb or may be very large. They seem to anchorthe hair bundle to the overlying (otolithic) membrane. They are present incochlear hair cells only during development.•An unansered question: What is the functional significance of differences inbundle shape?

• Stereocilia act as rigid, pencil-like rods that bend at the base about the rootlet.

Images: IHC/OHC (Promenade website), Turtle VHC (Ellengene Peterson, unpublished), FrogVHC (Strassmaier & Gillespie, 2002)

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Stereocilium Structure

Tilney et al.(1983)

Tiln

ey e

t al.

(198

0)

Stereocilia are composed of a paracrystalline array of tightly (hexagonally) packed actinfilaments with fimbrin cross-bridges.From alligator lizard (Tilney et al., 1980)

• >3,000 actin filaments per stereocilium• ~18-30 form rootlet and extend into cuticular plate• The rootlet extends as a cone into the cuticular plate, increasing in diameter thefarther it penetrates. Rootlet filaments are interconnected by fine 3-nm filaments andare presumably anchored by myosins among other proteins.•The actin core is suitable for myosin motility (Shepherd et al., 1990). Demembranedhair bundles were blotted and the movement of myosin coated beads were recorded.Myosin freely moved along the actin complex, seemingly uninhibited by the presenceof fimbrin cross-bridges. This observation is critical for later discussion of myosindependent adaptation.

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Hair Bundle Motion

Chick Tall Hair CellWater-jet Stimulation500 Hz15º displacementStroboscopic Lamp(Keith Duncan)

This figure illustrates the movement of a stereocilia bundle of an isolated chickhair cell with fluid-jet, projecting a fluid wave onto the bundle. All stereociliamove together as a compact, stiff structure.

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Fettiplace, Ricci and Hackney, 2001

Tip Links

That the stereocilia bundle moves as a unit is due to the fact that a variety oflinking proteins connect the stereocilia at different heights of the bundle.

Tip-links are upward pointing links that connect the tip of shorter stereocilia tothe shafts of adjacent stereocilia in the next taller row. Lateral links connectthe shafts of adjacent stereocilia, and ankle links are specialized lateral links atthe base of stereocilia (not shown here). Tip-links and lateral links are presentin all hair bundles, but the extent of lateral link connectivity is highly variable(i.e. making horizontal connections along the entire length of the stereociliashafts or making dense interconnections just below the stereociliary tips).

The mechanotransducer channels are thought to be located close to the tip ofthe stereocilia, where the tip links contact the stereocilia. Deflection of thestereocilia bundle stretches the tip links or structures connected to the tip linksand thereby opens transduction channels.

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Mechanotransduction,based on studies byCorey, Crawford, Eatock,Fettiplace, Gillespie,Hudspeth and colleagues

Stephan Blatrix

+ 80 mV

-60 mV

dV = 140 mV

A very simple view on how mechanotransduction may work:the deflection of the stereocilia opens mechanotransduction channels,unspecific cation channels, permeable for Na, Ca and K. Due to the high Kconcentration in the endolymph, mainly K enters through the channel into thecell. The driving force is 140 mV. Proof for this theory will be presented later inthis lecture after introducing methods how transduction currents have beenrecorded.

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Transduction: Methods – 1Frog Sacculus

Hudspeth & Corey, 1977

Corey & Hudspeth, 1983

Recording from hair cells is no trivial task due to the unique fluid environment in vivo, thelocation of these cells within the bony labyrinth, and the necessity for micromechanicalstimulation of the hair bundle. Here, we will describe several recording techniques.

Single-electrode voltage recording (left)An epithelial preparation of the frog sacculus is pinned in an experimental chamber. Hair cellsare penetrated using a single fine tipped microelectrode, measuring the cell’s membranepotential (note: not a voltage or current clamp configuration). A glass fiber holding thestereocilia bundle from the top is moving the bundle.

Transepithelial preparation (right)An entire vestibular organ (most often the sacculus) is dissected and a portion of the otolithicmembrane (overlying hair cells) is removed (OM). The preparation is mounted across a holein a nonconducting surface (W). Thus, there are now two separate fluid chambers (simulatingthe in vivo environment). Electrodes are placed in the upper and lower chambers, and theapical and basolateral surfaces are clamped to 0 mV using a voltage-clamp circuit. Hairbundles are displaced en mass (SP); transduction currents flowing in through transductionchannels and out through the basolateral surfaces are measured by the clamp circuit. Theintracellular membrane potential is not clamped using this method, and large changes inintracellular potential will alter transduction currents.

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Receptor Potential in the Frog Sacculus

Preparation: Bullfrog sacculusMethods: Apical surface, single electrode recording

(A) Receptor potentials from a 10-Hz triangle wave stimulus. Upward displacements indicatemotion toward the tallest stereocilia. Deflections are parallel to an axis of bilateralsymmetry (along the graded heights of the bundle). Greater deflections result in greaterchanges in receptor potential. Note the rectification for large, negative stimuli.

(B) Input-output curve, V(x), for curves as in (A). Peak changes in receptor potential are lessthan 10 mV. Saturating displacements are less than 1 µιχρον (or 10º deflection). Thecurve is asymmetric (greater changes for positive displacements than negative) andapproximates a Boltzman relationship. This suggests that some transduction relatedcurrent is present in hair bundles at rest. Note: statistically significant changes inmembrane potential for photoreceptors is 10 µV. This would correspond to adisplacement of 500 picometers. Estimates for auditory hair cells are as low as 1 pm!

(C) Hyperpolarizing square current pulses were injected into the hair cell during triangle-wavestimulation and recording of membrane potential. V = I R. Thus, for the constant currentpulses, when changes in V are reduced during deflection toward the tallest stereocilia, theinput resistance into the hair cell is also reduced. Presumably, conductance changesfrom the opening and closing of an ion channel are responsible for the change in inputresistance. Therefore, transduction currents result from transduction channels whosegating is triggered by hair bundle displacement.

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Transduction: Methods – 2Receptor Potential in Mammalian Hair Cells

Intracellular Recording in vivo

Dallos et al. 1982 Russell & Sellick 1978

The schematic on the left is showing two approaches to intracellular recordingfrom cochlear hair cells in the guinea pig cochlea in vivo. In the lateralapproach (Dallos et al 1992), the electrode passes through a fenestra in thecochlear bone and approaches the organ of Corti through the scala media.This method has been used to collect data from the three low frequency turnsof the cochlea. In the scala tympani approach (Russell and Sellick, 1978), theelectrode passes into the organ of Corti through the basilar membrane fromthe opened scala tympany. The approach is suitable to the high frequencyregion of the cochlea. (Figs. from The Cochlea: Dallos et al. eds., Springer,pages 27, 28).The Figure on the right shows intracellular recordings from a fourth-turn OHC.The peak receptor potential amplitude is plotted as a function of peak soundpressure at the ear drum. Note that the curve rectifies, the voltage change tothe negative halfwave is smaller than to the positive halfwave of the soundsignal.

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Transduction: Methods – 3Frog Sacculus and Mouse Utriculus, voltage clamp

Howard & Hudspeth, 1987

the stimulator is connectedto the kinocilium

Patch clamp, used by variousgroups, also used in themammalian vestibular organ(Jeffrey Holt and others)

Probe

PatchPipette

Apical surface voltage clamp (top)The two-electrode voltage clamp (top left) was the first methodology allowing voltage clamp ofa single hair cell, and thus tight control over basolateral conductances. In this way, the currentthrough transduction channels could be directly measured (rather than inferred from changesin membrane potential). This was used in epithelial preparations and was extremely difficult,requiring two recording electrodes and a stimulating probe.Apical surface whole-cell recording techniques (top right) allow for fast and easy single-electrode recordings. Unfortunately, the apical surface of hair cells is notoriously difficult torecord from using patch electrodes (recall that the cuticular plate is a dense matrix and ispositioned here).

Whole-cell and perforated patch recordings (bottom)More conventional patch-clamp recordings are currently in use, involving whole-cell recordingson either dissociated cells or epithelial preparations. In the latter case, adjacent cells must becleared away from the hair cell of interest in order to expose the basolateral surface to thepatch pipette. Often, hair cells are dissociated through mechanical or enzymatic treatments,but one might imagine the toll taken on delicate hair bundle structures and the integrity ofbasolateral ion channels. A variety of semi-intact epithelial preparations are currently in useby many labs. In some cases, neural elements remain, offering the chance to ask broaderquestions regarding transduction and transmission.

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Transduction: Methods – 4Mammalian Cochlea- voltage clamp

In the mammalian cochlea the first recordings from transduction currents wemade in cultured explants of 1-3 day old mice cochleae. The stereociliabundles were stimulated by a waterjet. The bundles are too short to bestimulated by a stiff probe.At first receptor potentials were recorded (Russell, Cody, Richardson 1986)and later also the patch clamp technique was implemented for voltage clamprecordings, in order to record transduction currents (Kros, Ruesch andRichardson, 1992). For the patch clamp recordings the basolateral membraneof OHCss had to be cleared as demonstrated in Fig. B for IHCs.

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Transducer Currents in Outer Hair Cells

driv

er v

olta

gecu

rrent

/ pA

Kros, Ruesch and Richardson, 1992

Voltage clamp recordings form hair cells made it possible, to isolate thetransduction current. Looking at the isolated current allows to understand,which features of the receptor potential are due to properties of thetransduction current and which are due to other elements in the signalingpathway of the cochlea.On the left: Five recordings of transduction currents in the neonatal mousecochlea in response to 5 different stimulus intensities (waterjet, sinosoidalstimulation). The positive driver voltage corresponded to fluid flow that movedthe bundle towards the kinocilium and opened transduction channels causinginward currents. Fluid movement in the other direction closed transducerchannels that were open at rest. The membrane potential was clamped to -84mV.On the right: B. Transfer function of the transducer conductance (currentdivided by the driving voltage).C. Transducer conductance as a function of bundle displacement. This cellwas stimulated with force steps.

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Location of Transduction Channels - 1

Jaramillo & Hudspeth, 1991

Preparation: Isolated hair cells from bullfrog sacculusMethods: Whole-cell patch clamp during displacement and iontophoretic application ofchannel blocker (gentamicin)

Aminoglycoside antibiotics (e.g. gentamicin) act as open-channel blockers of transductionchannels. The blocker (at 500 mM) was applied by iontophoresis, a method in which currentpassed through a high-resistance pipette pushes positively-charged ions/drugs out of thepipette.Left, Top: In the control condition, current relaxation is due to adaptation mechanisms. A briefpulse of blocker was applied following bundle displacement, and a rapid reduction in currentwas seen. The slow return of transduction current after block results from diffusion of the(reversible) blocker away from the hair bundle.Right: The location of drug application was carefully varied around the profile of the hairbundle. The maximum effect was consistently at the tip with little effect at the base of the hairbundle. Block at “1a” demonstrates extent of diffusion, therefore some block at base (nearshortest stereocilia) is expected.Left, Bottom: (A) To control for possible movement artifacts during drug application, theblocker was applied to hair bundles at rest. Application of the blocker at the top of the bundlereduced resting transduction current (recall that 10-20% of channels are open at rest).Application at the bottom of the hair bundle did not affect resting current. (B) Block wasapplied at the top of the bundle, bottom, and while advanced into the base of the hair bundle.This was done to control for possibilities of transduction channels being located within thebase of the bundle (with the bundle acting as a diffusion barrier). This control further supportsthe location of channels at the tip eliminating the chance that the hair bundle acts as adiffusion barrier.

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Tip-Link Structure

Kachar et al., 2000

Left:Helical structure of the tip link. (A) Proposed model for tip-link structure. Twohelically intertwined protofilaments (Inset) make up the tip link, attaching attwo points to the taller stereocilium and contacting three filaments emanatingfrom the shorter stereocilium. Note the dense plaques (red color) at theconnection points. (B) Freeze-etch image of tip link from guinea pig cochlea.Note the thick carbon coat forming a halo around the tip link and thestereocilia surface. (C) Higher magnification view of the tip link in B. (D)Surface plot of the pixel intensities of the digitized image of the tip link shownin B created with National Institutes of Health IMAGE. The pseudo-three-dimensional image helped visualize the helical configuration and the possibleperiodic substructure of the protofilaments. (Scale bars: B = 50 nm; C and D =10 nm.)

Right:Upper and lower attachments of the tip link. (A and B) Freeze-etch images oftip-link upper insertions in guinea pig cochlea (A) and (left to right) two fromguinea pig cochlea, two from bullfrog sacculus, and two from guinea pigutriculus (B). Each example shows pronounced branching. (C and D) Freeze-etch images of the tip-link lower insertion in stereocilia from bullfrog sacculus(C) and guinea pig utriculus (D); multiple strands (arrows) arise from thestereociliary tip. (E) Freeze-fracture image of stereociliary tips from bullfrogsacculus; indentations at tips are indicated by arrows. (Scale bars: A = 100 nm,B = 25 nm; C–E = 100 nm.)

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Assad et al., 1991

Location of Transduction Channels – 2Tip Link Destruction

Preparation: Bullfrog sacculusMethods: TEM/SEM quantification of tip-link presence as well as measure of transductioncurrent via whole-cell patch clamp following BAPTA treatment.

133 nm movement forward after break, due to pre-tensioning of tip-links (note inward tilt ofmost hair bundles).

Trace above seems to indicate a large increase in inward current after BAPTA. More recentevidence supports the notion that transduction channels remain open after breaking tip-linkswith BAPTA or elastase treatment. This result throws a minor curve at the gating-springhypothesis, in that breaking the gating-spring should result in closure of gates and eliminationof resting transduction current. However, it is conceivable that both BAPTA and elastasemodify the transduction channel as well and quite possibly destroy the gate along with the tip-link.

Incubation of hair bundles with any tetracarboxylic calcium chelator (e.g. BAPTA) results in thedestruction of tip links and transduction currents. At one time, it was thought that the lowcalcium condition created by the chelators was responsible for tip-link destruction, the key nowseems to be in the chelator itself (particularly tetracarboxylic chelators). Neither low calciumalone nor chelators in other families break tip-links.

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Location of Transduction Channels – 3Tip Link Regeneration

Preparation: Chick basilar papilla (cochlea analog), in culture for 0-24 hours.Methods: Whole-cell patch clamp of isolated hair cells as well as imaging of tip-links.

Incubate tissue in control media with or without a 15 minute pretreatment with BAPTA.Quantify tip-links (from SEM or TEM micrographs) at various time points. Electrophysiologyconducted on isolated hair cells in whole-cell patch clamp while hair bundles were displacedby a pipette attached to a piezoelectric bimorph (+/- 1.2 µm).

Tip-links regenerate within 12 hours after BAPTA treatment (top panel).• After 24 hours, the number of tip links in treated bundles approaches 90% of those incontrol bundles.• Small percentage of these tip links are abnormal (attached to wrong stereocilia,different angles).• Regeneration on this time scale is not dependent on protein synthesis.• Regeneration is dependent on intracellular calcium concentration, where a low [Ca]is apparently a signal for regeneration.

The regeneration of tip links is associated with the return of transduction currents.• Although transduction returns it is significantly altered (e.g. lower peak currents,slower adaptation, and lower extent of adaptation).

_____________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________

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Denk et al., 1995

Localization of transductionchannels at both ends of tiplinks

Location of Transduction Channels – 4

Preparation: Bullfrog sacculusMethods: Epithelial preparation with whole-cell patch clamp and CG-1 Fluorescence with two-photon laser scanning microscopy

(A-C) Expected patterns of deflection-dependent fluorescence if (A) channels are located onlyat lower ends, (B) channels are located at upper ends, and (C) channels are located at bothends.(D) Fluorescence of a representative cell. Left: undeflected, Right: deflected, -90 mV holdingpotential.(E) A second cell, left: undeflected and right: deflected at -90 mV. Some stereocilia in shortestand tallest row were responsive in the resting state, but more so in deflected state. Thus,channels possibly located at both ends of the tip link.(F) A third cell, deflected in both cases with left: +60 mV and right: -90 mV. Less fluorescencein the +60 mV condition since this approximates the reversal potential for calcium. This panelis supportive of the change in fluorescence resulting from changes in calcium entering throughthe transduction channels.__________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________

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Transduction Channels Gate Fast

Preparation: Bullfrog sacculusMethods: Transepithelial voltage clamp

Top: A prepulse of -0.4 µm closes all transduction channels. The onset of current inactivation steps is slightly delayed, but curves are fit by a single exponential. The delay ofactivation from a resting position is approximately 25 µs. Such a delay in photoreceptors isabout 2 orders of magnitude greater! This delay in hair cells is extremely short and excludesthe involvement of a second messenger system. Instead, these kinetics suggest the directmechanical gating of transduction channels.

Bottom: A prepulse of 1.0 µm opens all transduction channels. Current relaxation requirestwo exponential components. The closing rate saturates for large negative stimuli.

---------------------------------------------------------------------------------------------------------------------------Corey and Hudspeth, 1979, and Lumpkin et al., 1997Relative permeabilities:

NH4 (1.3), K (1.0), Rb (1.0), Cs (1.0), Na (0.9), Li (0.9), TEA (0.4), Ca (5-200)Ca required for transduction (> 10 µM), but it also blocks at high concentrations. Thus,there is likely a calcium binding site within the pore of the transduction channel.

Pore diameter:At least 0.54 nm

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Fast gating of the Transduction Channel Ledto the Gating-Spring Hypothesis

In 1982 and 1983, it became clear that the gating kinetics of the transduction channels wereextremely fast, precluding the involvement of a second messenger system. Instead, it wassuggested that a direct mechanical gating of the channel would be necessary. The gating-spring hypothesis proposes that transduction channels are physically blocked in a trap-doorfashion, where gating of the trap-door involves the action of an attached gating-spring (right,top). Tension in the gating-spring increases during excitatory stimulation until passing athreshold for opening the gate (i.e. imagine a rubber-band attached to a mouse-trap…pullingon the rubber-band will eventually cause the clamp on the mouse-trap to open).These ideas were formed prior to experiments localizing transduction channels to the tip of thebundle and prior to observations of fine filament links located at the tip of the hair bundle (tip-links) (left). At the beginning of the lecture, we pointed out the presence of specialized linkslocated between the tip of one stereocilium and the shaft of an adjacent taller stereocilia. Thisfine filament is in a unique position to sense mechanical displacement along the axis ofsymmetry in the hair bundle.Thus, the gating-spring model places transduction channels at one or both ends of the tip-link,where positive or excitatory displacement builds tension in the tip-link and opens the channelwhile negative or inhibitory displacement slackens the tip-link and allows for channels to close(right, bottom). Some resting tension in the tip-link must be responsible for opening 10-20%of the channels in an unstimulated hair bundle.

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• non-selective cation channel permeable for Na+, K+, Ca2+ with substantialCa2+permeabiliy

• large single channel conductance (100 pS)• blocked by low concentrations of aminoglycoside antibiotics• blocked by amiloride (like epithelial sodium channels αENaC)• blocked by tubocurarine (like Ach receptors)• blocked by Ca2+ -channel antagonists like nifedipine• located at the top of the stereocilia

• These properties are very unspecific and none of the known channel typescompletely fits this profile

• former candidate: αENaC; amiloride blocks and immunogold labeling wasfound close to the tip links. However: αENaC KO mice still transduce.

• former candidate: ATP-activated channels (P2X receptor). Is localized at the tipof stereocilia bundles; similar pharmacological profile as transduction channel.However: a detailed pharmacological analysis found differences between thosetwo channels. If both channels are activated, their currents are additive,suggesting two distinct currents.

The Search for the Molecular Identity of the Transduction Channel:1 - Transduction Channel Properties

Molecular identity of the transduction channel is still unknown. One approachto identify the transduction channel is to characterize it’s features extensivelyand compare with the features with other known ion channels to find the genefamily it may belong to. This approach has been unsuccessful as there are nospecific features of the transduction channels that would distinguish them frommost classes of unspecific cation channels.Therefore laboratories now choose genetics as their strategy to search for thetransduction channel gene.

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• Invertebrate species may havetransduction channels that also use amechanism with a the gating spring.

• Some invertebrate species can bereadily approached with geneticsbecause of their fast generation time.

• Mutations can be induced and mutantswith mechanosensory defect can beidentified.

• In these mutants the defect genes canbe isolated.

The Search for the Molecular Identity of the Transduction Channel:2 - Invertebrate Mechanoreceptor Models

From Gillespie and Walker (2001).

From Gillespie and Walker (2001).

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The Search for the Molecular Identity of the Transduction Channel:3 - the Nematode Worm Caenorhabditis elegans

The microtubule arraymay be deflectedrelative to the mantleand this deflection maybe detected by thetransduction channel

Transduction by theDegenerin / ENaCfamily

From Gillespie and Walker (2001).

Genetic screens identified C. elegans mutants (mec mutants) that weredefective in mechanosensation.Mutant worms that responded inappropriately or not at all to a simple touch ofan eyelash were selected and most of the responsible genes have beenidentified. Mec4 and Mec10 (socalled degenerins, also related to Epithelialsodium channels) are candidates to be part of a transduction channel,however, attempts to elicit mechanically induced currents from heterologouscells expressing these channels has been unsuccessful. Also up until now ithas not been possible to record receptor currents from C. elegans touchneurons.

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The Search for the Molecular Identity of the Transduction Channel:4 - Drosophila melanogaster

Movement of thebristle displacesthe dendrite ofthe mechano-sensory neuron

NompC is part ofthe transductionchannel. Itbelongs to theTRP family ofion channels

Like in C. elegans, through the screen from flies for mechano-insensitivemutants two genes have been identified, that are involved inmechanotransduction, nompA and nompC (no mechanoreceptor potential).nompA probably serves as an extracellular mechanical link.nompC has been shown to be part of the mechanotransduction channel(Walker et al, 2000). Receptor currents can be recorded in the fly bristles andone nompC allele was shown to not just interrupt transduction, but to changethe properties of the transduction channel. The receptor currents in thesemutants had amplitudes close to those in wildtypes, however, noticable fasteradaptation. This experiment put nompC on the map as a possible subunit of atransduction channel!!nompC is part of the TRP channel family (transient receptor potential family).This family is very diverse and right now members of this family are underdetailed research as they are likely candidates for vertebrate transductionchannels.

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The Search for the Molecular Identity of the Transduction Channel:5 - The Vertebrate Hair Cell

For vertebrate hair cells a number of elements in the transduction apparatushave been identified, but the search for the transduction channel is still on….

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• TRP (transient receptor potential)A member of this family, nompC (no mechanoreceptorpotential C: fly mutant), may be part of thetransduction channel in drosophila. The TRPsuperfamily is extensive with large variations insequences, pharmacology, selectivity, etc. Channelsin this superfamily remain strong candidates.Sidi et al. (2003) found the zebrafish ortholog ofdrosophila nompC in zebrafish hair cells and havepostulated that it may be part of the mechanoreceptorin these vertebrate hair cells. The evidence is not asstrong as for drosophila.

The Search for the Molecular Identity of the Transduction Channel:5 – the strongest candidate