The Auditory Periphery

2 – Structure and Transduction

Dr. Elisabeth Glowatzki 955-3877 [email protected] 521 Traylor Building

Websites: Promenade ‘round the (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, 1998 An Introduction to the Physiology of , J. O. Pickles, New York: Academic Press, 1982 Fundamentals of Hearing: An Introduction (3rd ed.), W. A. Yost, San Diego: Academic Press, 1994 Hackney CM, Furness DN (1995) in vertebrate hair cells: structure and function of the stereociliary bundle. Am. J. Physiol. 268:C1-C13.

1 The

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 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 , 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).

2 Cross sections of Organ of Corti of guinea pig. Upper from apex, lower from base of cochlear spiral. R. Pujol.

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

3 Deflection of the Stereocilia Bundles

Transduction process:

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

4 Transduction and Synaptic Transmission at the Inner Hair Cell

Stephan Blatrix

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 through transduction channels. The transduction current generates a receptor potential. Depolarization of the hair cell opens voltage gated Ca2+-channels at the base of 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 afferent fiber 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 induce an increase in the firing rate in auditory nerve fibers. Deflection in the opposite direction induce a reduction in firing rate. At rest (when no signal is applied to the hair cells), there is still some influx of K+ into the hair cell (about 10 % of the maximal current), some transmitter is released causing ‘spontaneous activity’ in the auditory nerve fibers.

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

g transduction g (ATP)

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

6 Hair Cell & Hair Bundle Examples

IHC OHC

Turtle VHC

Turtle VHC Bullfrog VHC

Hair cell and hair bundle examples in electron-microscopic images. Note: in vivo, the stereocilia and (the tallest stereocilium in vestibular hair cells) are rigid and upright (not curved, as shown particularly in the turtle vestibular hair cell bundles due to 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 anchor the hair bundle to the overlying (otolithic) membrane. They are present in cochlear hair cells only during development. •An unansered question: What is the functional significance of differences in bundle 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), Frog VHC (Strassmaier & Gillespie, 2002)

7 Stereocilium Structure

Tilney et al. (1983) Tilney et al. (1980)

Stereocilia are composed of a paracrystalline array of tightly (hexagonally) packed actin filaments 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 the farther it penetrates. Rootlet filaments are interconnected by fine 3-nm filaments and are presumably anchored by myosins among other proteins. •The actin core is suitable for myosin motility (Shepherd et al., 1990). Demembraned hair bundles were blotted and the movement of myosin coated beads were recorded. Myosin freely moved along the actin complex, seemingly uninhibited by the presence of fimbrin cross-bridges. This observation is critical for later discussion of myosin dependent adaptation.

8 Hair Bundle Motion

Chick Tall Hair Cell Water-jet Stimulation 500 Hz 15º displacement Stroboscopic Lamp (Keith Duncan)

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

9 Tip Links

Fettiplace, Ricci and Hackney, 2001

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

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

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

10 + 80 mV

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

dV = 140 mV

-60 mV Stephan Blatrix

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 K concentration in the , mainly K enters through the channel into the cell. The driving force is 140 mV. Proof for this theory will be presented later in this lecture after introducing methods how transduction currents have been recorded.

11 Transduction: Methods – 1 Frog Sacculus

Corey & Hudspeth, 1983

Hudspeth & Corey, 1977

Recording from hair cells is no trivial task due to the unique fluid environment in vivo, the location of these cells within the , and the necessity for micromechanical stimulation 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 cells are penetrated using a single fine tipped microelectrode, measuring the cell’s membrane potential (note: not a voltage or current clamp configuration). A glass fiber holding the stereocilia 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 (overlying hair cells) is removed (OM). The preparation is mounted across a hole in a nonconducting surface (W). Thus, there are now two separate fluid chambers (simulating the in vivo environment). Electrodes are placed in the upper and lower chambers, and the apical and basolateral surfaces are clamped to 0 mV using a voltage-clamp circuit. Hair bundles are displaced en mass (SP); transduction currents flowing in through transduction channels and out through the basolateral surfaces are measured by the clamp circuit. The intracellular membrane potential is not clamped using this method, and large changes in intracellular potential will alter transduction currents.

12 Receptor Potential in the Frog Sacculus

Preparation: Bullfrog sacculus Methods: Apical surface, single electrode recording

(A) Receptor potentials from a 10-Hz triangle wave stimulus. Upward displacements indicate motion toward the tallest stereocilia. Deflections are parallel to an axis of bilateral symmetry (along the graded heights of the bundle). Greater deflections result in greater changes 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 less than 10 mV. Saturating displacements are less than 1 µιχρον (or 10º deflection). The curve is asymmetric (greater changes for positive displacements than negative) and approximates a Boltzman relationship. This suggests that some transduction related current is present in hair bundles at rest. Note: statistically significant changes in membrane potential for photoreceptors is 10 µV. This would correspond to a displacement 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-wave stimulation and recording of membrane potential. V = I R. Thus, for the constant current pulses, when changes in V are reduced during deflection toward the tallest stereocilia, the input resistance into the hair cell is also reduced. Presumably, conductance changes from the opening and closing of an ion channel are responsible for the change in input resistance. Therefore, transduction currents result from transduction channels whose gating is triggered by hair bundle displacement.

13 Transduction: Methods – 2 Receptor 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 recording from cochlear hair cells in the guinea pig cochlea in vivo. In the lateral approach (Dallos et al 1992), the electrode passes through a fenestra in the cochlear bone and approaches the organ of Corti through the scala media. This method has been used to collect data from the three low frequency turns of the cochlea. In the scala tympani approach (Russell and Sellick, 1978), the electrode passes into the organ of Corti through the basilar membrane from the opened scala tympany. The approach is suitable to the high frequency region 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 sound pressure at the drum. Note that the curve rectifies, the voltage change to the negative halfwave is smaller than to the positive halfwave of the sound signal.

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

Howard & Hudspeth, 1987

the stimulator is connected to the kinocilium

Probe Patch clamp, used by various groups, also used in the mammalian vestibular organ Patch (Jeffrey Holt and others) Pipette

Apical surface voltage clamp (top) The two-electrode voltage clamp (top left) was the first methodology allowing voltage clamp of a single hair cell, and thus tight control over basolateral conductances. In this way, the current through transduction channels could be directly measured (rather than inferred from changes in 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 to record from using patch electrodes (recall that the cuticular plate is a dense matrix and is positioned here).

Whole-cell and perforated patch recordings (bottom) More conventional patch-clamp recordings are currently in use, involving whole-cell recordings on either dissociated cells or epithelial preparations. In the latter case, adjacent cells must be cleared away from the hair cell of interest in order to expose the basolateral surface to the patch 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 of basolateral ion channels. A variety of semi-intact epithelial preparations are currently in use by many labs. In some cases, neural elements remain, offering the chance to ask broader questions regarding transduction and transmission.

15 Transduction: Methods – 4 Mammalian Cochlea- voltage clamp

In the mammalian cochlea the first recordings from transduction currents we made in cultured explants of 1-3 day old mice cochleae. The stereocilia bundles were stimulated by a waterjet. The bundles are too short to be stimulated 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 clamp recordings, in order to record transduction currents (Kros, Ruesch and Richardson, 1992). For the patch clamp recordings the basolateral membrane of OHCss had to be cleared as demonstrated in Fig. B for IHCs.

16 Transducer Currents in Outer Hair Cells driver voltage pA current /

Kros, Ruesch and Richardson, 1992

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

17 Location of Transduction Channels - 1

Jaramillo & Hudspeth, 1991

Preparation: Isolated hair cells from bullfrog sacculus Methods: Whole-cell patch clamp during displacement and iontophoretic application of channel blocker (gentamicin)

Aminoglycoside antibiotics (e.g. gentamicin) act as open-channel blockers of transduction channels. The blocker (at 500 mM) was applied by iontophoresis, a method in which current passed through a high-resistance pipette pushes positively-charged ions/drugs out of the pipette. Left, Top: In the control condition, current relaxation is due to adaptation mechanisms. A brief pulse of blocker was applied following bundle displacement, and a rapid reduction in current was 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 hair bundle. The maximum effect was consistently at the tip with little effect at the base of the hair bundle. Block at “1a” demonstrates extent of diffusion, therefore some block at base (near shortest stereocilia) is expected. Left, Bottom: (A) To control for possible movement artifacts during drug application, the blocker was applied to hair bundles at rest. Application of the blocker at the top of the bundle reduced 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 was applied 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 the base of the bundle (with the bundle acting as a diffusion barrier). This control further supports the location of channels at the tip eliminating the chance that the hair bundle acts as a diffusion barrier.

18 Tip-Link Structure

Kachar et al., 2000

Left: Helical structure of the tip link. (A) Proposed model for tip-link structure. Two helically intertwined protofilaments (Inset) make up the tip link, attaching at two points to the taller stereocilium and contacting three filaments emanating from the shorter stereocilium. Note the dense plaques (red color) at the connection 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 the stereocilia 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 shown in B created with National Institutes of Health IMAGE. The pseudo-three- dimensional image helped visualize the helical configuration and the possible periodic 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 of tip-link upper insertions in guinea pig cochlea (A) and (left to right) two from guinea pig cochlea, two from bullfrog sacculus, and two from guinea pig utriculus (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 the stereociliary tip. (E) Freeze-fracture image of stereociliary tips from bullfrog sacculus; indentations at tips are indicated by arrows. (Scale bars: A = 100 nm, B = 25 nm; C–E = 100 nm.)

19 Location of Transduction Channels – 2 Tip Link Destruction

Assad et al., 1991

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

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

Trace above seems to indicate a large increase in inward current after BAPTA. More recent evidence supports the notion that transduction channels remain open after breaking tip-links with BAPTA or elastase treatment. This result throws a minor curve at the gating-spring hypothesis, in that breaking the gating-spring should result in closure of gates and elimination of resting transduction current. However, it is conceivable that both BAPTA and elastase modify 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 the destruction of tip links and transduction currents. At one time, it was thought that the low calcium condition created by the chelators was responsible for tip-link destruction, the key now seems to be in the chelator itself (particularly tetracarboxylic chelators). Neither low calcium alone nor chelators in other families break tip-links.

20 Location of Transduction Channels – 3 Tip 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. Electrophysiology conducted on isolated hair cells in whole-cell patch clamp while hair bundles were displaced by 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 in control 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). ______

21 Location of Transduction Channels – 4

Localization of transduction channels at both ends of tip links

Denk et al., 1995

Preparation: Bullfrog sacculus Methods: 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 only at lower ends, (B) channels are located at upper ends, and (C) channels are located at both ends. (D) Fluorescence of a representative cell. Left: undeflected, Right: deflected, -90 mV holding potential. (E) A second cell, left: undeflected and right: deflected at -90 mV. Some stereocilia in shortest and 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 fluorescence in the +60 mV condition since this approximates the reversal potential for calcium. This panel is supportive of the change in fluorescence resulting from changes in calcium entering through the transduction channels. ______

22 Transduction Channels Gate Fast

Preparation: Bullfrog sacculus Methods: Transepithelial voltage clamp

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

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

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

23 Fast gating of the Transduction Channel Led to the Gating-Spring Hypothesis

In 1982 and 1983, it became clear that the gating kinetics of the transduction channels were extremely fast, precluding the involvement of a second messenger system. Instead, it was suggested 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-door fashion, 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 a threshold for opening the gate (i.e. imagine a rubber-band attached to a mouse-trap…pulling on 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 the bundle 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 links located between the tip of one stereocilium and the shaft of an adjacent taller stereocilia. This fine filament is in a unique position to sense mechanical displacement along the axis of symmetry 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 channel while 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.

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

• non-selective cation channel permeable for Na+, K+, Ca2+ with substantial Ca2+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 types completely fits this profile

• former candidate: αENaC; amiloride blocks and immunogold labeling was found close to the tip links. However: αENaC KO mice still transduce. • former candidate: ATP-activated channels (P2X receptor). Is localized at the tip of stereocilia bundles; similar pharmacological profile as transduction channel. However: a detailed pharmacological analysis found differences between those two channels. If both channels are activated, their currents are additive, suggesting two distinct currents.

Molecular identity of the transduction channel is still unknown. One approach to identify the transduction channel is to characterize it’s features extensively and compare with the features with other known ion channels to find the gene family it may belong to. This approach has been unsuccessful as there are no specific features of the transduction channels that would distinguish them from most classes of unspecific cation channels. Therefore laboratories now choose genetics as their strategy to search for the transduction channel gene.

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

• Invertebrate species may have transduction channels that also use a mechanism with a the gating spring.

• Some invertebrate species can be readily approached with genetics because of their fast generation time.

• Mutations can be induced and mutants with mechanosensory defect can be identified.

• In these mutants the defect genes can be isolated.

From Gillespie and Walker (2001).

From Gillespie and Walker (2001).

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

The microtubule array may be deflected relative to the mantle and this deflection may be detected by the transduction channel

Transduction by the Degenerin / ENaC family

From Gillespie and Walker (2001).

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

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

Movement of the bristle displaces the dendrite of the mechano- sensory neuron

NompC is part of the transduction channel. It belongs to the TRP family of ion channels

Like in C. elegans, through the screen from flies for mechano-insensitive mutants two genes have been identified, that are involved in mechanotransduction, 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 and one nompC allele was shown to not just interrupt transduction, but to change the properties of the transduction channel. The receptor currents in these mutants had amplitudes close to those in wildtypes, however, noticable faster adaptation. This experiment put nompC on the map as a possible subunit of a transduction 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 under detailed research as they are likely candidates for vertebrate transduction channels.

28 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 apparatus have been identified, but the search for the transduction channel is still on….

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

• TRP (transient receptor potential) A member of this family, nompC (no mechanoreceptor potential C: fly mutant), may be part of the transduction channel in drosophila. The TRP superfamily is extensive with large variations in sequences, pharmacology, selectivity, etc. Channels in this superfamily remain strong candidates. Sidi et al. (2003) found the zebrafish ortholog of drosophila nompC in zebrafish hair cells and have postulated that it may be part of the mechanoreceptor in these vertebrate hair cells. The evidence is not as strong as for drosophila.

30