The Auditory Periphery
2 – Hair Cell Structure and Transduction
Dr. Elisabeth Glowatzki
955-3877
[email protected]
521 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, 1998
An Introduction to the Physiology of Hearing, 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) Mechanotransduction in vertebrate hair
cells: structure and function of the stereociliary bundle. Am. J. Physiol.
268:C1-C13.
1
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 rodshaped.
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).
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 basilar membrane 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 (ATP)
g transduction
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
Bullfrog VHC
Turtle VHC
Hair cell and hair bundle examples in electron-microscopic images.
Note: in vivo, the stereocilia and kinocilium (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 nonmammalian) 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
Tilney et al.
(1983)
Tilney et al. (1980)
Stereocilium Structure
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 endolymph, 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 bony labyrinth, 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 otolithic
membrane (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 ear 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
Pipette
Patch clamp, used by various
groups, also used in the
mammalian vestibular organ
(Jeffrey Holt and others)
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 singleelectrode 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
current / pA
driver voltage
Transducer Currents in Outer Hair Cells
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-threedimensional 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) Freezeetch 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 tiplink.
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).
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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 twophoton 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.
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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 gatingspring 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 (tiplinks) (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 mechanosensation.
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 mechanosensory 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