Intro and Auditory Transduction 1, 2 + 3

?
  • Created by: Sarah
  • Created on: 19-05-19 11:51
what is one of the most common sensory disorders in humans and one of the most chronic diseases even above diabetes?
age related hearing loss
1 of 278
why is this so importnat?
we're an ageing population so the number of people affected by age related hearing loss is increasing
2 of 278
what problems do we have with age related hearing loss?
we don't have a cure and we don't undertand the causes of it
3 of 278
what are spontenaoulsy active with ca spikes in early development
hair cells
4 of 278
what is mechanotransduction?
the conversion of sound into an electrical signal
5 of 278
what does optogenetics use?
transgenic animals with light sensitive channels to activate neurons with light- modulate AP activity
6 of 278
where is mechanotransduction occuring?
at the sensory receptor = the sensory hair cell
7 of 278
what do ribbon synapses convert information into? where?
ribbon synapses convert the sound information into an electrical signal in the afferent nerve
8 of 278
where are ribbon synapses present?
in the auditory, vestibular system and the photoreceptor in the retina
9 of 278
what does the vestibular system detect?
propicepton and movement
10 of 278
what is synaptopathy?
defects in synapsis- caused by loud sound noise eg damage ear and lose synapsis in adferent fibre
11 of 278
why study the auditory system?
how biological systems orchestrate their development and function is crucial to disease, energy+ dimenstion in wuantum physics range
12 of 278
what does it mean that teh auditory system can perform biological task with an energy and dimension that reaches the range of quantum physics?
can detect sound induced displacement in the hair like structure in less than 2nm in the order of picometer
13 of 278
what is the frequency range detected in human hearing?
20Hz to 20,000 Hz
14 of 278
why can we detect sound in space?
because a micorsencond difference in the arrival of sound between 2 ears
15 of 278
why is the auditory system better than other system,s?
can detect a huge frequency, it can amplify sound by several hundred fold, detects sound from both ears and coordinates- precision and timing is amazing
16 of 278
why do we want to undertsnad causes of deafness and hearing loss?
improve quality of life, socially isolated, depressed, cognitive decline, more at risk of dementia
17 of 278
best cure for hearing loss atm?
cochlea implants or hearing aids
18 of 278
what is sensory perception?
the processing of external stimuli- gives a perception of the outside world so your brain can make informed decisions
19 of 278
why do you need sound transduction why can't the brain perceive sound and light directly?
integrate information and filter out unwanted, different modalities but brain only understands an electrical signal so need sensory transduction for brain interpretation
20 of 278
what are all sensory receptors?
transducers
21 of 278
what do transducers do?
detect the stimulus and concert it into an electrical signal
22 of 278
what is generated when sound displaces hair like structures?
displaces the bundle and opens a channel then membrane permeability of the cell changes, ions come into the cell and geenrate a receptor current
23 of 278
what is the ion that flows inside the hair cell?
K
24 of 278
what is the scala media high in?
K
25 of 278
what fluid does the scala media contain?
endolymph
26 of 278
what does K moving into the hair cell do?
depolarises the cell, activates ca channels fo rvesicle fusion and NT release onto the auditory fibre- activate afferent nerve APs
27 of 278
what 4 types of information do sensory receptors transmit?
1) modality- type of stimulus 2) location- where it is 3) intensity 4) duration
28 of 278
what are examples of different receptors transmitting different modalities?
mechanoreceptor- hearing, balance, pressure and temp. Photorecepotr- light. Chemoreceptor- olfaction
29 of 278
2 mechanisms for sound localisation (location)?
1) interaural timing differences 2) interaural level differences
30 of 278
mechanisms for sound localisation (location) mainly for LOW frequency sound?
1) interaural time differences- difference of arival time of sound from 2 ears as small as 10 microseconds, reaches 1 ear 10microsecs faster
31 of 278
mechanisms for sound localisation (location) mainly for HIGH frequency sound
interaural level differences- the difference in intensity of sound at the 2 ears as small 1-2 decibels - sound louder on closer ear
32 of 278
which strategy do most species use?
both
33 of 278
what one do bats use more?
interaural level differences because hearing range of species is very high frequency
34 of 278
what is the intensity information part that sensory receptors transmit?
larger stimulus = stronger response of rec --> stronger APs in auditory neurons reaching threshold
35 of 278
how is duration encoded?
tonic receptors or phasic receptrs
36 of 278
what do tonic receptors encode?
slow adapatation
37 of 278
what do phasic receptors encode?
burst, fast adaptation
38 of 278
what receptors are auditory hair cells
tonic mechanotranducers as they need to respond to sound continually not at the beginning and then become deaf
39 of 278
do auditory hair cells have a low or high adaptation?
low adaptation
40 of 278
what is sound?
sound is produced by vibrating particles which causes changes in air pressure which travel as waves through air and produce sound
41 of 278
does the particle int he air travel/?
no sound is travelling in space, air is not travelling its disturbance within air that travels
42 of 278
what determines the frequency?
the number of cycle rarefraction and compession per second
43 of 278
what is frequency measured in
hertz
44 of 278
what is frequency recived in?
pitch
45 of 278
what is the amplitude? whats it expressed in?
amplitude of waves- is expressed in decibels perceived as loudness
46 of 278
what do we still not know the identity of?
the MET channel
47 of 278
what is the auditory organ?
the cochlea
48 of 278
what forms the inner ear in the temporal bone?
the cochlea and vestibular system attached together
49 of 278
what is sound transmitted to the ear via?
the middle ear
50 of 278
what is the problem with sound getting transmited to the ear?
has to be transmitted from air to water so acoustic information is reflected by water, allows sound in water to travel 4 times faster than sound in the air
51 of 278
it takes much more energy to srart sound in water as sound is reflected as it hits the water surface how is this overcome?
by having the outer, inner and middle ear, sound comes in the outer ear vibrates the tympanic membrane energy transferred to ossicle- malleus, incus and stapes
52 of 278
what does this change the sound waves into?
into a mechanical movement transfers energy from air to solution
53 of 278
how is sound transmitted from air to water?
the tympanic membrnae vibrates the ossicles, the stapes is connected to the oval window in the inner ear which causes solution inside the cochlea to move
54 of 278
what are the smallest bones you have in your body?
ossicles
55 of 278
what 3 compartments are there in the inner ear?
scala media, scala vestibula and scala tympani
56 of 278
where is the cochlear?
between the scala media and scala tympani
57 of 278
what brings the vibrations in?
scalia vestibuli
58 of 278
what sends the vibrations out
scala tympani
59 of 278
how is the cochlea a frequency analyser?
low frequency sounds detected at apex and high frequency at the base
60 of 278
what is the most important mechanical property that allows this tonotopic gradient
the basilar membrane
61 of 278
what sits on top of the basilar membrane?
the organ of corti
62 of 278
how does the basilar membrane change from apex to base?
thinner + smaller = apex base = wider + bigger
63 of 278
what does this difference in width of basilar membrane do?
changes the stiffness of the membrane from apex to base
64 of 278
where is the BM more stiff?
towards the ase big structure hard to move but pin like projection at apex allows it to be less stiff
65 of 278
what does this difference in stiffness allow?
tonotopic organisation- allows it to work as a freqnecy analyser so different freuqncy of sound will activate a psecific portion of the basilar membrane
66 of 278
how does the basilar membrane get moved?
vibration is passed by stapes pulling in and out attached to the oval window
67 of 278
what is happening in the compression part of the sound wave?
you push everything forward so the stapes pushed the solution inside the oval window and this causes a downward movement of the BM
68 of 278
which way is the BM moving when you have rarefraction in the sin wave?
BM moves upwards
69 of 278
when you get sound how does this cause BM to change hair cells?
sound -> rarefraction -> stapes moves outwards, BM moves upwards -> hair cell bundles move towards taller stereocilia -> activates hair cells
70 of 278
how is the mechanotransducer channel gated?
by a tip link so by moving the stereocilia bundle you open the tip links
71 of 278
the IHCs are not attached to the tectorial M so how do they move?
they move because the solution inside their moves so they follow the movement of the solution
72 of 278
how do you get inhibitory movement of the channel?
sound compression-> stapes moves inwards, BM moves downwards and the opposite movement of the sterociliary bundle closes the channel
73 of 278
what do the IHC have projecting from the top of them?
stereociliary bundle
74 of 278
what are the primary sensory receptors of the ear?
IHCs
75 of 278
what are IHCs connected to?
auditory afferent fibres so send info to the auditory cortex
76 of 278
how many rows of OHCs are there?
3
77 of 278
why are OHCs important?
they enhance the sensitivity and frequency selectivity along the length of the cochlea
78 of 278
what do OHCs contribute?
movement- they shrink and elnogate and change the RMP
79 of 278
what do OHCs do when they are activated?
change their legnth and amplify the signal because they express a motor protein called prestin
80 of 278
what is the hair cell responsible for mechanical amplification?
OHCs
81 of 278
what happens to hair bundle development in embryonic stages?
have a lot of microvilli (undifferentiated projections), the kinocilium migrates on one side
82 of 278
what does the kinocilium migrating on one side do?
determines the polarity of the sterociliary bundle where the bundle will start to grow as all sterocilia organised in the same pattern = they're polarised
83 of 278
what is kinocilium made of?
tubulin
84 of 278
what happens in later embryonci stages?
the kinoclium is still there, start to have some some strerocilia but t alot are still undifferentiated microvilli
85 of 278
end of embryonic, beginning of postnatal what happens?
get different rows of stereocilia- growth of sterociliary bundle is highly regulated
86 of 278
how do you get the staircase structure?
shorter row of stereocilia keep growing up to PN day 5 then stop and then longer rows keep growing up to PN day 12 in mice
87 of 278
when is the onset of hearing in mice?
PN day 12- so taller row grows up until then and organisation is pretty much complete
88 of 278
differences in shape between HC and OHC?
shape, width and height of sterocilia different- IHC- U shaped, OHC- V shaped
89 of 278
what can change depending on the species?
the number of sterocilia rows
90 of 278
what are sterocilia supported by?
bundles of polarised actin filaments
91 of 278
why is polarised actin important and different from filopodia etc?
because they maintain a constant height throughout life in stereocilia
92 of 278
how are sterocilia held together as one structure?
tip links
93 of 278
what are tip links important for?
development and function of the stereociliary bundle
94 of 278
what happens at the bottom part of the stereocilia where they are connected to the hair cell?
there is tapering, stereoiclia get smaller where they insert into the hair cell = unique to stereocilia
95 of 278
why is there this narrowing of the bundle?
it allows movement- if width of the bundle was the same throughout when the bundle is moved by solution or the tectorial M they'd bend but wan movement
96 of 278
how do ypou get a good control of the movement?
make the width of the sterocilia narrower where it inserts into the hair cell
97 of 278
what needs to be regulated to get hair bundles to grow to the right amplitude in development?
polymerisation and depolymerisation as they have to be a constant height all the time
98 of 278
the length and width and polymerisation and depolymersiation is coordinated activity to get the staircase structure how?
proteins- Eps8 and Eps8L2
99 of 278
What else needs to be coordinated other than height, width and poly and depolymerisation?
the same within the hair bundle and the same as the hair cells around it such coordinated development
100 of 278
where is EPS8 primarily localised?
at the tip of the long stereocilia, [faintly labelled on the shorter ones but mainly top]
101 of 278
this protein localised to the top of long stereocilia suggests it might be important for what?
regulating the length
102 of 278
what was shown in the Eps8 KO?
tallest sterecoilia most affected (where Eps8 is expressed) there's a correlation between the level of expression and the regulation of stereocilia height- only one row of stereocilia
103 of 278
what is Eps9 needed for?
to control the normal maturation and growth of the stereociliary bundle- in the absence of Eps8 bundles unable to develop
104 of 278
where is Eps8L2 localised?
shorter rows of stereocilia
105 of 278
what happened in Eps8L2 KO?
development of stereociliary bundle was completely normal- no difference in height or width, could hear normally but fewer shorter sterocilia and different shape
106 of 278
what happens to the mice at 6 months of age?
sterociliary bundle degenerated- progressive hearing loss
107 of 278
what was Eps8L2 therefore important for?
maintenance of the stereociliary bundle- doesn't have a ole early on development but is required to keep them for life
108 of 278
what happened to Eps8 KO mice hearing?
they were profoundly deaf- never able to hear
109 of 278
why were they profoundly death?
the bundle needs to be able to be attached to the tectorial membrane to move them in the excitatory direction because they're short won't reach tectorial M so no coupling stereocilia not deflected
110 of 278
where is the MET channel localised?
at the top of the shorter stereocilia
111 of 278
what is the MET channel gated by?
tip links that run between the long and short stereocilia
112 of 278
what happened when they used low calcium to break the tip links? what does it show?
theres no opening of the channel, tip links are required for transduction
113 of 278
2 key molecules involved in mechanoelectrical transduction?
cadherin 23 and protocadherin 15
114 of 278
how do we look at the properties of the channel in the lab?
dissect an entire mammalian cochlear from apex to base put at the bottom of a recovery chamber and do electrophysiology and 2 photon imaging under microscop
115 of 278
how does the mechanically gated channel opened by sound open?
tip links
116 of 278
how did they address the question of how the channel opens what experimental setup?
made device- fluid filled jet which is a chamber inside and a buzzer next to a chamber filled with solution in a pipette, pipette solution is the same as the solution in the bath
117 of 278
what does the buzzer mimic in this situation?
a sound wave as it causes the movement of solution which is what we have in the cochlear
118 of 278
how can we drive this as a sine wave?
by sucking and pulling- fluid jet will stimulate the bundle here
119 of 278
what does the patch pipette do?
electrophysiological recordings attached to the bottom of the cell to record current flow through the channel
120 of 278
what is an important characterstic of the tip links?
they're kept under tension- so they keep the channel partially open
121 of 278
how much percent is the transducer channel usually always open?
20%
122 of 278
why is the channel being partially open all the time an advantage?
allows you to modulate the channel in an excitatory or inhibitory way
123 of 278
what do we measure first?
a baseline current with the patch pipette
124 of 278
what happens when we apply a stimulus in the positive side of the sine wave?
the solution will go out, bundle is pushed and record a large current going into the cell
125 of 278
how do you move the bundle in the other direction?
**** the solution
126 of 278
what happens when you **** the solution?
the tip links get slack as the tension has been released so you close the channel and go back to resting current
127 of 278
what do you get if you sitmulate a hair bundle with a fluid jet in an embryonic bundle?
you get nothing as the bundle is not mature, the channel isn't there yet so you stimulate and nothing happens
128 of 278
what happens to the MET current early postnatal?
you start to see a current that gets larger with development then as more mature the current gets bigger
129 of 278
how do you do an experiment to check if the MET channel is actually open eg if its still open in a mutant mouse?
imaging- have a dye that goes through the channel and image, if open- dye goes into the cell and is labelled. if channel is non-functional/closed because a mutated protein then no dye into cell
130 of 278
what is the dye used to see if the MET channel is open? what does it give us an idea of
FM1-43- gives an idea of how much current is going through the channel
131 of 278
why do you have an excitatory and inhibitory direction depending on which way the bundle moves?
planar cell polarity- it ensures a coordinated response from all the hair cells at the same time
132 of 278
what happens if you stimulate the bundle from the wrong side so the bundle moves towards hte inhibitory direction?
can't stimulate the bundle, tip links not stretched so nothing is going to happen channel won't happen
133 of 278
why do you need planar cell polarity?
to stimulate all of the cells and bundle at the same time- coordinated response
134 of 278
how sensitive does the transducer need to be to detect sound?
highly sensitive- quantum physics range peak and non meters
135 of 278
why don't you want super super sensitive hearing?
would get constant noise eg would hear blood inside your ear
136 of 278
how do we study mechanotransduction in vitro?
dissect out cochlear, place it on a mesh, cochlear at bottom of chamber in EC solution to keep it alive, then 1) fluid injector- stimulates bundle 2) patch pipette attached to cell measures current
137 of 278
what does the patch pipette record?
a receptor current
138 of 278
what is the fluid gel?
fluid jel is in the fluid jet it is the same solution as whats in the bath- so continuity, buzzer vibrates move the solution out of the pipette and moves the bundle in one direction
139 of 278
what happens when you move the buzzer backwards?
you **** the solution into the fluid injector- bundle moves in the opposite direction
140 of 278
depending on whether you **** or blow the solution you move the bundle in what direction?
an excitatory or inhibitory direction- either open or close the channel
141 of 278
when you move the bundle towards the taller sterocilia do you open or close the channel?
open the channel so have a large transducer current flowing through the channel
142 of 278
what drives K moving into the cell?
electrical current
143 of 278
the organ of corti is localised in the middle of what?
scala tympani and scala media
144 of 278
why is the reticular lamina important?
provides a barrier between the solution in the scala media and the scala tympani- provides a filter dor diffusion
145 of 278
what solution is the scala media?
endolymph
146 of 278
what solution is in the scala tympani?
perilymph
147 of 278
endolymph scala media is similar to what?
IC solution because it's high in K and low in Ca
148 of 278
what part of the hair cell is in contact with endolymph? what part with perilymph?
sterocilia bundle is surrounded by endolymph. rest of the hair cell is in contact with the scala tympani solution = perilymph
149 of 278
what is perilymph high in?
like EC- high in Na and Ca
150 of 278
because of these 2 different solutions in the different chambers you have what potential in the scala media?
+80mV
151 of 278
why is the driving force for K into the cell electrical current?
scala media- +80mV, inside hair cell- -60mV RMP, so huge electrical driving force of 140mV to drive K into the cell
152 of 278
the electrical driving force making K go into the cell generates what?
a receptor current -> receptor potnetial
153 of 278
how is the shape of the hair bundle specialised in turtles?
lots of rows of sterocilia and greater adhesion- good to detect low sound stimulus low frequency sounds
154 of 278
how is the bat hair bundle specialised to detect high frequency?
only 2 rows of stereocilia, lots of space in between so decreased visocosity resistance so can move very fast
155 of 278
what is the OHC taller rows of stereocilia attached to? why?
OHCs taller rows of stereocilia attached to the tectorial membrane - displacement of this bundle driven by movement of tectorial M
156 of 278
what is the major difference in the shape of the bundle of the IHC and OHC?
outer hair cells = V shaped. Inner hair cells = U shaped
157 of 278
on the IHC what is different about it?
the taller row of sterocilia is much taller than the the OHCs- not attached to the tectorial M so don't have an active mechanism to move need to be more flexible to be moved by the solution moving
158 of 278
what is likely to be the transducer channel pore?
TMC1
159 of 278
has the development of being able to sense sound in air been developed from a common ancestor?
no hair cells we use for vestibular balance likely to be from a common ancestor but the ability to sense sound has developed completely independently
160 of 278
why is sound important?
communication, emotion, navigation (tell which way to go), recognise diff objects around us, create a topographic view of the auditory world, survival
161 of 278
what are OHCs important for?
amplification
162 of 278
where are the transducer channels located?
on the stereocilia of the IHCs and OHCs
163 of 278
what different channels do IHCs have in there membranes to shape how they respond to stimulation?
have ca channels that elicit the release of NT onto the afferent fibres that send APs to the brain in the IHCs
164 of 278
what do OHCs have in their CM to give a different response to stimulation to the IHCs?
have prestin- a motor protein which allows the cells to contract
165 of 278
resting transducer current thats depolarising the cells at rest, what is it in IHCs and OHCs?
-55mV = IHCs, -40mV- OHCs
166 of 278
what does excitatory bundle deflection do for the IHCs?
transducer channels open more so depolarises cells to 30mV- increases NT release as activates ca channels that cause NT vesicle fusion
167 of 278
what does excitatory bundle deflection do for the OHCs?
depolarises them to -20mV- causes hem to contract
168 of 278
what happens to the transducer current when the hair bundle goes in the inhibitory direction?
transducer current shuts off, cell hyperpolarises, -65mC in IHC and -50mV OHCs
169 of 278
what happens with inhibitory bundle movement for IHCs?
NT release reduces
170 of 278
what happens with inhibitory bundle movement for OHCs?
OHCs elongate
171 of 278
where are tip links in between?
longer and shorter sterocilia
172 of 278
what molecules is the tip link made up of?
cadherin 21 and protocadherin 15, mysoin motor at upper end, transducer channel at the lower end
173 of 278
what is the upper end of the tip link bound to?
a mysoin motor- myosin 7a in mammals auditory hair cells
174 of 278
mutations in these proteins mostly causes what?
Ushers syndrome- causes deafness and blindness
175 of 278
how do we investigate the MET channel in vitro?
dissect out cochlear, put under microscope and patch the hair cell, stimulate it with a fluid jet and ecord currents
176 of 278
what happens when you push the bundle?
fluid comes out of the fluid jet and pushes the bundle in the excitatory direction- opening of transducer channels and large inward transducer current
177 of 278
what happens when you pull the bundle back in the opposite direction?
**** fluid back intot he fluid jet and close the transducer channels current reduces compared to rest as turned off resting current
178 of 278
what can we apply as well as a step stimulus?
a step movement of fluid out of the fluid jet- keeps bundle in excitatory direction
179 of 278
what do you get in response to a step movement of fluid out of the fluid jet?
get a step inward current that adapts/reduces over time
180 of 278
what can you generate from a step displacement of the bundle?
a current displacement curve- a property of the current
181 of 278
what does the current displacement curve tell us?
how the transducer current varies with how much you're moving/displacing the bundle
182 of 278
what do you plot to get the current dispalcement curve?
tranducer current against displacement
183 of 278
what relationship is there?
sigmoidal relationship
184 of 278
why does the current open with no displacement?
channels are partially open at rest- inward resting current
185 of 278
what increases the more you displace the bundle?
the current
186 of 278
where is the most sensitive part of the transducer current?
the sloping part of the current displacement relationship
187 of 278
where do we want to maintain the hair bundle position ideally? why?
at this sloping part as its the most sensitive part of the transducer current so it can respond to change
188 of 278
how does it maintain its position around the sloping, most sensitive part/
they use adaptation
189 of 278
what is adaptation?
a decrease in response to a constant stimulus shown here so in response to step displacmeent the current decreases over time doesn't respond as much
190 of 278
where is the myo7a motor?
at the top of the tip link
191 of 278
what is the myosin motor always trying to do? why?
climb up the actin filaments so it keeps the tension on the tip links to maintain the resting position of the bundle
192 of 278
what does the tension of the tip links do the transducer current?
the tension of the tip links keeps the channels partially open at rest so you get the resting transducer current
193 of 278
what happens to the tension of the tip links when the hair bundles are deflected in a positive direction?
the tension of the tip links increases, open the transducer current and you get an influx of K and Ca ions
194 of 278
what is fast adaptation of the transducer current caused by?
Ca
195 of 278
how does ca cause fast adaptation?
a binds to the pore of the tranducer channel and blocks the channel
196 of 278
what causes the slow adaptation?
ca causing the myosin motor (myosin 7a) to slip on the actin filaments, reduces tip link tension which flaps the channel closed so slower adaptation of the current reducing
197 of 278
how is adaptation reversed?
channels close in the hair bundle so less ca goes in, so the myosin motor can begin to climb back up and produce tension to open the tip link, pulling the hair bundle back towards it resting position even though it's still being stimulated
198 of 278
Function of transducer channel adaptation?
reset the operating range of the hair bundle so a current can still be elicited but it needs a larger stimulus = maintains sensitivity and prevents saturation
199 of 278
how is adaptation mostly achieved?
by the myosin motor maintaining tension on the tip links and keeping the resting position of the hair bundle in the most sensitive range
200 of 278
time for slow and fast adaptation?
fast- less than 10msecs slow- lower than 100ms
201 of 278
are slow and fast adaptation independent?
yes independent and occur at different locations
202 of 278
what would low calcium cause in theory like in endolymph?
need less displacement to elicit a current as not as much adaptation
203 of 278
what does a high ca concentration do the amount of displacement needed?
shifts need larger displacement as you have adaptation
204 of 278
when does the elevation in ca happen?
during bubdle deflection ca triggers adaptation and causes a right shift in the displacement curve so you need more displacement
205 of 278
why does the lower ca concentration occur in vivo?
endolymph- low ca, lower ca releives the ca block of the MET channel and reduces adaptation- shift left
206 of 278
what is the MET channel highly permeable to?
more permeable to ca than Na or K
207 of 278
how do you compare the different ca permeabilities between cells or mouse genotypes?
have only ca in the EC so a really high conc and only cesium IC and measure the MET current reversal potential so comparing the permeability of these 2 ions through the channel
208 of 278
how is the experiment done?
have a isne wave bundle displacement thats superimposed on a voltage ramp so the voltage changes from -120mV to +120mV so you can see where the transducer current reverses direction so the reversal potential
209 of 278
how do you tell if it's more permeable to ca?
if it's more permeable to ca it will be closer to the reversal potential (+40mV)
210 of 278
what HCs were more permeable to ca- apex middle or base outer hair cells?
apical cells more permeable to ca because their reversal potential is closer to vrev
211 of 278
what does this mean?
there's a gradient in ca permeability along the cochlear for OHCs
212 of 278
is there a gradient of calcium permeability in the IHCs?
no the gradients the same all the way along
213 of 278
what else is there a gradient in in OHCs?
actual size of the transducer current so apex cells = smaller current compared to base cells
214 of 278
is there a gradient for IHCs transducer current along the cochlea?
no
215 of 278
how do people look at a single channel in HCs?
destroy the tip links using a chemical so there's just one tip link length so you're only stimulating one channel
216 of 278
is the current higher in base or apex OHCs?
in basal cells the current is higher- is also a grad in single channel conductance along cochlear from apex to base
217 of 278
is the channel small or large?
large they have an average conductance about 100 picosiemens (much larger than K channels)
218 of 278
where are MET channels?
only at the lower ends of the tip links
219 of 278
what experiment did they use to show MET channels are only at the lower end of the tip links?
confocal imaging and ca fluorescence in rat hair cells
220 of 278
what is the idea behind this experimnent?
pushing the bundle in diff directions and opening the channels, hold the hair bundle in the deflective position and have no current flowing into the cell and holding the bundle in the smae position cause an inward flow of ca ions
221 of 278
what is the hair bundle having down to it?
having the hair bundle fixed in one position you can see which rows of stereocilia calcium was entering in- because hard to tell when bundles moving
222 of 278
how did they make sure there wasn't any inward flow of ca into the cell?
depolarised the cell to a really positive potential so the transducer current would be outward and not inward, then pushed hair bundle in +ve direction to open channel so channels open but no influx of ca
223 of 278
what did they then do after the hair bundle was maintained in this positive deflection?
hyperpolarise the cell to -80mV so ca ions could now flow into the cell and cause an increase in fluorescence
224 of 278
where was the large ca influx?
only in 2 rows- row 2 + 3 of stereocilia NOT row 1 so there's no ca influx into taller stereocilia
225 of 278
what did this suggest?
that transducer channels are located at the bottom end of the tip links
226 of 278
when do you get ca entry?
when they brought the MP back down hyperpol it but channels open because bundle fixed in a positive deflection position
227 of 278
do you get the same thing with OHCs?
yes- moves, get ca entry and it moves back to the resting position
228 of 278
what could the MET channel potentially by?
a single channel with a vestibule area or it could consist of a channel plus accessory subunits
229 of 278
what does the MET channel mostly transmit? why?
is non-selective in vivo mainly to allow K into the cell because of the high K conc in the endolymph
230 of 278
what are the activation kinectics of a MET channel like?
really rapid so hair cells respond to sound within microseconds
231 of 278
what is the conductance like in MET channels?
channels are really high, the conductance is around 80 to 140pS which is graded tonotopically in OHCs but not IHCs
232 of 278
what are MET channels highly permeable ot?
Ca- at least 4 times more than K
233 of 278
why does the channel have slow and fast adaptation?
to keep the bundle operating in the most sensitive region
234 of 278
what is tonotopically organised in the OHC MET channel properties?
current amplitude, single channel conductance and ca permeability
235 of 278
what has been debated for many years?
what the molecular identity of the channel is
236 of 278
why do we want to know the molecular identity of the MET channel?
drug discovery- can see how it interacts with drugs and new treatments
237 of 278
what new treatments is becoming a good possibility but is limited because we don't know what the MET channels made up of?
gene therapy- need to know what the gene is in order to replace a faulty version
238 of 278
why has it taken us so long to know what the MET channel consists of (size)?
small number of cells in each cochlear- only 4,000 IHCs and 12,000 OHCs in each cochlear and each HC has 100 transducer channels but in other systems eg photorecs have millions
239 of 278
why has it taken us so long to know what the MET channel consists of (in vitro)?
we can't expand the cells in vitro- have no hair cell line to get a decent amount of material for molecular analysis
240 of 278
what has been used to understand the MET channel?
different strains of KO mice that are missing proteins- can see function of protein and what happens to mouse
241 of 278
how are techniques becoming avaliable to see what aas the pore region is made up of?
can specifically target parts of a molecule change = gene editing. Specific aas and see how that impacts function so target aas in pore region we think are important and see how it affects conductance
242 of 278
what are the 3 main candidates for the MET channel?
1) TMC1 (TM channel like protein 1) 2) TMIE- TM inner ear expressed protein 3_ LHPL5- lipoma HMFIC fusion partner like 5
243 of 278
what have all these proteins shown to be important for?
the function of the transducer channel
244 of 278
what criteria would a protein need to fufill to be a candidate for the MET channel?
1) Should cause deafness when absent 2)MET current abolished when pro absent 3) if mutated it should affect properties associated with the channel pore 4) located right place- lower end of tip links 5) does it form an ion channel
245 of 278
what would a candidate for the MET channel need to be able to do in another system?
form a MET channel in another system
246 of 278
what would a potential MET channel candidate need to bind to?
other channel complex proteins- bind to protocadherin 15 which is in the tip link to form a functional channel
247 of 278
most well studied protein?
TMC1
248 of 278
what do you get from auditory brainstem responses from a TMC1 KO?
mouse no waveform - mice are completely deaf even with the loudest sound
249 of 278
how do you record auditory brainstem responses?
surface electrodes put into the ear and record the activity of the auditory nerve = get ABR response
250 of 278
what are Beethoven mice?
mice that have a poitn mutation in TMC1 that affects its function
251 of 278
what happens to hearing in Beethoven mice?
they have progressive hearing loss
252 of 278
what is the Beethoven point mutation the same as?
the same mutation in human form of deafness
253 of 278
how many mutations in TMC1 cause human deafness?
35 diff mutations- one of the most affected proteins in deafness- very important
254 of 278
what does TMC1 affect?
current size and properties of the transducer currents- in the KO TMC1 not response to displacement of SC bundle no currents
255 of 278
what happens to ca permeability in the Beethoven mice?
ca permeability is reduced- (can't look at full TMC1 KO ca permeability because is no ca current)
256 of 278
is TMC1 present in the right place?
yes it localises to the tips of the shorter rows of stereocilia and not the long
257 of 278
how did they see where TMC1 was?
used a protein tagged with m cherry a fluorescent mlecule- see TMC1 at the tip of shorter rows of SC in IHCs+OHCs
258 of 278
is TMC1 able to form a channel?
yes TMC1 dimerises into a channel structure so purified proteins show 2 TMC1 molecules seem to form this channel based on symettery- unsual as channesl usually 4 subunits
259 of 278
when they compared the structure of TMC1 with other dimer channels what did they shw it's likely to have?
10 TMDs (rather than normal 6)
260 of 278
can TMC1 form a pore like structure
yes computer simulation shows how TMC1 molecules are likely to interact with water to form a pore
261 of 278
what is the downside of TMC1?
no ones been able to express it at the cell surface in heterologous cells so not been able to express it in another cell and get it transported to the M to form a functional channel
262 of 278
why can't you study the function of TMC1 in Cos7 cells?
it's expressed in the cells but there's no expression at the M it's restricted to the ER- something else needs to transport the channel from the ER to CS
263 of 278
Structure of TMIE?
small protein so by itself unlikely to be big enough to form a channel pore so it could only form a pore if combined with other proteins
264 of 278
if TMIE is mutated or KO what does it cause?
deafness - transducer current completely abolished (like TMC1)
265 of 278
where is TMIE localised?
like TMC1 located to the tips of shorter stereocilia
266 of 278
what is TMIE likely to be? why?
could be part of the MET channel but its unlikely to be the whole thing- has not been shown to form channels in other systems
267 of 278
what is the other candidate for the MET channel?
LHFPL5- lipoma HMGC Fusion partner like 5
268 of 278
what is LHFLP5?
a TMD protein
269 of 278
what happens when LHFPL 5 is mutated or KO?
deafness
270 of 278
where does it localise like all the rest?
tips of the shorter rows of stereocilia
271 of 278
model of point mutation in TMC1?
beethoven mice
272 of 278
what happens to the transducer current in LHFPL 5 KO?
not completely eliminated- unlikely to be alone responsible for the channel
273 of 278
what is LHFPL 5 likely to be interacting with?
TMC1 and TMIE- likely to be a component of the MET channel complex
274 of 278
what is the transducer channel complex formed of?
TMC1, TMIE, LHFPL5 - these are bound to protocadherin
275 of 278
how many TMD does TMC1 have?
10 TMD
276 of 278
what are all these proteins needed for?
the proper function of the MET channel, a mutation in any of them causes deafness
277 of 278
what is it very likely that TMC1 forms?
the channel pore either alone or in combo with other proteins- so could be a heteromeric pore with TMIE and LHFPL5
278 of 278

Other cards in this set

Card 2

Front

why is this so importnat?

Back

we're an ageing population so the number of people affected by age related hearing loss is increasing

Card 3

Front

what problems do we have with age related hearing loss?

Back

Preview of the front of card 3

Card 4

Front

what are spontenaoulsy active with ca spikes in early development

Back

Preview of the front of card 4

Card 5

Front

what is mechanotransduction?

Back

Preview of the front of card 5
View more cards

Comments

No comments have yet been made

Similar Biology resources:

See all Biology resources »See all Sensory neuro resources »