Outer hair cell Outline electromotilitybioewhit/courses/bioe592/mat/Brownell... · 1.06 1.08 1.1...
Transcript of Outer hair cell Outline electromotilitybioewhit/courses/bioe592/mat/Brownell... · 1.06 1.08 1.1...
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Outer hair cell electromotility
W.E. [email protected]
713-798-8540
Sensory Neuroengineering, Rice1 October 2003
Outline1. Mammalian hearing
2. The cochlear amplifier
3. OHC electromotility
4. OHC piezoelectricity
5. Membrane bending
6. The role of prestin – molecular motor vs. modulation of membrane properties
Inner Ear Mechanoreceptor OrgansVestibular system common to all vertebrates > 400 million years old 0-102 Hz
Cochlea found onlyin mammals ~ 220 million years old 100-105 Hz
Allman, 1999
Mammalian Hearing -A Higher Frequency Range Than Other Vertebrates
ElephantHuman
BatsDolphins
Mice
Heffner & Heffner (1980)
Relation Between Interaural Time and High Frequency Limit
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Sound Propagation in theInner Ear
The Traveling Wave: Passive Hearing
Tonotopic mapping of frequency
Frequencyincrease
fkm =Θ+Θ+Θ &&& η
Mechanical models
Filtering
Passive
Frequency
Active
The Swing A Passive Filter The Swinger
An Active Filter
Passive vs. Active
Thomas Gold, Professor Emeritus of Astronomy at Cornell University. First to propose an active mechanism (1947). His proposal was ignored till the discovery of otoacoustic emissions (1979) and electromotility (1983).
George von Békésy (1899 – 1972), received Nobel Prize in 1961 for his discoveries concerning the physical mechanisms of stimulation within the cochlea. Characterized passive filtering in the dead cochlea.
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Organ of Corti
The outer hair cell is the cochlear amplifier
Electromotility& the Cochlear Amplifier
200 msec pulses from a holding potential of –60 mV. Initial pulse is hyperpolarizing and each successive pulse +10 MV from that.
OHC displacements (∆L)
-200 -150 -100 -50 0 50-2.0
-1.5
-1.0
-0.5
0.0
0.5
∆L (µ
m)
V (mV)
Data of Santos-Sacchi, 1992
1. ∆L ≠ f (current)
2. ∆L ≠ f (calcium)
3. ∆L ≠ f (ATPt)
4. ∆L = f (voltage)
E (membrane) ~ 100 x 10-3 / 5 x 10-9
= 20 MegVolts/m
E (lightning) < 10 KVolts/m
The largest electrical gradient is across the membrane
Membrane potential = ΘMembrane thickness = tElectrical gradient = E = Θ/t
Θ
tΘ
E is even greaterat membrane interface
Surface charge and membrane dielectric properties influence polarization
Petrov & Sachs, 2002
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A couple of aspirins will diminish OAEs
8 aspirins will result in ~ 40 dB hearing loss and significant problems with speech discrimination.
Salicylate reduces electromotility
Male (60 yrs) with hearing loss
Myer & Bernstein, 1965
Salicylate blocks otoacoustic emissions - McFaddden & Plattsmier, 1984
Aspirin Ototoxicity
Chlorpromazine (Thorazine™)
• Anti-psychotic• No documented
effect on hearing• Alters membrane
biomechanics
Voltage (mV)
-100 -80 -60 -40 -20 0 20
Cel
l len
gth
(mic
rons
)
45.5
46.0
46.5
47.0
47.5
48.0
Pre-treatmentCPZ
61.0
60.5
60.0
59.5
59.0
58.5
CPZ affects voltage-displacement in isolated cells
-20 0 20 40 60 80
0.0
0.2
0.4
0.6
0.8
1.0
2.0
-ω +ω
Flu
ores
cenc
e
bleaching pulse
E
D
C
B
A
Frac
tiona
l Flu
ores
cenc
e
Time (sec)
C
E
B
D
A
-10 -5 0 5 10 15
2ω
ω=5.35µm
Distance (µm)
Flu
ores
cenc
e
Fluorescence Recovery After Photobleaching
-120 -100 -80 -60 -40 -20 0 20 40 60
2.0
3.0
4.0
5.0
D (
10-9
cm
2 /sec
)
Holding Potential (mV)
V1/2 = -36 mVdx = 12.3 mV
2.18 x 10-9 cm2/sec
4.44 x 10-9 cm2/sec
Voltage-Dependence of lateral diffusion
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Control Salicylate Both Chlorpromazine Control
No consistent change in OHC morphology
The Bilayer Couple Hypothesis and the Outer Hair Cell: Drug-Application
1.0
2.0
3.0
4.0
5.0
6.0
BothChlorpromazineSalicylateControl
D (
10-9
cm
2 /sec
)
Bathing Medium
*
*
n=12
n=12
n=12
n=5
Drug-Dependence of the Diffusion Coefficient
Crenators:•Salicylates•2,4-Dinitrophenol•Bilirubin•Furosemide•Barbiturates
Cup- Formers:•Chlorpromazine•Local Anesthetics
(Lidocaine, Tetracaine, etc.)•Antihistamines
(Bromopheniramine, etc.)•Propranolol•Verapamil•Chloroquine Deuticke, 1968; Sheetz and Singer, 1974
Outward Inward
Amphipath Families The motor-mechanism is located in the cell membrane
1. Electromotility requires prestin – a membrane protein
2. Drugs that alter membrane mechanics alter electromotility
3. Electromotility and lipid lateral diffusion are both dependent on the transmembrane potential
Flat frequency response
Frank et al., 1999
The problem:Normally, membrane capacitance short circuits high frequency potentials resulting in a low pass filter.
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The solution:
Piezoelectricity - the strong coupling of electrical and mechanical energy in the lateral wall results in high frequency charge movements
PiezoelectricityStrong coupling
of electrical polarization & mechanical stress
Pierre & Jacques Curiediscovered
piezoelectricity
Gabriel Lippmannproposes
converse effect
OHC piezoelectricity (mechanoelectrical transduction)
Dong et al., 2002
EdTDdETsS
T
E
ε+=
−=
OHC piezoelectric coefficient is very large
STRUCTURE PE COEFFICIENTQuartz 2.3 e-12 C/N
PZT 400 e-12 C/NCow femur .08 e-12 C/N
OHC 26000 e-12 C/N
Microchamber equivalent circuit
Effect of piezoelectricityon the OHC
Weitzel et al. 2003
• Improved high-frequency responses
• Resonances
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Do bats and whales use PE resonance to echolocate?
Russell & Kossl (1999)
Extracellular µ-domain voltage divider
Resonance in OHC admittance
µEIS
Resonance in OHC Electromotility
Frank et al. 1999
Electromotile response of unloaded OHCs is best fit with a 2nd order resonant system with resonant corner frequencies that are similar to those predicted by PE.
Resonance in basilar membrane
Frequency (kHz)
0 20 40 60 80 100
Nor
mal
ized
BM
vel
ocity
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Electrical SV-STElectrical RWAcoustic
0 20 40 60 80 100
BM
vel
ocity
pha
se (d
egre
es)
-1440
-1260
-1080
-900
-720
-540
-360
-180
0
180
Electrical SV-STElectrical RW
Acoustic
B
A
Grosh, Zheng, de Boer and Nuttall
Dieler et al., 1991
The OHC lateral wall is a composite
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The OHC lateral wall: a self assembling, trilaminate, nanoscale composite
Fractals
Lateral wall - 100 nm
Organ of Corti - 100 µm
Parthenon - 40 M
Holley et al., 1992
• Actin - circumferential• Spectrin - longitundinal• Pillars - radial
The OrthotropicCortical Lattice
Trilaminate Structures
Dieler et al., 1991
Plasma membrane folding Pipette aspiration
Morimoto et al. 2001
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HEK electromotilitymeasured under voltage clamp with AFM
Zhang et al., 2001Mosbacher et al., 1998
V(+)
Electromotility in native HEK 293 cells
Mosbacher et al. 1998
Mechanical
Electrical
Voltage dependentpressure changes in squid axon
Terakawa, 1984
Phospholipids:the forgotten molecules
Don’t forget water
Membrane self assembly
Marrink, Lindahl, Edholm & Mark, 2001
Surface tension
attr
actio
n←
Ener
gy →
repu
lsio
n
Intermolecular distance
warmer
cooler
(
µ,
∂γ=−∂G
)TA
the energy required to increase the surface area of a liquid by a unit amount
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Electrical potential changes γ:Lippmann mercury voltmeter
G. Lippmann, Ann. Phys. 149 (1873) DC Grahame (1947)
Pressure/tension changes inmembranes and an interface
Lippmann equation
Gabriel LippmannNobel Prize, physics 1908
A Gibbs adsorption equation for a polarizable interface,
∂γ=− ∂ −Γ∂µ−σ ∂oAS T E
:
o
-2 )
-2surface charge (Cm )-1: surface tension (Nm )
: electrical potential difference (V): chemical potential (V): temperature ( K ):surface concentration one component (moles m
:interfacial entr
σ
γ
µ
Γ
o
A
E
T
S 2-1opy per unit area (JK m )−
contains the observed relation between surface charge and the ratio of the change in surface tension to the change in electrical potential,
( , )
ο
µ
∂γσ −∂
=TE
Differential tension leads to bending
Petrov & Sachs, 2002
Includes a differential change in surface tension at the two membrane interfaces
Circumferential Ripples and
Electromotility
Active bending in ripples
pillar
Plasma membrane
Spectrin
∆V
cF ∝
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OHC electromotility – voltage induced change in membrane curvature
-200 -150 -100 -50 0 50 100 150
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
∆L (µ
m)
V (mV)
Membrane based bending motor
))()1(
( oo
eff
of VVkT
pk
bfNL −
−=∆
λL
Paul Langevin(1872-1946)
with salicylate
Outer membrane ripples on motile cells: Coincidence or functional roles?
OHC -Dieler et al. 1991
Oscillatoria -Adams et al. 1999
Flexibacter BH3 -Dickson et al. 1980
Trilaminate Walls
Oscillatoria –Adams et al. 1999
OHC
Edge of a Myxococcus xanthus colony - individual bacteria showing adventurous gliding motility, time lapse 600x speed (Kaiser lab website - Stanford).
Adventurous Motility
We are not alone Common MotifsOuter Hair Cell / Gliding Bacteria
Both are cellular hydrostats with turgor pressure Both are vulnerable to aminoglycocidesTrilaminate wall
plasma membrane / outer membranecortical lattice / peptoglycan layer (pillars / TonB)subsurface cisterna / cytoplasmic membrane
Rippled plasma/outer membrane ⇒ excess membrane
Ripple orientation - circumferenential / spiraling
Low cholesterol in membrane / no cholesterol
No f-actin inside the axial core / no cytoskeleton
Electromotility / gliding motility both are blocked by lanthanides
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Prestin - a protein involved in electromotility
Zheng et al, 2000
Has homology with sulfate transporters
Prestin associated nanoscalemovements at acoustic frequencies
Zheng et al, 2000
Small anions also required
Oliver et al., 2001
Transport in a Flat Membrane
Figure 11-9; Molecular Biology of the Cell -1994
Membrane Bending and Transport? Membrane based mechanisms
Iwasa, 2001
Sachs & Woolf, 2003Does not consider the influence of intracellular anions
Protein based Anions and membrane lipids
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Hofmeister series
Clarke & Lüpfert, 1999ClO4 > SCN > I > NO3 > Br > Cl > F > SO4
anion adsorption at membrane interface
Oliver et al., 2001I > Br > NO3 > Cl > HCO3 > F > SO4
Flexoelectricity:coupling of membrane curvature of with the electric field
characterized in biological membranesby Petrov
Todorov, 1993
The flexoelectric effect
pillar
Plasma membrane
Spectrin
∆c
Vm1 Vm2
+≈
L
D
W
Cb
fffcUεεε0
D Cb
o w L
c membrane curvature; f flexoelectric coefficient (dipole); f flexoelectric coefficient (charge);: permittivity of free space; : dielectric constant of water; :dielectric constant of m: : :
ε ε ε embrane
Phosphatidylserine(PS)
Phosphatidylethanolamine(PE)
Phosphatidylcholine(PC)
Sphingomyelin(SM)
Outerleaflet
Innerleaflet
Lateral diffusion
Flip-flop
Plasma membrane as a Liquid Crystal
The bilayer self assembles, cellular machinery adds proteins
Liquid Crystal Nature of BiomembranesProtein and lipid molecules comprising biomembranes
possess dipole moments
Dipoles contribute to the flexoelectric effect• curvature deformation changes membrane polarization
As c is increased, dipoles become more aligned increasing the polarization of the membrane
EP
PE
Hyperpolarization/Elongation:Disorder in the liquid crystal
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Depolarization/Shortening:order & bending
OHC Length Affects Diffusion
Is Prestin Required for Normal Cochlear
Mechanical Amplification ?
Auditory Brainstem Response Exps. Show Mutants have Decreased
Sensitivity in vivo
Distortion Product OtoacousticEmissions Exps. Show a Decrease in Sensitivity for Heterozygous Mice in
vivo
f1
f2
The ABR-DPOAE Combo Platter
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Cochlear Microphonic Amplitude Exps. Suggest Electromechanical Transduction Remains Intact in
Mutants in vivo
0.98
1.0
1.02
1.04
1.06
1.08
1.1
1.12
0 6 93
OH
C+V
esic
le P
ost/P
re S
urfa
ce a
rea
Vesicles number
Control(n=23)
SAL(n=36)
CPZ(n=27)
SAL+CPZ(n=28)
Excess plasma membrane
HEK electromotilitymeasured under voltage clamp with AFM
Zhang et al., 2001Mosbacher et al., 1998
V(+)
Prestin reverses the polarity of native HEK electromotility
AChR transfection(control for transfection)
0 50 100 150 200 250-2.5
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
Dis
plac
emen
t (nm
)
Time (ms)
Prestin transfection
0 50 100 150 200 250
-0.4
-0.2
0.0
0.2
0.4
0.6
Time (ms)
Phase reversal with prestin
Ludwig, et al. 2001
Non-linearcapacitance
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Non-linearcapacitance
requires prestin
Ludwig, et al. 2001
Reduced chloride restores HEK native polarity in prestin
tranfected cells
Native HEK cellKF in pipette
Prestin transfectionKF in pipette
Dis
plac
emen
t (nm
)
Time (ms)0 50 100 150 200 250
-3
-2
-1
0
1
2
3
0 50 100 150 200 250
-1.0
-0.5
0.0
0.5
1.0
1.5
How might prestin reverse membrane tension polarity?
1. A Lippmann Poisson Boltzmann analysis requires the surface charge on the inner leaflet be more positive than the outside
2. A positive charge is consistent with a role for cytoplasmicanions - they become the counterions in the electrical double layer
3. The effectiveness of anions in altering the Lippmann tension is the same as that for OHC plasma membrane capacitance, a Hofmeister series
I- > Br- > Cl- > F- > SO42-
Intensity - invariance
From Recio & Rhode, 2000Modified by Shera, 2001 Anderson, 1971
RC analysis revealsimpossible response Dissecting the lateral wall
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Membrane based electromechanical force transduction
Stereocilia, recent hint of rapid,voltage dependent movement
Synaptic transmission? rapid, intensity invariant
YES! – piezoelectric like, > 50 kHzfeedback results inintensity invarianceof the fine structure of BM mechanics