1

Outer hair cell electromotility

W.E. Brownellbrownell@bcm.tmc.edu

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

2

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.

3

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

4

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

5

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.

6

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

7

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

8

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

9

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

10

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 ∝

11

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

12

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

13

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

14

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

15

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

16

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

17

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