Electric signals to reveal ion pump function

Biological Research Center of the Hungarian Academy of Biological Research Center of the Hungarian Academy of SciencesSciences

Institute of BiophysicsInstitute of Biophysics

P. O. B. 521, H-6701 Szeged, HungaryP. O. B. 521, H-6701 Szeged, Hungary

András DérAndrás Dér

Electric signals

Crucial role in life functions:

Signal and energy transduction

Nobel prize (1963)

Hodgkin, Huxley, Katz

Signal transduction

Propagation of the nerve impulse

chemiosmosis: P. Mitchell, Nobel prize, 1978

ATP-ase: Boyer, Walker, Skou, Nobel prize, 1997

mitochondrial electron transfer

Energy transduction

Why should we measure electric signals?

Direct information about kinetics, ion specificity

Together with other methods: details of the molecular mechanism is expected to

be revealed

Physicist's approach: atomic level description

- chance to design molecules for biotechnology

How to measure electric signals?

Microelectrode techniques fail for most pump proteins

Patch clamp; Nobel prize 1991: Neher and Sackmann

Alternative methods. Prerequisite: electrically asymmetric sample

1. Surface methodsBLM method (Dancsházy et al., 1976; Bamberg et al., 1980)

SSM method (Fendler et al., 1992)

Advantage: ion specificity

Disadvantage: limited spatio-temporal information

2. Bulk methods

Advantageous for kinetic experiments

Suspension method (Keszthelyi and Ormos, 1980)

Gel method (Dér et al., 1985)

Dried samples (Nagy, 1978; Váró, 1983)

Bacteriorhodopsin

bR plays a model role among ion-transporting membrane proteins

stability, absorption changes, photoelectric effects

polarizerexciting laser 

sample detector transient recorder

computer

trigger

input 1

input 2

amplifier

electrodes

detectormonochromator

monochromatorcont. light source(mirror)

(mirror)

Gel method

Correlation between electric and optical signals

)(~)( txdt

dt i

i

ki

k

Conditions: speed, linearity, insensitivity to geometric details

Bi

Eii rrr

trEqr iiiiE )(

k

kSDki

Bi err

Modeling the electrolyte

0.0 0.5 1.0 1.5 2.0 2.5 3.00

200

400

600

800

1000

1200

1400

bulk

Na+

Cl-

Con

cent

ratio

n (a

.u.)

Time (µs)

Ionic relaxation

Properties of ionic relaxation

1. speed

2. anisotropy (F)

3. linearity (E)

4. insensitivity to geometric details (B,C,D)

Temporal superposition solved

)(~)( txdt

dt i

i

ki

k

ki

kk ttu )()(

How can we use this?

k0 k

1 k2 kkk

n qd 0

kdq q

q

q

…

…

Detection of the 3D electric signals

Dér et al. (1999)

Measurement Model

Testing MD models

The function of a bR molecule

Further application examples of the bulk methods

Cl- pumping

(halorhodopsin, bacteriorhodopsin)

Signal transduction

(Chlamydomonas rhodopsin, squid rhodopsin)

Primary processes of photosynthesis

(plant and bacterial photosynthetic reaction centers)

Bioelectronics - fast photodiode, motion sensitive camera

(bacteriorhodospin)

Acknowledgements

Lajos Keszthelyi

Pál Ormos

György Váró

Rudolf Tóth-Boconádi

László Oroszi

László Fábián

Szeged

Stefka Taneva

Sofia

Sándor Suhai

Nicoleta Bondar

Heidelberg

Walther Stoeckenius

San Francisco