General Anaesthetics and Membrane Interactions

P.-L. Chau

Pasteur Institute, Paris

O

H

HH

HOH

Oalphaxalone

O

diethyl ether

Cl C Cl

H

Cl

chloroform

BrHC CF3

Cl

halothane

N

N

O

O

O

H

H

pentobarbital

HC C O CH

F

F

F

F

Cl

F

enflurane

F C CH

O CH

F

F

ClF

F

isoflurane

CF3HC O CH

F

FF

desflurane

F3C CH

O

CF3

CH2

F

sevoflurane

ClHC C O CH3

F

FCl

methoxyflurane

OH

propofol

F.H. Johnson andE.A. Flagler (1951)Journal of Cellular and Comparative Physiology, 37,15-25.

Pressure reversal

• Johnson and Flagler (1950) - reversal of anaesthesia at 130 atm

• Lever et al. (1971) - reversal at 140 atm, Miller et al. (1973) - reversal at about 200 atm

• Dundas (1979) - Royal Navy divers

• disappearance of pressure reversal at about 300 atm

ESR experiments on membrane

• J.R. Trudell et al. (1973) used electron spin resonance spectroscopy

• ESR experiments on spin-labelled phosphatidylcholine and halothane

• changes in membrane structure when [halothane] increased, but when pressure increased, these changes were reversed

Free energy change

• free energy change of inserting halothane into hydrated DMPC

• breakdown into enthalpic and entropic components

• method of energy representation (N. Matubayasi and M. Nakahara (2000) J. Chem. Phys., 113, 6070-6081.)

0

1

-1

2

-2

nm

region I

region II

region III

region IV

hydratedDMPCbilayer

� 20 0 20distance from xy-plane / Å

0

0.01

0.02

0.03

0.04z-

dens

ityz-density of atoms of hydrated DMPC

105 Pa108 Pa2.108 Pa4.108 Pa

region

-30

-20

-10

0

10

20

∆µ /

kJ m

ol-1

free energy change of insertion of halothane

105 Pa2.108 Pa4.108 Pa

I II III IV bulk water

0 1e+08 2e+08 3e+08 4e+08pressure / Pa

-50

-40

-30

-20

-10∆H

or T

∆S /

kJ m

ol-1

enthalpic and entropic changes of insertion of halothane into water

∆HT∆S

Insertion of halothane into hydrated DMPC

• Pressure increase makes halothane less water soluble and ‘drives’ it into the membrane (P.-L. Chau et al. (2012) Mol. Phys., 110, 1461-1467)

• What really happens to halothane inside the membrane at high pressure?

x

y

z

Simulation of pressure reversal

0 10 20 30 40distance from xy plane / Å

0e+00

1e-04

2e-04

z-de

nsity

/ Å

-3distribution of halothane in DMPC at different pressures

phosphorus, 105Paphosphorus, 2.107Paphosphorus, 4.107Pahalothane C, 105Pahalothane C, 2.107Pahalothane C,4.107Pa

No effect in z-direction, but aggregation in x-y plane:

1 atm 200 atm 400atm

Cross-over simulations

• could this be a statistical quirk? confirm by “cross-over” simulations

• take final configurations at 200 atm and 400 atm, and reduce pressure to 1 atm

• re-analyse results

Halothane in POPC

• Repeat simulations but with [halothane] = 2 x [halothane]clinical

• use a different phospholipid for the membrane

-20 0 20 40distance from xy-plane / Å

0

0.001

0.002

0.003ρ(

r)z-density of different atom types

N(POPC)C(hal)1atmC(hal)200atmC(hal)400atm

0

10

20

0 0.5 1 1.5

g(r)

r / nm

105 Pa

2×107 Pa

0

10

20

0 0.5 1 1.5

g(r)

r / nm

105 Pa

4×107 Pa

0

10

20

0 0.5 1 1.5

g(r)

r / nm

105 Pa

2×107→ 105 Pa

0

10

20

0 0.5 1 1.5

g(r)

r / nm

105 Pa

4×107→ 105 Pa

Simulation and experimental results

• halothane aggregates at 200 atm but not at 400 atm (Tu et al. (2012) Chem. Phys. Letts., 543, 148-154)

• previous experiments show that GAs bind as monomers (Jenkins et al. (2001) Journal of Neuroscience, 21, RC136)

Pressure reversal hypothesis

• halothane aggregates at 200 atm; lower monomeric concentration leads to pressure reversal (P.-L. Chau et al. (2007) Chem. Phys. Letts., 438, 294-297; K.-M. Tu et al. (2012) Chem. Phys. Letts., 543, 148-154)

• But is this effect true for other general anaesthetics?

0 10 20

r / Å

0

1

2

3

4

5g

(r)

g(r) of isoflurane C2 in 512 POPC : NPT 100 ns data

p = 105

Pa

p = 2.107

Pa

p = 4.107

Pa

0 10 20

r / Å

0

1

2

3

4g

(r)

g(r) of isoflurane C2 in 512 POPC : NVE, 100 ns data

p = 105

Pa

p = 2.107

Pa

p = 4.107

Pa

Isoflurane does not aggregate

• Isoflurane does not aggregate at 200 atm

• This effect cannot account for pressure reversal

• Recent results suggest involvement of the GABA type A receptor

Acknowledgements

• Tim Forester, Bill Smith and Ilian Todorov (Daresbury Laboratory)

• Ruth Lynden-Bell, Mike Payne and Gábor Csányi (University of Cambridge)