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)