Effects of halothane on channel activity of N-acetyl gramicidin

Vol. 101, No. 3,198l August

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 963-969

14, 1981

EFFECTS OF HALOTHANE ON CHANNEL ACTIVITY R. J.

Bradley*,

OF N-ACETYL GRAMICIDIN

G. Parenti-Castelli'

D. W. Urry+,

and G. Lenaz'

*

Neurasciences Program, Department of Psychiatry and 'Laboratory of Molecular Biophysics, University of Alabama in Birmingham, Birmingham, Alabama 35294. §On leave from the Instituto di Chimica Biologica, Bologna, Italy. Received

June

11, 1981

SUMMARY: The kinetics of channel formation for N-acetyl gramicidin black lipid membranes in the presence and absence of halothane events and by performing spectral analysis of membrane noise. of the general anesthetic reduced the single channel lifetime the single channel conductance.

were measured in by observing single Low concentrations but did not change

INTRODUCTION: A variety tics

affect

of different membrane

layer

and thereby

Other

possibilities

tein

or alter Channel

tion.

are

B:;$-helix

supports

interpretation

cidin

would

form

at the formyl for

dimer

conductance pared

which

that

end would

formation.

Indeed

is

dramatically

to normal

gramicidin

it

(11)

proteins.

bilayers

is a well

some relevance

to real molecule

(3)

membrane forms

by forming

Much experimental proposed

because

the hydrogen

has been shown that

reduced

bi-

lipid

It was also

with

of the lipid

on the pro-

(3).

channel

interfere

anesthe-

directly

tne membrane

two molecules

a more unstable

general

of membrane

act

the gramicidin

across

(4-10).

that

(1,2).

may bear

and dimerizes

ends of the

anesthetics

in artificial

proposed

at the formyl this

the general

by gramicidin

was originally

the behavior

interaction

system

propose the structure

or destabilizing that

formation

essentially by altering

lipid-protein

model

It

stranded

permeability

changing

the

characterized

theories

that

suggesting

that

now

N-acetyl

grami-

the macroscopic

the rate

of dimer

bonds

evidence

which

in the case of N-acetyl-gramicidin

a single

hydrogen

the additional bonding

func-

methyl is

group

required bilayer as com-

formation

is

0006-291X/81/150963-07$01.00/0 963

Copyright I& 198I b-v Academic Press, Inc. AN rights of reproducrion in any form reserved.

BIOCHEMICAL

Vol. 101, No. 3,198l

lower.

In addition

nearly

two orders

was selected nearly the

for

effects

channel

lifetimes

of magnitude

shorter

than

studies

to those

of the general containing

significantly

and in a manner

BIOPHYSICAL

single

our

comparable

of membranes are

the

AND

because values

for

altered

membrane

gramicidin

gramicidin (9).

channels.

added

to the

the

derivative

are more We report

and demonstrate within

are

This

and conductance

halothane

membrane

N-acetyl

gramicidin

lifetime

at concentrations

decreases

for

for

real

anesthetic N-acetyl

that

its

RESEARCH COMMUNICATIONS

bathing that

here

solution

the

kinetics

physiological

dose

range

permeability.

METHODS AND MATERIALS: N-acetyl gramicidin was the same as used in earlier studies (9). Black lipid membranes were formed as previously described (8) on a 0.2 mM diameter aperture separating two teflon chambers each filled with 10 ml of electrolyte containing picomolar concentrations of N-acetyl gramicidin. The lipid solution used to form the membrane consisted of 2% (W/V) diphytanoyl phosphatidylcholine in n-decane. All experiments were carried out at room temperature (23+l°C). Membrane potential was clamped at 100 mV and single events were capturgd on a storage oscilloscope after amplification by a Kiethley 427 current amplifier. For noise analysis, the signal obtained from bilayers containing many channels was digitized at a rate of 1024 points per 400 ms epoch, after low pass filtering (10,000 Hz). The power spectrum was obtained via the fast Fourier transform and averaged over 512 epochs. Each averaged spectrum was fitted by a Lorentzian function of the form S(f) = 2yuT/[1+(2rfT)'] where f is the frequency, p is the mean conductance of the membrane; T is the mean lifetime of the channel and y is the single-channel conductance. In experiments on the effects of halothane, the drug was pipetted directly into both chambers while stirring, during the lifetime of an active bilayer. The chambers were covered to slow down evaporation of the halothane. The stated halothane concentrations are maximal values and are not corrected for evaporation.

RESULTS AND DISCUSSION: The conductance cidin

was similar

value to those

potassium

chloride

sizes

considerably

are

was found

less

It

reduced

the channel

reduced

some 40% by addition times

of the model

values

single

reported

before

channel (9),

and 30 pS in 1 M Cesium chloride

(8,9).

Relaxation

(v)

that lifetime

and single

than

the

the addition

corresponding of

T in a dose

of 1 mM halothane channel

(Figure

halothane

conductances

964

to the

1). for

N-acetyl (pS)

These native

bathing

manner.

gramiin 1 M

channel gramicidin

electrolyte

In 1 M KC1 T was

to the aqueous were

for

26 picosiemens

values

dependent

size

measured

bath from

(Table the

I). power

BIOCHEMICAL

Vol. 101, No. 3,198l

’

Hz

5

”

AND

BIOPHYSICAL

‘I

I”‘.1

IO

50

Hz

100

(Figure

1).

tration

of halothane.

current

The single

lipid

upon the

formed

shorter

therefore is

be thicker

proposed

that

of the membrane

the thus

to the

lipid

chains

decane

there

is very

as well

formed number

alkanes than

as from

conductance

measurements

bilayers

is dependent

noise

channel

Based on capacitance

using

Halothane

Channel fluctuations recorded in the presence of 1 M KC1 with an applied potential of 100 mV. m - The power spectrum of current fluctuations obtained from membranes with many active channels was computed from the fast Fourier transform of 1024 points digitized from a 400 msec epoch. The spectrum was averaged over 512 epochs. Each spectrum could be approximated by a single Lorentzian function. Arrows depict the half-power frequency (f,) and T was calculated from the relationship 'I = l/2 nfc. The control value was f, = 12.9 Hz (T = 12.3 msec) and in 5 mM halothane fc = 38 Hz (T = 4 msec). Bottom - Single channel events under the same conditions as above. The channel lifetimes averaged for many single events were similar to the values computed from the power spectrum. The single channel conductance y, 26 picosiemens (pS), was unchanged by halothane.

1:

of membrane

of planar

50 100

IO

5mM

Control

spectrum

5

200msec

200msec

FIGURE

RESEARCH COMMUNICATIONS

has been proposed

dispersions

of carbon

atoms

such as decane bilayers

shorter

formed

alkanes

tnicltening

it,

are

The

conclusion

little

alkane

present

in the

tetradecane

located

between

longer is

that

alkanes for

in the bilayer

965

the thickness

in N-alkane

solvents

(12-14).

Bilayers

N-alkane

using

recordings

by any concen-

that

of lipids

have a lower

whereas

(15,16).

channel

y was not changed

it

from

single

capacitance

and may

or hexadecane. the are

It

two lipid

layers

aligned

parallel

alkanes

(17,18).

longer

than

The linear

BIOCHEMICAL

Vol. 101, No. 3,198l

AND

BIOPHYSICAL

TABLE Halothane

membrane

is

hexadecane.

12.3

1

K

7.4

5

K

4.0

10

K

2.5

0

CS

21.1

1

CS

12.6

5

CS

4.5

10

cs

2.1

active

This

rate

of channel

which in lipid

gramicidin

anesthetic

properties

thickening

of the membrane

anesthetics

yet

alcohol

of lipid

nor

drug

dimerization. Regardless concentrations

to the

smaller

that bilayer

would is

formed

based

bathing from

open

lecithin

capacitance in these

the addition bathing

significantly

changes,

caused

bilayers

up until

the data

the

a

(18). of

a decrease

in

However,

altered

by

Notwithstanding report,

it

concentrations

had any effect

decrease

is

(20).

this

show clearly

have

addition

is not

(23,24).

of physiological solution

where

in tetradecane

reduced

alkanes

ion channels

electrolyte

the is

of anesthesia

on experiments

of solvent-free

of the mode of action of halothane

destabilize

to

dissociation

As small

basis

the

In fact,

bilayer.

(19). the

across

instability

of channel

alkanes

solubilayers poten-

as compared

increased

decane

that

channel

decane

the

and the rate

to the

is the

and solvent

to be demonstrated

in the thicker

theory

the thickness

(21-23)

anesthetic

which

an ion

using

from

has been suggested

of membranes that

formed

to result

using

of this

benzyl

is reported

the role

it

presentation

the capacitance it

formed

fluctuations in 1M chloride of the power spectrum in in n-decane. The applied

to form

bilayers

is decreased

in bilayers

the anesthetic

dimerizes

dimer

formation

increased

Another

N-acetyl gramicidin the cut-off frequency phosphatidylcholine

has been proposed

of the conducting

T (msec)

K

gramicidin less

(1 M)

0

Time constant (T) for tions calculated from formed from diphytanoyl tial was 100 mV.

pentadecapeptide

I

Ion

(mM)

RESEARCH COMMUNICATIONS

had of an

on gramicidin that

probability

anesthetic of channel

Vol. 101, No. 3,1981

formation lipid

BIOCHEMICAL

by N-acetyl

gramicidin

concentration

as 77 mM which

of halothane would

correspond

AND

BIOPHYSICAL

in these

lipid

required

for

of membrane

capacitance

using

allows

the mode of action

solvent

membrane must

that

and a subsequent

now "dimple"

explanations

Anesthetics

increase

might

the

alter

and an effect Other

theories

around or could

alter

relevant

concentrations

lation

acid

liposomes

results

suggest

surrounding

that

lipids

lipid-protein

It

are

release

required

membrane

proteins

annulus

conformation that

(27) clinically

the rotational

corre-

and mitochondrial

to produce

from

the

lipid

has been found

(1)

which

translocation

the

lower

extracted

possible.

by dimpling.

fluidize

in synaptic

lipids

the anesthetics by integral

in ion

significantly

labels

However,

an effect

an inactive

(28).

lipid

comparable

these

membranes.

immobilization

induced

and thereby

(2)

These on

destabilize

the

interactions.

Anesthetics by inducing

could

also

a conformational

latter

effect

drugs

at the

gramicidin,

has been postulated nicotinic

bonding

known to hinder by halothane

in the

interior

bilayer

increased

rate

must

protein

open channels.

as the

mode of action

for

receptor occur

on the

of dissociation

on the membrane

or by blocking

explain

(20,31,32).

in order

formation

could

effect

(29)

acetycholine

hydrogen is

have a direct change

Such an effect

the

from

involved

the

are

the destabilization

separations

spin

(18,20).

formed

of the

because

(26),

might

as high

in bilayers

environment

them to adopt

concentrations

formed

lipid

anesthetics

of anesthetics

but mugh higher in

that

channel

to form

proteins

are

of 5.9 mM (25).

drugs

membranes

to decrease

causing

phase

of stearic

effects

Halothane

thus

lateral

times

membranes

act

have suggested

such proteins

a channel

of the

be a thickening

of the

of membrane

could

concentration

could

of lipid

Estimates anesthesia

by these

to the

fluidity

behavior

which

for

pertaining the

general

caused

destabilization

more in order

alternative

bilayers.

to an aqueous

The reduction

RESEARCH COMMUNICATIONS

to form

of hydrogen

hydrogen

bonding

the decreased which

bonds

rate

The of uncharged

In the case of N-acetyl the

conducting

in model

dimer.

systems

of two gramicidin

we have measured.

967

a variety

either

of dimerization

(33).

molecules and

BIOCHEMICAL

Vol. 101, No. $1981

Regardless the protein, ing

of the mode of action we have found

electrolyte

channel

system

these

effects

tion

should

This

basis

work

RESEARCH COMMUNICATIONS

of anesthetics,

the

containing

to elucidate

of halothane

ACKNOWLEDGMENT: Grant

help

BIOPHYSICAL

a low concentration changes

in solvent

on the molecular

Health,

that

significantly

formation,

model

AND

the role

and may be expected

on the

of halothane

reaction bilayers.

whether

rates Further

of the

lipid

to provide

lipid

in the bath-

of N-acetyl studies

gramicidin of this

and solvent additional

or

in informa-

of anesthesia.

was supported

in part

by the National

Institutes

of

No. GM-26898.

REFERENCES: 1. 2. i: 2 7. 8. 1:: i:: 13. 14. 15. 16. 17. 18. 19. 20. 21.

Lenaz, G., Curatola, G., Mazzanti, L., Bertoli, E. and Pastuszko, A. (1979) J. Neurochem. 32, 1689-1695. G., Mazzanti, L., Parenti-Castelli, Lenaz, G., Curatola, G. and Bertoli, E. (1978) Biochem. Pharmacol. 27, 2835-2844. Urry, D. W. (1971) Proc. Nat. Acad. Sci. U.S.A. Qs, 672-676. Urry, D. W., Goodall, M. C., Glickson, J. D. and Mayers, D. F. (1971) Proc. Natl. Acad. Sci. U.S. A. 68, 1907-1911. Bamberg, E. and Janko, K. (1977rBiochim. Biophvs. Acta 465, 486-499. Bamberg, E., Apell, H.-J., -Alpes, H. (1977) Pro& Natl. Acad. Sci. U.S.A. 74, 2402-2406. Apell, H.-J., Bamberg, E., Alpes, H., LBuger, P. (1977) J. Memb. Biol. 31, 171-188. Bradley, R. J., Urry, D. W., Okamoto, K. and Rapaka, R. S. (1978) Science 200, 435-436. Szabo, G. and Urry, D. W. (1979) Science 203, 55-57. Weinstein, S., Wallace, B., Blout, E. R., Morrow, J. S. and Veatch, W. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4230-4234. Goodall, M. C. (1971) Arch. Biochem. mophys. 147, 129-135. Haydon, D. A., Hendry, B. M., Levinson, S. R. and Requena, J. (1977) Biochim. Biophys. Acta 470, 17-34. White, S. H. (1977) Ann. N.Y. Acad. Sci. 303, 243-265. Benz, R., Frohlich, O., Lluger, P. and Montal, M. (1975) Biochim. Biophys. Acta 394, 323-334. Andrews, D. M., Manev, E. D. and Haydon, D. A. (1970) Spec. Disc. Faraday Sot. 1, 46-56. McIntosh, T. J., Simon, S. A. and MacDonald, R. C. (1980) Biochim. Biophys. Acta 597, 445-463. Fettifice, R., Andrews, D. M. and Haydon, D. A. (1971) J. Memb. Biol. 5, 277-296. Haydon, D. A., Hendry, B. M., Levenson, S. R. and Requena, J. (1977) Nature 268, 365-358. Hendry, B. M., Utbaw, B. W. and Haydon, D. A. (1977) Biochim. Biophys. Acta, 513, 106-116. Ashcroft, R. G., Coster, H.G.L. and Smith, J. R. (1977) Nature 269, 819820. Franks, N. P. and Lieb, W. R. (1978) Nature 274, 339-342.

968

Vol. 101, No. 3,198l

24. 25. 26. 27. 28. 29.

32. 33.

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH COMMUNICATIONS

Turner, G. L. and Oldfield, E. (1979) Nature 277, 669-670. Ebihara, L., Hall, J. E., MacDonald, R. C., McIntosh, T. J. and Simon, S. A. (1979) Biophys. J. 28, 185-196. Reyes, J. and Latorre, R. (1979) Biophys. J. 8, 259-279. Kaufman, R.D. (1977) Anesthesiology 46, 49-62. Trudell, J. R., Hubbell, W. L. and Cohen, E. N. (1973) Biochim. Biophys. Acta 29J, 321-327. Lee, A. G. (1976) Nature 262, 545-548. Trudell, J. R. (1977) Anesthesiology 46, 5-10. Eyring, H., Woodbury, J. W. and D'ArrGo, J. S. (1973) Anesthesiology 38, 415-424. Adams, P. R. (1976) J. Physiol. 260, 532-552. Bradley, R. J., Dreyer, F., Muller, K.-D., Peper, K., and Sterz, R. (1978) Pflugers Arch. Ges. Physiol. 373, 64. Ogden, D. C., Siegelbaum, S. H. and Colquhoun, D. Nature, in press. Sandorfy, C. (1978) Anesthesiology 4&, 357-359.

969