Vol. 88, No. 2, 1979

BIOCHEMICAL

RESEARCH COMMUNICATIONS Pages 433-439

AND BIOPHYSICAL

May 28, 1979

A DIFFERENT KIND OF HEMOCYANIN CHANNEL IN OXIDIZED

CHOLESTEROL MEMBRANES

G. Menestrina Dipartimento Received

March

di

Fisica,

22,

1979

and R. Antolini

Universith

di Trento,

38050

- Povo (Trento)

Italy

SUMMARY

A new kind of hemocyanin channel in oxidized cholesterol black lipid membranes has been characterized using the protein extracted from the mollusc Paludina Vivipara. The channel has constant conductance versus applied voltage and is subjected to open-closed fluctuations that follow fairly well a binomial distribution, independent from the applied potential. Single channel electrical properties are in good agreement with those of many channels membranes. This channel is completely different from that already studied formed by Keyhole Limpet Hemocyanin and for some aspects reminds other channels particularly those obtained with Excitability Inducing Material and Alamethicin. Several channels

works

(1,

through

2, 3) have

black

lipid

membranes.

the interesting

analogies

doped

membranes

and excitable

copper

protein

hemolymph

of a number

hemocyanins obtained

all from

purchased,

of another para, Limpet;

which

million,

is

for

in fact,

as 100s (4),

so far

purified

around

and a hollow

reported

from

that

Limpet

dissolved

abundance

performed

obtained

the blood

structurally

they

cylindrical

We found

Keyhole

transporting

freely

of this

were

of hemocyanin

an oxigen

occurs

out

with

Megatura

in the

of different the protein

Crenulata

and

by Calbiochem.

pH 7, they

means that

which

some results

many aspects

which

35 nm of length.

form,

is

to open

was to point

properties

Despite

of the Giant

we present

hemocyanin,

weight,

of Hemocyanin

main purpose

tissues.Hemocyanin

experiments

work

Their

of invertebrates.

liophilized

the ability

the electrical

molecular

the blood

in

In this

between

of great

described

also

this

to that

interaction

Paludina

weight

state of about

30 nm of external

protein

interacts

Vivi-

of Keyhole

in the aggregation

a molecular

shape with

the

of the mollusc similar

are both have

studying

known eight

diameter

with

and

bilayer

0006-291X/79/100433-07$01.00/0

433

Copyright All rights

@ 1979

by Academic Press, Inc. in any form reserved.

of reproduction

BIOCHEMICAL

Vol. 88, No. 2, 1979

membranes

giving

channel

is

rise

completely

The ability quite

general

state,

property

each

it

of channels,

in the with

but

membranes

of gastropod

also

with

channel

yet

the nature

of the

two cases.

artificial

of hemocyanins

observe

Hemocyanin,

formation

different

to interact

we could

Pomatia

to the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Busycon shows

appears

in the

Canaliculatum individual

now to be a

100s aggregation and @Helix

electrical

characteristics. MATERIALS

AND METHODS

Black lipid membranes were formed between two electrolytic solutions with the usual technique (5) and were comprised of oxidized cholesterol prepared following the procedure of Tien (6) with cholesterin and n-octane Fluka puriss. p.a. Paludina Vivipara Hemocyanin, directly purified from the blood of the at a final concentration of about 30 mg/ml, buffered at pH 7 and animal, stored at -2O'C with 180 mg/ml of Sucrose, was a generous gift of Prof. B. Salvato (Center for the study of Physiology and Biochemistry of Hemocyanins, Padova). Before use, it was dialized overnight at 4'C against buffer TRIS, pH 7.2; after dilution the state of aggregation, the degree of oxigenation and the concentration of the protein was determined by means of an U.V. vis. spectra. Small amounts of a 3 mg/ml stock solution were added to one side of the membrane, to get a final concentration of about 20,ug/ml. The electrolytic solution, 0.2 M KC1 buffered with TRIS, at pH 7.2, was then magnetically stirred for about one minute. In these conditions we could observe few channels for long periods. The electrical set-up was composed of a D.C. battery power supply, two Ag-AgCl electrodes and an electrometer (Keithley mod. 602). The output signal was controlled on an x-t chart recorder and through a low noise amplifier (P.A.R. mod. 113) it was recorded on a magnetic F.M. recorder (Tandberg series 115). The statistical analysis of current fluctuations was carried out subsequently on the recorded traces. To this purpose the signal was sampled through a linear switch (Siliconix DG 201) at a typical frequency of 100 Hz and analyzed by a multichannel (Silena system 27). A scheme of the set-up is given in fig. 1. The statistical analysis gave Gaussian curves (n + 1, when n channels were present in the membrane) corresponding to the possible conductance states of the system. These curves were equally spaced, each space reflecting the single channel conductance, and had an area proportional to the time the system had spent in that conductance state. In the text positive voltages mean that the protein free compartment is positive with respect to the compartment containing the protein. RESULTS AND DISCUSSION Adding solution

small

of a black

amounts lipid

of Paludina membrane

Hemocyanin one can observe,

434

to one of the bathing keeping

membrane

voltage

BIOCHEMICAL

Vol. 88, No. 2, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

X-t

chart I

recorder

I

I

Magnetic

J

tape

Multicharnel a”a’yzer

J-l

w

Pulse

fig.

Block diagram of the of current fluctuations channels.

1

a step

constant,

increase

correspond

amplitude, 70 pS.

wise

of a channel. to fluctuations, fluctuations, observed difference

Excitability

in the

fluctuations.

by these

2, are quite

scale.

jumps

conductance

changes

see (8))

are quite

evident

a mean channel

except

are

call

subjected

already a little

them ON-OFF

membrane membrane

formation

These

for

as a function

and allow

conductance

jumps

to those

we shall

at different

of about

as due to the

current

(7),

at various

are of equal

conductance

similar

conductance

3 shows some traces

which

of channels.

Material

For analogy

channel

formation

these

and closing

Inducing

The single

example,fig.

method

time

steps

may be interpreted

appearance

in fig.

These

the statistical analysis with few hemocyanin

in the membrane

opening

presented with

measured

their

indicating

up used for in membranes

of current.

in conductance

Soon after

generator

set

to an increase

Such increase

100 Hz

of voltage potentials.

potentials.

us to calculate

can be As an

The discrete (for

of 68 f 7 pS, independent

the of the

potential. Informations

on the probability

drown

by the statistical

given

time.

n

In fact

channels

are

distribution if

few channels

open at a given P(n)

Where

f

a number 3 typically)

is

of a single

=

n!

the probability

of different at various

time

N! (N-n)!

of the number are present,

traces,

potentials

to be open can be

of channels

open at a

say N, the probability

that

is: fn(l-f)N-n

of each channel

current

channel

(7) to be open.

in the presence in order

435

to test

We have analyzed

of few channels whether

f, also

(2 or called

BIOCHEMICAL

Vol. 88, No. 2, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

I

1 I

V,=+lOmV

$2 - ON 5 w E 2

'

- OFF

0

------lOOsec

--------*

fig.

2

4

TIME Formation and subsequent ON-OFF fluctuations of a single channel of Paludina Hemocyanin. The discrete conductance jump corresponds to about 73 pS. Protein concentration was 20 pg/ml and salt solution was 0.2 M KCl.

Vi=+30mV 5pA

0 -sosec~--.-.---‘-

_---.

-

.---

1 ’

-5pA

V,=-3omV

-2OpA

I 66pS

k--J

1

60pS

Va=-70mV

74 ps

100sec 0

-.-_~-.-.-____-.-_-_---

-1OpA

-

-2OpA

Va=-9OmV 74,s

69pS

fig.

3

Current fluctuations in oxidized cholesterol membranes modified with Paludina hemocyanin. One can observe the current jumps resulting from the formation of some channels at different membrane potentials. Applied voltage and changes in membrane conductance are reported. Protein concentration was 20 ~g/ml and salt solution was 0.2 M KCl.

436

Vol. 88, No. 2, 1979

BIOCHEMICAL

-40

AND BIOPHYSICAL

a) smgle

0

--80

-40--

RESEARCH COMMUNICATIONS

channel ~~

4.

.-

so

-

I

APPLIED

4

fig.

(mV)

VOLTAGE

Conductance versus applied voltage in the two cases: channel (protein concentration a> single ZOpg/ml) b) many channels membrane (protein concentration 200 pg/ml>. Salt solution was 0.2 M KCl.

TABLE I Comparison between the electrical properties formed by two different kinds of hemocyanins: and Paludin'a Vivipara. Keyhole Single (0.2

Limpet

Paludina

channel conductance M KCl) 200 pS

Single channel exhibits conductance levels Statistical conductance dependent

many

distribution levels

Single (0.2

built is

of time

of

Fraction voltage

voltage

up in this a pure

and that with

open,

is

way (see

of the

an example

in fig.

one,

f = l/2.

The values

a random

deviation

which

not

implies of related

of time open not dependent

Many channels membrane have linear I-V characteristic

a function

binomial

channel conductance MKCl) 70 pS

Single channel fluctuates between open and closed state

Many channels membranes have not linear I-V characteristic

fraction

of the channel Keyhole Limpet

f

applied

voltage.

5) showed

that

that

the channels

act

resulted

always

to the applied

437

between potential.

Histograms the

distribution

independently 0.50

f 0.15

Vol. 88, No. 2, 1979

BIOCHEMICAL

4channels

(PA)

-0

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

----~

IOOsec

a)

4

c)

35

3 1

Channels fig.

Statistical analysis of the time spent by the system in each conductance state: trace analyzed a> current b) photograph of the screen of the multichannel analyzer displaying 5 Gaussian curves corresponding to any possible number of open channels, when 4 channels are present c) histogram obtained normalizing the areas of the curves of part b (solid line) compared with a binomial distribution (dotted line).

5

The fact are not channels membranes

open

that

dependent

both

channel

on the applied

membranes with

single

should

about

protein

and waiting

potential

as is

voltage,

allows

have a linear

I-V

.

4x10' longer)

shown

conductance

in fig.

channels exhibited

a constant

438

us to foresee

characteristic,

(obtained

4.

and fraction

by adding conductance

of time that

open

also

Actually ten times versus

more applied

many

Vol. 88, No. 2, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CONCLUSIONS The channel different

from

formed that

by Paludina

formed

by Keyho>e

stresses

the

differences

systems.

Its

new characteristics

the

different

in the

channels

ii)

As in

remind

cholesterol

f,

of time

open,

channel

(9)

Alamethicin

the case of all

perfect

account

electrical

Hemocyanin

is

Hemocyanin

(2,

properties

it

completely 8).

between

in an intermediate

Table

I

the two

position

between

known:

in oxidized fraction

Limpet

put

already

ON-OFF fluctuations

i)

Vivipara

the Excitability

membranes

channels,

of many channels

Material

channel

(7)

independent

studied

Inducing

of applied

single

membrane

potential

channel

reminds

properties

give

a

properties.

ACKNOWLEDGEMENTS We are greatful to Dr. Sandra manuscript and to Prof. B. Salvato

Gliozzi for for helpful

a critical discussions.

reading

of the

REFERENCES 1.

Pant,

M.C.,

Conran,P.

(1972)

2.

Latorre, R., Alvarez,O., Ehrenstein,G., J. Membrane Biol. 25, 163-182.

3.

Ehrenstein,G.,

4.

Van Holde,K.E., Van Bruggen,E.F.J. (1971) Series (Timasheff, S.N. and Fasman, G.D., Marcel Dekker, New York.

5.

Szabo,G.,

6.

Tien,H.T. (1974) Bilayer 14arcel Dekker, New York.

7.

Bean,R.C. (1973) in: vol. 2, pp. 409-477,

8.

Antolini,R.,

9.

Eldridge,C.A., 264-272.

Lecar,H.

(1977)

Eisenman,G.,

Ciani,S. lipid

J. Membrane

Biol.

8, 357-362.

Espinoza,M.,

Q. Rev. Biophys.

in: Biological Macromolecules eds), Vol. 5, pp. l-53,

(1969)

J. Membrane

membranes

(BLM),

Morowit2,H.J.

G. FEBS letters, (1978)

Biol.

theory

1, 346-382. & practice,

(Eisenman,G.,

ed.)

in press.

Biochem.

439

(1975)

10, l-34.

Membranes. A series of advances Marcel Dekker, New York.

Menestrina,

Reyes,J.

Biophys.

Res.

Commun. 85,