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,