Vol. 168, No. 2, 1990 April 30, 1990
BIOCHEMICAL
Stretch-activated Mario
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 443-450
composite Zoratti*,
ion
Valeria
channels
Petronilli
in Bacillus and
Ildiko
subtilis
Szabo
Centro CNR Fisiologia Mitocondri, Istituto di Patologia Genera/e, Dipartimento di Biologia, Via Trieste 75, 35121 Padova, Italy Received
February
19,
1990
The
presence
of
ion-conducting
subtilis
giant
protoplasts
technique. conductances
with
presence
of substate
values
in
a
exhibited
electrical
phenomena of
presumably
have
the
been
the exist
the
of the
object
they powerful
direct
E.cwli
[4,5]
and
protoplasts
[6,73
exhibit
several
Evidence
has
negative
bacteria
these
[83.
The
pores the
among various
several *To
questions bacteria?
conductances
whom
correspondence
is
known
that
permeation
recent
application
as
“patch-clamp” derived
gram-positive
also
channels closely
in
in the has
related
molecular
substate-rich the
case
begun,
widespread are
species
from
S.faecalis
cytoplasmic
now
how
to
glycerol
the
the
about
proposed
spheroplasts
from
answered:
many
stretch-
bacteria
been
known
Both
to
How
little
as (e.g.1
may reside
How
species,
high-conductance,
be
the
suggesting
giant
had
until
of these
procaryotes?
Cl],
technique
presented
need
the
the of
gram-negative
They
such
obtained
characterization
cycles,
in a manner
of
hypothetical
channels
indicate behaving
of
attention
observation.
been
observations of channels
lower-conductance
membrane.
stretch-activated,
pores.
several
characteristics
in time
membrane
electrophysiological
their
preliminary
outer
largely
of
Inc.
in phenomena
131 allowed
The
Bacillus
patch-clamp
stretch
blocks
Press,
cytoplasmic
remained
The
into
of considerable
of the
the
repeated
building
Academic
to be Involved
i-23, but of
the
and
01990
range.
changed
of
activation
of aggregates
channels to
membrane
using
activity.
giant
porins
channels
nS and
afterwards
channels.
While
the
spontaneous the
the
the
Following
corresponding
activated
caused
levels
manner.
channels decay
in
in
discovered
stretch
the
the
was
Membrane
cooperative
pores
the
of
membrane and
several
are
pores give
gram-
rise
these
found
in
to
the
observed? should
be addressed. 0006-291X/90 443
$1.50
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
168, No. 2, 1996 In this as all
BIOCHEMICAL
communication
gram-positive
membrane.
The
we
report
bacteria, results
AND BlOPHYSlCAL on
offers
provide
our the
a first
RESEARCH COMMUNICATIONS
work
with
8. subtilis,
advantage
tentative
of
which,
having
answer
a single
to the
questions
above. MATERIALS
AND METHODS
Bacillus subtilis ceils were treated as described for S. faecalis 171 to obtain giant (2-3 urn) protoplasts. Patch-clamp micropipettes were drawn from Hllgenberg 11411 glass to a tip dlameter of about 0.5 urn and used without coating. To activate the channels, transmembrane pressure gradients were created by applying suction to the pipette interior by mouth or with a syrlnge. The depression was monitored with a mercury manometer. Data could be obtained in the cell-attached conftguration or from excised patches obtained by wlthdrawing the pipette or upon disintegration of the protoplast. Results did not differ in these cases. Data reported here were collected from excised patches symmetrically bathed in 350 mM KCI, 1OmM CaCi2, 1mM MgC12, 5mM Hepes/K+, pH 7.2. Steady transmembrane voltage gradients were applied and controlled by a List EPC7 unit and the current response due to membrane channel openings was visualized on an oscilloscope. Signal output from the EPC7 current/voltage converter was filtered at 3 KHz (b-pole Bessel, Frequency Devices 902LPF), recorded on a Racal 4 analogical tape recorder and subsequently digitized and processed using an Indec L11/73-70 data acquisition system. Current amplitude histograms were constructed as described in C91. Voltages glven are those of the bath (cytoplasmic side of the membrane), zero being conventionally assigned to the pipette voltage. Currents (cations) entering the bath side (or anions leaving it) in response to a negative applied potential are Conductances stated assigned a negative sign and plotted downward. were calculated assuming ohmic current-voltage relationshlps. RESULTS The
straightforward
membrane
patch
observation
of
however,
upon
interior.
The
patch
and
activation thus
activity
as well
lower
conductance
increased
as wlth
which observed
died
out often
4.3-4.8
Channel of
suction
to the
applied
form
the
or
one
5.7-6.1
was
few
parameters
(upward)
contact
was of nS,
bursts
partially
three but
of
[lo].
In many
of
cases
the
openings the
This current
The ranges, lower
as
towards
of
Increased. discrete
and
same
membrane
drifted
resistance
released.
from
the
the
baseline
area
pipette
determinations.
history
the
intermedlate
the for
later
a the
varied
to another
the
conductance
444
to
high-conductance
because of
Hg)
In
the
in
evident,
differential
suction
of
across
resulted invariably
cm
cycle
was applied,
appearence in
actlvlty
on
a train
gradient
rare1 y
(a
lower
membrane/glass
as pressure fell
of
voltage
pressure
depends
1: as suction
a only
stretch
geometrical
values
the
one
requiring clearly
the
in Fig.
coincided
steps.
from
as on its
assumed
illustrated
formation
also
with
of
seal
application response
response
patch
2.8-3.4,
current
to patch
patch, The
application
after
current namely values
seal
drift spikes, steps about were
Vol. 168, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Fig. 1. Membrane stretch-induced channel activity. The intenslty of the electrical current flowing through the membrane patch is plotted vs. time. Applied voltage, V = -25 mv. The events measure approx. 70 PA (2.8 nS). Continuous trace. An upward drlft of the baseline lndlcates that suction (i.e., membrane stretch) was Increaslng.
also
observed.
In
other
cases,
signals,
due
(Fig.
Rarely,
suction
caused
the
lower
(
BIOCHEMICAL
Stretch-activated Mario
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 443-450
composite Zoratti*,
ion
Valeria
channels
Petronilli
in Bacillus and
Ildiko
subtilis
Szabo
Centro CNR Fisiologia Mitocondri, Istituto di Patologia Genera/e, Dipartimento di Biologia, Via Trieste 75, 35121 Padova, Italy Received
February
19,
1990
The
presence
of
ion-conducting
subtilis
giant
protoplasts
technique. conductances
with
presence
of substate
values
in
a
exhibited
electrical
phenomena of
presumably
have
the
been
the exist
the
of the
object
they powerful
direct
E.cwli
[4,5]
and
protoplasts
[6,73
exhibit
several
Evidence
has
negative
bacteria
these
[83.
The
pores the
among various
several *To
questions bacteria?
conductances
whom
correspondence
is
known
that
permeation
recent
application
as
“patch-clamp” derived
gram-positive
also
channels closely
in
in the has
related
molecular
substate-rich the
case
begun,
widespread are
species
from
S.faecalis
cytoplasmic
now
how
to
glycerol
the
the
about
proposed
spheroplasts
from
answered:
many
stretch-
bacteria
been
known
Both
to
How
little
as (e.g.1
may reside
How
species,
high-conductance,
be
the
suggesting
giant
had
until
of these
procaryotes?
Cl],
technique
presented
need
the
the of
gram-negative
They
such
obtained
characterization
cycles,
in a manner
of
hypothetical
channels
indicate behaving
of
attention
observation.
been
observations of channels
lower-conductance
membrane.
stretch-activated,
pores.
several
characteristics
in time
membrane
electrophysiological
their
preliminary
outer
largely
of
Inc.
in phenomena
131 allowed
The
Bacillus
patch-clamp
stretch
blocks
Press,
cytoplasmic
remained
The
into
of considerable
of the
the
repeated
building
Academic
to be Involved
i-23, but of
the
and
01990
range.
changed
of
activation
of aggregates
channels to
membrane
using
activity.
giant
porins
channels
nS and
afterwards
channels.
While
the
spontaneous the
the
the
Following
corresponding
activated
caused
levels
manner.
channels decay
in
in
discovered
stretch
the
the
was
Membrane
cooperative
pores
the
of
membrane and
several
are
pores give
gram-
rise
these
found
in
to
the
observed? should
be addressed. 0006-291X/90 443
$1.50
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
168, No. 2, 1996 In this as all
BIOCHEMICAL
communication
gram-positive
membrane.
The
we
report
bacteria, results
AND BlOPHYSlCAL on
offers
provide
our the
a first
RESEARCH COMMUNICATIONS
work
with
8. subtilis,
advantage
tentative
of
which,
having
answer
a single
to the
questions
above. MATERIALS
AND METHODS
Bacillus subtilis ceils were treated as described for S. faecalis 171 to obtain giant (2-3 urn) protoplasts. Patch-clamp micropipettes were drawn from Hllgenberg 11411 glass to a tip dlameter of about 0.5 urn and used without coating. To activate the channels, transmembrane pressure gradients were created by applying suction to the pipette interior by mouth or with a syrlnge. The depression was monitored with a mercury manometer. Data could be obtained in the cell-attached conftguration or from excised patches obtained by wlthdrawing the pipette or upon disintegration of the protoplast. Results did not differ in these cases. Data reported here were collected from excised patches symmetrically bathed in 350 mM KCI, 1OmM CaCi2, 1mM MgC12, 5mM Hepes/K+, pH 7.2. Steady transmembrane voltage gradients were applied and controlled by a List EPC7 unit and the current response due to membrane channel openings was visualized on an oscilloscope. Signal output from the EPC7 current/voltage converter was filtered at 3 KHz (b-pole Bessel, Frequency Devices 902LPF), recorded on a Racal 4 analogical tape recorder and subsequently digitized and processed using an Indec L11/73-70 data acquisition system. Current amplitude histograms were constructed as described in C91. Voltages glven are those of the bath (cytoplasmic side of the membrane), zero being conventionally assigned to the pipette voltage. Currents (cations) entering the bath side (or anions leaving it) in response to a negative applied potential are Conductances stated assigned a negative sign and plotted downward. were calculated assuming ohmic current-voltage relationshlps. RESULTS The
straightforward
membrane
patch
observation
of
however,
upon
interior.
The
patch
and
activation thus
activity
as well
lower
conductance
increased
as wlth
which observed
died
out often
4.3-4.8
Channel of
suction
to the
applied
form
the
or
one
5.7-6.1
was
few
parameters
(upward)
contact
was of nS,
bursts
partially
three but
of
[lo].
In many
of
cases
the
openings the
This current
The ranges, lower
as
towards
of
Increased. discrete
and
same
membrane
drifted
resistance
released.
from
the
the
baseline
area
pipette
determinations.
history
the
intermedlate
the for
later
a the
varied
to another
the
conductance
444
to
high-conductance
because of
Hg)
In
the
in
evident,
differential
suction
of
across
resulted invariably
cm
cycle
was applied,
appearence in
actlvlty
on
a train
gradient
rare1 y
(a
lower
membrane/glass
as pressure fell
of
voltage
pressure
depends
1: as suction
a only
stretch
geometrical
values
the
one
requiring clearly
the
in Fig.
coincided
steps.
from
as on its
assumed
illustrated
formation
also
with
of
seal
application response
response
patch
2.8-3.4,
current
to patch
patch, The
application
after
current namely values
seal
drift spikes, steps about were
Vol. 168, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Fig. 1. Membrane stretch-induced channel activity. The intenslty of the electrical current flowing through the membrane patch is plotted vs. time. Applied voltage, V = -25 mv. The events measure approx. 70 PA (2.8 nS). Continuous trace. An upward drlft of the baseline lndlcates that suction (i.e., membrane stretch) was Increaslng.
also
observed.
In
other
cases,
signals,
due
(Fig.
Rarely,
suction
caused
the
lower
(
