(Received for publication, March 29, 1995; and in revised form, May 22, 1995)
From the
The large mechanosensitive ion channel (MscL) of Escherichia
coli was expressed on a plasmid encoding MscL as a fusion protein
with glutathione S-transferase in an Escherichia coli strain containing a disruption in the chromosomal mscL gene. After purification of the fusion protein using
glutathione-coated beads, thrombin cleavage allowed recovery of the
MscL protein. The purified protein was reconstituted into artificial
liposomes and found to be fully functional when examined with the
patch-clamp technique. The reconstituted recombinant MscL protein
formed ion channels that exhibited characteristic conductance and
pressure sensitivity and were blocked by the mechanosensitive ion
channel inhibitor gadolinium. The recombinant MscL protein was also
used to raise specific anti-MscL polyclonal antibodies which abolished
channel activity when preincubated with the MscL protein.
Mechanosensitive ion channels have been found in organisms of
different phylogenetic origin including animals, plants, fungi, and
bacteria(1, 2, 3, 4) . Although
exclusively documented in patch-clamp experiments, the ubiquity of
mechanosensitive channels suggests that they have important
physiological functions in various types of biological cells.
Increasing evidence indicates that the physiological role of these
channels is to modulate cell responses to mechanical stimuli such as
stretch, contraction, or osmotic
stress(5, 6, 7) . Microorganisms, such as the
enterobacterium Escherichia coli, are constantly exposed to
changes in environmental osmolarity. A recent study by Berrier et
al.(7) demonstrated that the loss of metabolites
following osmotic down-shock was blocked by gadolinium, the well
documented inhibitor of mechanosensitive channels(4) . Patch-clamp studies of giant spheroplasts of E. coli have
revealed the presence of two distinct types of mechanosensitive ion
channels in the bacterial cell envelope: a small weakly anion-selective
mechanosensitive channel (MscS) ( In the present study, we have used a common method for expressing
recombinant proteins in E. coli to produce significant amounts
of purified MscL protein(14, 15) . The recombinant
protein was found to be fully functional when reconstituted into
artificial liposomes and was used to raise polyclonal anti-MscL
antibodies. This work was presented in preliminary form(16) .
Channel activation was normally
achieved by applying pressure steps of -20 to -30 mmHg by
mouth over a period of several seconds every 5-10 s. For rapid
pressure steps ( Single-channel currents were filtered at 1 kHz,
recorded using a patch-clamp amplifier (List Electronics, Darmstadt,
Germany), and digitized at 5 kHz by a computer running WinTida analogue
to digital acquisition software (Heka Electronics, Heidelberg,
Germany). Data files were analyzed off-line using commercial software
or programs written in this laboratory.
Figure 1:
Plasmid map of pGEX1.1
encoding the GST-MscL fusion protein. The expanded region shows the DNA
sequence and corresponding amino acids of the fusion between the two
domains. The thrombin cleavage site with the expected additional
N-terminal amino acid residues of the recombinant MscL protein are
shown. The mscL ATG start codon and corresponding methionine
residue are shown in boldface.
Figure 2:
A, SDS-polyacrylamide gel electrophoresis
protein patterns during purification steps of the GST-MscL fusion
protein from E. coli. B, Western blot using
immunoaffinity-purified polyclonal anti-MscL antibodies. Lanes: 1, total E. coli cells before
induction with IPTG; 2, total E. coli cells after
induction with IPTG; 3, glutathione-Sepharose beads absorbed
material; 4, thrombin cleavage of glutathione-Sepharose beads
absorbed material; 5, purified MscL protein. Numbers on the left indicate positions of molecular weight
markers. Arrows indicate the positions of the fusion protein (FP), GST, and MscL.
Figure 3:
Activation of the purified recombinant
MscL after reconstitution into artificial liposomes. Upper trace shows pressure applied to the interior of the pipette, and the lower trace shows current. The holding potential was -10
mV. C denotes the closed state and O
Activation of the MscL by pressure
ceased following removal of the stimulus as shown in Fig. 3. As
pressure is increased in small steps, the threshold of activation is
crossed, and the channel activated. Furthermore, if the applied
negative pressure is maintained at a constant level, the channel
activity in many patches slowly increased with time (Fig. 3). In
patches where the number of active channels was low, MscL activity
occurred as a burst of single openings followed by a long inactivation
period. Following this inactivation, channel activity was not recovered
either by repeated application of voltage (-40 mV to +40 mV)
or by application of negative pressure up to 200 mmHg.
Figure 4:
A,
pressure sensitivity of the MscL in response to normal pressure
application recorded from an isolated patch of artificial liposome
membrane. Traces are 20-s recordings at a holding potential of
-10 mV from the same patch, as pressure applied to the interior
of the pipette was increased. C denotes the closed state and O
In response to a more rapid (1-2 s)
change in pressure, the MscL exhibited rapid activation, followed by
adaptation (Fig. 5). At present, we are unable to examine this
phenomenon in more detail because of the relatively slow step change in
pressure attainable with our experimental apparatus. This activation
following a rapid change in pressure has also been observed for the
MscS in in situ recordings from giant E. coli spheroplasts(21) .
Figure 5:
Activation of the MscL following rapid
changes in pressure application. Pressure step rise time was
approximately 1 s. Upper trace shows pressure applied to the
interior of the pipette and the lower trace shows currents.
The holding potential was -10 mV. C denotes the closed
state and O
Figure 6:
Current-voltage plot of mechanosensitive
single-channel current amplitude and pipette potential. Recordings of
20-s duration were obtained from isolated patches of liposome membrane
in response to normal pressure application, and amplitudes were
estimated from the current-amplitude histograms. Data are presented as
mean ± S.D. from n patches for the following: +20,
+10, -10, -20 mV (n = 7), +30,
-30 mV (n = 3), and +40, -40 mV (n = 2).
Figure 7:
Effect of gadolinium on mechanosensitive
single-channel currents activated by normal pressure application in an
isolated patch of liposome membrane. Traces are 20-s recordings at a
holding potential of -10 mV from the same patch before and after
perfusion of 10
In this study, we have used a common method of expressing
recombinant proteins in E. coli as fusion proteins with GST (14, 15) to produce substantial amounts of purified
MscL protein. A plasmid expression vector was constructed encoding a
hybrid protein with fusion of MscL to the C terminus of GST separated
by a thrombin cleavage site. Induction of the hybrid gene resulted in
strong expression of the fusion protein followed by a rapid single-step
purification from E. coli cell lysates using
glutathione-coated beads. The recombinant MscL protein was further
purified by mild detergent extraction following thrombin cleavage of
the fusion protein bound to the beads. Proteolytic digestion of the
fusion protein by thrombin resulted in the presence of several amino
acid residues at the N terminus of the recombinant protein that are not
found in the wild-type MscL protein, as confirmed by N-terminal amino
acid sequence analysis. The experiments with the recombinant MscL did
not indicate any major effects of these additional amino acid residues
on channel properties. However, unlike the native MscL protein examined
following gel filtration and reconstitution, which does not exhibit
rectification (9) , at positive pipette voltages a slight
rectification was observed with the recombinant channel. At present, we
are unable to explain this observation, but one possibility may be that
the additional ten amino acids present on the recombinant MscL
interfere with the unidirectional passage of ions through the channel
pore. With these amino acids present, it is unlikely that the
N-terminal portion of MscL plays a major role in the transduction of
mechanical force used for activation of this channel, since the
activation pressures for the recombinant MscL were similar to those
observed for the native protein reconstituted in liposomes(9) . In the present study, we have used the purified recombinant protein
to generate specific polyclonal anti-MscL antibodies which showed
strong reactivity with both fusion protein and MscL in Western blot and
ELISA analyses. When incubated with MscL protein prior to
reconstitution, these antibodies abolished channel activity. These
anti-MscL antibodies should enable us to study MscL location in the
native E. coli cell envelope, as well as to identify
cross-reactive proteins in other organisms. Purified MscL protein
was reconstituted into liposomes and found to be fully functional,
exhibiting characteristic conductance and pressure sensitivity, similar
to that of the native channel(9) . In addition, following
incorporation into liposomes, the recombinant channel was blocked by
the common inhibitor of mechanosensitive channels,
gadolinium(4) , at concentrations similar to those reported to
inhibit the MscL of E. coli following reconstitution of
solubilized native membranes(7, 9) . Gadolinium
appeared to increase the activation threshold of the MscL, suggesting a
partial reversal of the inhibition by pressure. However, since in the
majority of cases the number of channels observed per patch was
relatively high (on average 3 to 6), this result may also reflect that
due to the increased open probability, there is an increased likelihood
of observing those channels in the patch not inhibited by gadolinium. The number of channels present in a particular patch of membrane
appeared to influence the type of activity displayed by the recombinant
protein. Where the number of active channels in the patch was
relatively low(1, 2) , openings occurred as a single
burst followed by long inactivation. In the majority of patches where
the number of active channels was higher, the MscL exhibited sustained
activity, and, furthermore, in many patches channel activity was
observed to slowly increase with time. A similar increase in activity
of the MscL from native E. coli membranes has been observed,
and, typically, channel activity continues to increase with time until
the patch ruptures. ( Activation by
pressure of mechanosensitive channels in E. coli can be
described by a Boltzmann distribution(8) . However, for the
recombinant MscL, in many patches continuous application of pressure
resulted in an increase in channel activity with time, and,
furthermore, where channel number was low, channel inactivation was
observed. Therefore, in these experiments, the Boltzmann distribution
could only be used as an approximate description of the channel
quasi-steady-state activity, since the channels were not truly in an
equilibrium state. However, the results for this apparent pressure
sensitivity of MscL did show that the reconstitution method decreases
the pressure required to activate these channels without altering the
activation profile with respect to pressure. A similar lowering in
activation pressure threshold has been reported for both MscS and MscL
when purified native channels were incorporated into
liposomes(9) . In conclusion, we have used a common method
for expressing recombinant proteins in E. coli to produce
significant amounts of purified MscL protein, and the recombinant
channel isolated was found to be fully functional when reconstituted
into artificial liposomes. Furthermore, the rapid protein purification
method described in this paper will enable us to examine mutagenized
MscL proteins and hence explore the role of specific regions of the
protein molecule involved in the mechanotransduction process.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)with a conductance of
approximately 1 nS (8, 9) and a large nonselective
channel (MscL) with a conductance of 2.5-3.0 nS(9) . Both
ion channels could be reconstituted into artificial liposomes either by
fusing bacterial membrane vesicles (9, 10) or by
reassembly of detergent-solubilized membrane
extracts(9, 11) , without loss of their
mechanosensitive properties. Furthermore, functional reconstitution was
used as an assay for the biochemical isolation of mechanosensitive
channel proteins (11) leading to the recent molecular
identification and cloning of the mscL gene encoding the large
mechanosensitive channel of E. coli(12, 13) .
Materials
E. coli strain DH5 was
from Life Technologies, Inc.; plasmids pGEM11Zf(+) and pGEX-2T
were from Promega and Pharmacia LKB Biotechnology (Uppsala, Sweden),
respectively. Bacto-tryptone and yeast extract were from Oxoid Ltd.
(Hampshire, United Kingdom). The following reagents were purchased from
Sigma: octyl glucoside, Tris, EDTA, lysozyme (L 6876), o-dianisidine, thrombin (T 7009), HEPES, SDS, MOPS, bovine
serum albumin (A2153), phosphatidylcholine (P 3644), and cholesterol.
Isopropyl-1-thio-
-D-galactopyranoside (IPTG) and Tween 20
were obtained from Aldrich. Ampicillin and chloramphenicol were from
Boehringer Mannheim (Mannheim, Germany). All other chemicals were
analytical reagent grade. Glutathione-Sepharose 4B beads and cyanogen
bromide-activated Sepharose beads were purchased from Pharmacia, and
Calbiosorb beads were obtained from Calbiochem-Novabiochem Corp.
Bacterial Strains, Plasmids, and Culture
Conditions
E. coli strain DH5 was used as the host
for recombinant plasmids and E. coli strain AW737-KO, carrying
a chromosomal insertion in the mscL gene(12) , was
used for the protein expression experiments. Plasmid p5-2-2 carrying
the entire open reading frame (ORF) of mscL on a XhoI
DNA fragment, generated by the polymerase chain reaction method, was
described previously(12) . Plasmids pGEM11Zf(+) and
pGEX-2T were used for generating pGEX1.1. Bacterial cells were grown at
37 °C in Luria-Bertani broth (10 g/liter Bacto-tryptone, 5 g/liter
yeast extract, 5 g/liter NaCl) with 50 µg/ml ampicillin for cells
carrying plasmids and 20 µg/ml chloramphenicol for the mscL knock-out E. coli mutant, added when required.
DNA Preparation, Manipulation, and
Analysis
Plasmid DNA was extracted from E. coli cells
using the alkaline lysis method (17) . Standard techniques (18) were used for the generation of recombinant plasmid
constructs described under ``Results.'' Restriction enzymes
and DNA ligase were purchased from Promega and used as specified by the
manufacturer. DNA restriction fragments were electrophoresed in
horizontal 0.8% agarose gels in 40 mM Tris, 1 mM EDTA, pH 8.0, and stained with ethidium bromide (0.5 µg/ml).
DNA fragments were excised from 1% low melting agarose gels (Promega),
melted at 55 °C, and used directly in ligation reactions.Protein Purification and Analysis
Recombinant
fusion protein was purified essentially as described
previously(14, 15) . Bacterial cells harboring the
plasmid pGEX1.1 were subgrown for 1 h at 37 °C (1 ml of an
overnight culture in 20 ml of broth), and fusion protein gene
expression was induced for 3 h with 0.1 mM IPTG. The cells
were harvested, resuspended in 5 ml of 150 mM NaCl, 1
mM EDTA, 50 mM Tris, pH 8.0, and lysed by addition of
lysozyme (0.1 mg/ml) and detergent (1.5% octyl glucoside). After bath
sonication for approximately 60 s (Unisonics Pty. Ltd., Sydney,
Australia), cell debris was pelleted (16,000 rpm, 20 min; J2-MI,
Beckman), and 0.5 ml of glutathione-Sepharose 4B beads were added to
the supernatant for 1 h at room temperature (20-22 °C). The
beads were then washed at least three times (by centrifugation using a
desk top centrifuge at 4,000 rpm for 5 min) in phosphate-buffered
saline (PBS) (137 mM NaCl, 2.7 mM KCl, 4.3 mM NaHPO
, 1.4 mM KH
PO
, pH 7.2, adjusted with NaOH) and
resuspended in PBS containing an additional 150 mM NaCl, 2.5
mM CaCl
, and 50 mM Tris. Thrombin was
added to a final concentration of
1 unit/µg of protein and
incubated for 1 h at room temperature. Octyl glucoside in PBS was added
to a final concentration of 1% (w/v) for 10 min, the beads were
pelleted (4,000 rpm, 5 min), and the supernatant was found to contain
the MscL protein. For reconstitution into artificial liposomes and
antibody generation, purified MscL protein was dialyzed for 16 h
against 4 liters of 10 mM Tris, 1 mM EDTA, pH 7.5,
with Calbiosorb beads to remove any octyl glucoside, and the protein
was concentrated in Amicon filtration units (Amicon, Inc.). Protein
concentrations were determined using the D
protein assay
(Bio-Rad), and protein samples were analyzed by 12% SDS-polyacrylamide
gel electrophoresis as described(18) . The N-terminal amino
acid sequence of the purified recombinant MscL protein was analyzed at
the University of Western Australia Centre for Molecular Biology, using
the Edman degradation procedure.
Reconstitution of Recombinant Proteins in Artificial
Liposomes
Liposomes were prepared using a method similar to that
described previously(9, 10) . Briefly,
phosphatidylcholine with 10% cholesterol was dissolved in chloroform.
Small aliquots of lipid were dried under nitrogen, resuspended in 5
mM Tris, pH 7.2, and bath-sonicated for 15 min. The liposomes
were collected by ultracentrifugation (TL-100, Beckman Instruments) at
105,000 g for 1 h and resuspended in 10 mM
MOPS, 5% ethylene glycol, pH 7.2. Purified protein was added at the
desired protein:lipid ratio. Aliquots of the liposomes were spotted
onto glass slides and allowed to dehydrate for several hours followed
by overnight rehydration (200 mM KCl, 5 mM HEPES, pH
7.2) under humid conditions.
Production and Purification of MscL-specific Polyclonal
Antibodies
Two female New Zealand albino rabbits were prebled
and injected with approximately 100 µg of purified MscL protein in
TiterMax adjuvant (Vaxcel, Inc., Norcross, GA). Antibody titers were
checked after 3 and 6 weeks by Western blot analyses essentially as
described(18) . Briefly, protein samples were electrophoresed
on 12% SDS-polyacrylamide gels and transferred to nitrocellulose in
transfer buffer (20 mM Tris, 150 mM glycine, 20%
methanol). The filters were reacted with rabbit antibodies (diluted
1:1100 for whole sera, 1:50 for affinity-purified antibodies),
incubated with horseradish peroxidase-conjugated goat anti-rabbit
immunoglobulin G (Sigma) (diluted 1:1,000), and developed with
substrate solution (0.2 mMo-dianisidine, 0.01%
HO
in 10 mM Tris, pH 7.4).
MscL-specific antibodies were purified by coupling purified MscL
protein to cyanogen bromide-activated Sepharose beads with subsequent
washes and elution conditions as described previously(19) .
Affinity-purified antibodies were dialyzed against 100-fold diluted PBS
and then concentrated 100-fold by lyophilization (Dynavac, Pty. Ltd.,
Sydney, Australia). For enzyme-linked immunosorbent assay (ELISA), the
wells of microtiter plates were coated with various concentrations of
purified MscL protein in PBS overnight at 4 °C. The coated plates
were then washed in PBS with 0.02% Tween, blocked with 2% bovine serum
albumin in PBS for 2 h at room temperature, washed again, and reacted
with antisera (diluted 1:1,000 for whole sera, 1:100 for
affinity-purified antibodies) at 4 °C overnight. The plates were
washed again and incubated with peroxidase-labeled secondary antibody
(diluted 1:5000) for 2 h at room temperature. Substrate was added, and
absorbance at 410 nm was measured on a microplate reader (Series 700,
Cambridge Technology, Inc., Watertown, MA).
Electrophysiological Recordings
The improved
patch-clamp techniques of Hamill et al.(20) were used
to record single-channel currents from isolated membrane patches.
Pipettes were made from borosilicate glass (Drummond Scientific Co.,
Broomall, PA) using a Flaming/Brown Micropipette puller (P-87, Sutter
Instrument Co., Novato, CA) and pulled to a diameter which gave bubble
numbers of 3.2-3.5 in 100% ethanol, corresponding to pipette
resistances in the range 6.4-5.3 M, respectively, when in
recording solution. Pipettes were coated with clear nail enamel (Super
Shine Top Coat, Sally Hansen, North Ryde, Australia) and filled with
recording solution (200 mM KCl, 40 mM
MgCl
, 5 mM HEPES, pH 7.2 adjusted with KOH). A
small aliquot (1-2 µl) of rehydrated liposomes was placed in
the 0.5-ml patch-clamp chamber containing recording solution, and the
chamber was situated on an inverted phase contrast microscope (IMT-2,
Olympus Optical Co., Tokyo, Japan). The reference electrode was a
Ag/AgCl pellet separated from the bath by an agar bridge (2% agarose in
1 M KCl). Pipettes were positioned with a Leitz
micromanipulator (Ernst Leitz Wetzlar GmbH, Wetzlar, Germany) and were
touched against unilamellar blisters arising spontaneously from the
liposomes as reported(9, 10) . Seals (>20 G
)
either formed immediately or following application of a brief
(1-2 s) pulse of negative pressure (<50 mmHg), applied to the
interior of the patch pipette.
1 s), suction was applied by mouth in a single step
from 0 mmHg to a pressure exceeding the activation threshold for that
particular patch.
Construction of a Plasmid Encoding a GST-MscL Fusion
Protein
The XhoI DNA restriction fragment containing
the entire ORF of mscL from plasmid p5-2-2 (12) was
first subcloned into plasmid vector pGEM11Zf(+) in the desired
orientation as determined by restriction enzyme analysis. The gene was
then excised and cloned into the expression vector pGEX-2T as a BamHI-EcoRI DNA fragment (now named pGEX1.1), thus
generating a continuous ORF with the GST gene (Fig. 1). This
genetic construct should lead to the production of a 41-kDa hybrid
protein consisting of a N-terminal GST portion (26 kDa) and a
C-terminal MscL portion (15 kDa) separated by a thrombin cleavage site.
Upon thrombin cleavage of the fusion protein, nine amino acids as well
as the initial methionine residue, which are not present in purified
wild-type MscL(12) , are expected to be present on the N
terminus of the recombinant MscL protein (Fig. 1). N-terminal
protein sequence analysis of the purified MscL protein confirmed the
presence of these ten amino acid residues.
Purification of the GST-MscL Fusion Protein
Upon
IPTG induction of E. coli cells carrying the plasmid pGEX1.1,
encoding the GST-MscL fusion protein, a major protein band of
approximately 40 kDa appeared in SDS-polyacrylamide gel electrophoresis
analysis (Fig. 2A, lane 2). The induced fusion
protein was purified in a single step by addition of
glutathione-Sepharose beads to E. coli cell lysates. Analysis
of the material absorbed by the glutathione-Sepharose beads revealed a
single major protein band of approximately 40 kDa (Fig. 2A, lane 3). After incubation of the
Sepharose-bound fusion protein with thrombin, two additional protein
bands of 26 and 17 kDa were generated, presumably representing GST and
MscL, respectively (Fig. 2A, lane 4).
Following thrombin digestion, the MscL channel protein was further
purified by removal of the GST portion of the fusion protein with the
beads fraction (Fig. 2A, lane 5). The
molecular weight of these protein bands corresponds well with those
predicted from the DNA sequence.
Generation and Effect of Anti-MscL Polyclonal
Antisera
Polyclonal anti-MscL antisera were generated by
injecting purified MscL protein into rabbits. Both animals showed
significant anti-MscL titers several weeks after injection, as
determined by Western blot and ELISA analyses of the collected sera.
MscL-specific antibodies were purified by affinity chromatography and
were used in the Western blot shown in Fig. 2B. Both
fusion protein and MscL showed strong reactivity with the antibodies (Fig. 2B, lanes 3 and 5), whereas no
significant reactivity was observed with any other E. coli proteins or GST (Fig. 2B, lanes 1 and 4). However, in E. coli cells strongly expressing the
fusion protein, several smaller molecular weight protein bands reacted
with the antibodies (Fig. 2B, lane 2), which
presumably represent proteolytic degradation products of the fusion
protein. Immune reactions of pre-sera, sera following MscL injection,
and affinity-purified anti-MscL antibodies, against purified MscL
protein were also examined in ELISA analysis. Pre-sera showed no
significant reactivity with MscL protein, whereas immune blood
exhibited strong reactivity; about half of the antibody titer was
recovered after purification over a MscL-affinity column (data not
shown).Electrophysiological Recordings of Reconstituted MscL
Protein
Purified MscL protein was reconstituted into liposomes
(protein:lipid ratio of 1:6000) and observed to be functional when
examined with the patch-clamp technique (Fig. 3). Single-channel
currents were recorded from excised patches of liposome membrane at
pipette potentials ranging from -40 mV to +40 mV and
pressures ranging from -50 to -200 mmHg. No channel
activity was observed in excised patches from liposomes not containing
MscL protein (n = 5 patches). In addition, no activity
was observed in patches from liposomes containing either GST-MscL
fusion protein (n = 8 patches; protein:lipid ratio of
1:3000) or GST protein alone (n = 4 patches;
protein:lipid ratio of 1:5900).
denotes the open state of n number of
channels.
Pressure Sensitivity of the MscL
Channels were
activated when negative pressure (suction) exceeded a threshold,
typically in the range of 50 to 100 mmHg. As the amount of applied
pressure increased, channel activity and hence channel open probability
also increased (Fig. 4A). We have used a Boltzmann
distribution curve to describe the apparent pressure sensitivity of the
reconstituted MscL protein. The open probability of the channels in a
particular patch was plotted against the applied suction, and the data
were fitted to a Boltzmann distribution (Fig. 4B). For
reconstituted MscL, an e-fold change in open probability was observed
following a change of 4.9 ± 1.4 mmHg (mean ± S.E., n = 3 patches) at a pipette potential of +10 mV, and 3.9
± 1.1 mmHg at a pipette potential of -10 mV (n = 2 patches). The average applied negative pressure
required to induce half-maximal activation of MscL was 72 ± 3
mmHg (n = 3 patches) at a pipette potential of +10
mV, and 71 ± 6 mmHg (n = 2 patches) at a pipette
potential of -10 mV.
denotes the open state of n number of channels. B, effect of pressure on the open
probability of the large mechanosensitive channel. Data are from the
same patch as in A. The open probability was estimated from
the area of the amplitude histograms (giving NP
)
divided by the maximum number of channels observed in this particular
patch (i.e. n = 3, see panel A, -70 mmHg
trace). The curve is a Boltzmann distribution relating applied
(negative) pressure and open probability, fitted by nonlinear
regression. The Boltzmann distribution has the form: where P
is the channel open probability, p is
the applied suction (mmHg), p is the suction (mmHg) at which
the channel is open half the time, and 1/S
is the slope of the plot of
ln[P
/(1 - P
)] versus suction. From this distribution, the sensitivity to
pressure of the channels in this particular patch was estimated to be
5.0 mmHg per e-fold change in open probability, and the pressure
required for half-activaction (where P
=
0.5) was estimated to be 65.2 mmHg.
denotes the open state of n number of channels. Note, there are up to 14 active channels
in this particular patch.
Conductance Measurements of Reconstituted MscL
The
conductance of the purified MscL was estimated from the amplitude of
the single-channel currents and the applied pipette voltage (Fig. 6). The MscL showed slight rectification at positive
pipette voltages. The conductance at negative potentials using a linear
regression fit to the data was 3,500 pS and, for positive potentials,
was 3,300 pS, with the reversal potential close to zero as expected for
this nonselective ion channel(9) .
Inhibition of the MscL by Gadolinium
In the
present study, reconstituted MscL was inhibited by gadolinium in a
reversible manner (Fig. 7). Complete blockade of channel
activity by 1.0 mM gadolinium was observed even at negative
pressures up to 150 mmHg. At a lower concentration (0.2 mM),
inhibition by gadolinium was still observed; however, this inhibition
could be reversed by increasing the applied pressure (data not
shown(9) ).
bath volume of 0.2 mM gadolinium and
following washout with 10
bath volume of control recording
solution. Pressure applied to the interior of the pipette for the
duration of each recording was -65 mmHg. C denotes the
closed state, and O
denotes the open
state of n number of channels.
Effects of Antibodies in Patch-Clamp
Experiments
Liposomes containing either purified MscL or MscL
which had been preincubated in a 1:1 molecular ratio of
affinity-purified anti-MscL antibodies for 1 h, were examined. In 11 of
the 16 patches examined containing MscL alone, single-channel currents
were observed under standard conditions of voltage and pressure.
However, in 16 patches examined where MscL was preincubated with
anti-MscL antibody, no single-channel opening events were observed at
pressures up to -200 mmHg.
)Taken together, the results suggest
that upon application of negative pressure there may be cooperativity
between MscL molecules, either with regard to activation or association
of the channel monomers with one another. It is tempting to speculate
that the possible mechanism of MscL activation by lipid bilayer tension
consists of assembly of pore-forming multimers from dispersed channel
monomers in response to mechanical force. A multimeric form (possibly a
tetramer) of the functional channel (9) is suggested from the
observation that the native channel purified from the E. coli cell envelope has an approximate molecular mass of 60-80 kDa
compared to that of the monomer of 15 kDa. A possible indication of an
association mechanism for MscL activation comes from the results of the
polyclonal antibody experiments. When mixed with the channel protein
prior to incorporation into liposomes, anti-MscL antibodies prevented
any channel activity from being observed. However, another more trivial
explanation for the effect of the antibodies may simply be that the
MscL protein-antibody complex may not insert into the lipid bilayer in
a way which allows channel activation by pressure. Further evidence for
interaction between functional MscL derives from the observation that
the recombinant MscL was more responsive to rapid steps in pressure
than in response to a gradual increase in stimulus. A similar
phenomenon has been reported for mechanosensitive channels of hair
cells of the turtle and stretch-activated channels of Xenopus oocytes(22) . A possible physiological role for this rapid
activation may be in providing part of a defense mechanism against
rapid changes in osmotic pressure(7) .
-D-galactopyranoside; GST, glutathione S-transferase; PBS, phosphate buffered saline; ELISA,
enzyme-linked immunosorbent assay.
We thank Dr. C. Kung and Dr. P. Blount, University of
Wisconsin-Madison, for helpful discussions and the generous donation of
the E. coli strain AW737-KO and mscL carrying plasmid
p5-2-2. We would also like to thank Dr. B. Chang for the donation of E. coli strain DH5 and plasmid pGEM11Zf(+), and Dr.
T. Ratajczak for the donation of plasmid pGEX-2T, both of The
University of Western Australia.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.