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J Biol Chem, Vol. 275, Issue 2, 1015-1022, January 14, 2000
,,§,
From the Laboratoire des Biomembranes, Unité
Mixte de Recherche CNRS 8619, Bâtiment 430, Université
Paris-Sud 91405 Orsay Cedex France and the ¶ Department of
Pharmacology, University of Western Australia, Nedlands,
Western Australia 6907, Australia
 |
ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
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MscL is a mechanosensitive channel that is gated
by tension in the membrane bilayer alone. It is a homo-oligomer of a
protein comprising two transmembrane segments connected by an external loop, with the NH2 and COOH termini located in the
cytoplasm. The contributions of the extramembranous domains of the
channel to its activity were investigated by specific proteolysis
during patch-clamp experiments. Limited proteolysis of the COOH
terminus or the NH2 terminus increased the
mechanosensitivity of the channel without changing its conductance.
Strikingly, after cleavage of the external loop of each monomer, the
channel was still functional, and its mechanosensitivity was increased
dramatically, indicating that the loop acts as a spring that resists
the opening of the channel and promotes its closure when it is open.
These results indicate that the integrity of most of the
extramembranous domains is not essential for mechanosensitivity. They
suggest that these domains counteract the movement of the transmembrane
helices to which they are connected, thus setting the level of
sensitivity of the channel to tension.
Mechanosensation and mechanotransduction, the processes by which
mechanical force is detected and transduced into electrical and
chemical signals by living cells, are at the basis of the physiology of
osmoregulation, touch, hearing, proprioception, as well as detection of
wind and gravity by plants. Since their discovery by patch-clamp
experiments (1, 2), mechanosensitive ion channels
(Msc)1 have been hypothesized
to play a major role in these processes. These channels gate in
response to changes in membrane tension and are present in animal cells
as well as in plant cells and bacteria (3-7). The molecular
identification of these channels proved to be difficult, and their
mechanism is not understood. The situation in this ion channel field
has thus been in sharp contrast with that known for voltage-gated or
ligand-gated channels for which a wealth of information is available today.
High conductance ion channels that are stretch-activated are present in
Gram-negative and Gram-positive bacteria (8) and in Archaea (9). They
have been proposed to catalyze the efflux of osmolytes and potassium
upon osmotic down-shock (10-13). In Escherichia coli,
patch-clamp experiments have revealed stretch-activated conductances
ranging from 100 to 1,500 picosiemens (in 0.1 M KCl). By
order of increasing conductances, three families of Msc can be
distinguished: MscM (M for mini), MscS (S for small), and MscL (L for
large) (14).
Prokaryotic Msc can be solubilized in detergent and functionally
reconstituted in giant liposomes amenable to patch-clamp recording.
This property has allowed purification of MscL, the channel of the
highest conductance, and cloning of its corresponding gene,
mscL (15). Expression of the mscL gene was shown
to be necessary and sufficient for the activity of this channel
(15-17). The gene encodes a 15-kDa small protein of 136 residues.
Cross-linking studies (17) and two-dimensional crystallization of MscL
at low resolution (18) suggested that the functional channel is a
homohexamer. Recently, three-dimensional crystals of the MscL homolog
from Mycobacterium tuberculosis were obtained (19), allowing
a determination by x-ray crystallography of its structure in the closed
conformation to 3.5 Å resolution. In the crystal, the channel is
organized as a homopentamer. Each subunit comprises two transmembrane
segments connected by an external periplasmic loop, with the
NH2 and COOH termini located in the cytoplasm. The closed
pore is lined by the five M1 helices (on the NH2-terminal side) tilted at an oblique angle with respect to the membrane. The M2
helix returns to the cytoplasm along the outside of the closed pore.
The COOH-terminal cytoplasmic domain consists of helices packed
together to form a helical bundle. Because MscL is the first purified
protein with unambiguous mechanosensitive activity, whose gene is
available and whose structure is known, it becomes a model system to
study the molecular mechanism of mechanosensation. It is a very simple
system: an oligomer of a small protein which is able to change its
conformation in response to a variation in membrane tension. However,
despite this simplicity, its molecular mechanism is still unknown.
In this study, we have examined the contributions of the
NH2 and COOH termini and the external loop of the channel
to its mechanosensitivity. This was achieved by proteolysis of these extramembranous domains (Fig. 1) during
patch-clamp experiments performed on the native membrane or in
liposomes in which the purified protein was reconstituted. We could
demonstrate that limited proteolysis of either the COOH terminus or the
NH2 terminus increased the mechanosensitivity of the
channel. Importantly, the channel remained functional after cleavage of
the only external loop of each subunit, such that its
mechanosensitivity was increased dramatically. The results are
interpreted within the framework of a molecular model for the
gating mechanism of MscL.
Preparation of Giant E. coli Protoplasts--
Giant E. coli protoplasts for which the plasma membrane is accessible to a
patch electrode were prepared form E. coli lpp ompA Preparation of Giant Proteoliposomes--
Recombinant MscL
proteins and mutant ( Electrical Recording--
Single-channel activity was measured
using the methods of Hamill et al. (22). Patch electrodes
were pulled from Pyrex capillaries (Corning code 7740) and were not
fire polished before use. Whether using cells or proteoliposomes,
recordings were performed in the excised patch mode, and the internal
face of the membrane patch could be superfused by solution coming from
the outlet of a manifold connected to a series of five piped inlets,
allowing easy change of solution. The flow rate of the solutions was
50-100 µl/min. Because a flow of solution can sometimes activate the
channels by itself, the patch was always superfused with a control
solution before application of protease. Trypsin (tosylphenylalanyl
chloromethyl ketone-treated), chymotrypsin
(N
Open probability Po multiplied by the unknown
number N of channels in a given patch, versus the
applied suction, were fitted with a Boltzmann distribution of the
form
For the native membrane as well as for proteoliposomes, the convention
for the membrane potential is the same and assigns zero level to the
pipette. The contents of all the pipette, bath, and perfusion solutions
are given in the figure legends.
Effects of Proteases on MscL Channels in the Native
Membrane--
The patch-clamp experiments were performed on giant
E. coli protoplasts (Fig. 6B in Ref. 14). After
excision, the inside-out patches were superfused with a control
solution similar to the bath solution. Negative pressure (suction) was
raised progressively until the activation of the MscL channels and was
then kept constant. The control solution was then exchanged for a
solution containing a protease. As shown in Fig.
2, the superfusion of the patch by trypsin (250 µg/ml) led to a sudden increase in channel activity at
constant pressure. However, the channels remained mechanosensitive and
closed immediately upon release of pressure. This effect was observed
in four experiments. A similar effect was observed when the patch was
superfused with chymotrypsin (300 µg/ml) (n = 2) (data not shown). In both cases, the activation by proteases did not
modify the unit conductance of the channels. When we attempted to study
the effect of proteases on the periplasmic side by adding them to the
pipette solution, the seals were highly unstable and were usually lost
upon application of pressure so that no meaningful results could be
obtained (but see below).
Effects of Proteases Applied in the Bath on Purified MscL
Reconstituted in Liposomes--
The MscL proteins alone form the ion
channel (15-17). However, it cannot be ruled out that in the plasma
membrane it may interact with other proteins which could modulate its
sensitivity. To examine whether the effects described above are caused
by the action of the proteases acting on the channel itself or on other
associated proteins, we turned to a reconstituted system. The purified
MscL was reconstituted in liposomes which were fused, by
dehydration-rehydration, into giant liposomes amenable to patch-clamp
recording. A first concern in such studies is the orientation of the
protein in the pure lipid bilayer compared with the native system. The
MscL channel is weakly voltage- dependent. In the plasma membrane, at a
given applied pressure, the channel tends to be more open at low
positive potentials than at low negative potentials (14). A first hint that the proteins were reconstituted with the right side orientation came from the observation of a voltage dependence of the same polarity
in liposomes as in the native membrane (not shown). This was confirmed
further by the experiments described in this paper.
After excision from a giant liposome, superfusion of the patch by
trypsin (250 µg/ml) resulted in an effect similar to that described
above for patches performed on the plasma membrane (n = 13). Examination of the amino acid sequence and topology of the MscL
monomer indicated that sites sensitive to trypsin are present in both
amino and carboxyl termini (Fig. 1). To test whether proteolysis of the
COOH terminus modifies mechanosensitivity, we examined the effect of
carboxypeptidase. As shown in Fig.
3A, superfusion of
carboxypeptidase Y (500 µg/ml) first led to a transient decrease in
channel activity (marked by an asterisk on the figure), which was systematically observed, followed by a rapid and significant increase in the channel activity (n = 12). There was no
change in unit conductance. Importantly, independent of the length of time during which carboxypeptidase was superfused on the patch (up to 5 min), no loss of channel activity was ever observed. Similarly, as
shown in Fig. 3B, superfusion of the patch by chymotrypsin (300 µg/ml) led to a rapid increase in the activity of the channels, at constant applied pressure, without a change in unit conductance (n = 11). The potential chymotrypsin cleavage sites
(aromatic residues) are present in the NH2 terminus (Fig.
1). However, hydrophobic residues, which are weaker potential targets
for chymotrypsin, are present in the COOH terminus. We therefore tested
the effect of aminopeptidase. As shown in Fig. 3C,
superfusion of the patch by aminopeptidase (500 µg/ml) also led to an
increase in the channel open probability at constant applied pressure
(n = 6). Taken as a whole these experiments indicate
that limited proteolysis of either the COOH or the NH2
terminus of the channel increased its sensitivity to pressure. In all
cases, the channels remained mechanosensitive and could be closed and
reopened at will upon decrease or increase of applied pressure.
Previous studies led to the conclusion that most of the COOH-terminal
extension plays no role in mechanosensitivity. Indeed, a mutant
(
We examined the effect of proteases on channel open probability
versus applied pressure described by a Boltzmann
distribution. In general, the pressure required for half-activation
(where Po = 0.5) varied from patch to patch. The
open probability was thus first determined before protease treatment,
by application of pressure increased stepwise every 15 s. The
protease was then superfused on the gating channels for 5-7 min, the
patch was washed with control solution, and increased pressure was
applied again for the open probability determination (Fig.
4, A, B,
C, and F). In control conditions, a full curve
could not always be obtained because of the fear of breaking the patch.
Nevertheless, a clear shift of the curve to lower pressures was
observed after treatment either by trypsin or chymotrypsin, or
carboxypeptidase, or chymotrypsin followed by carboxypeptidase. This
shift was a result of both a decrease in the pressure required for
half-activation and an increase of the sensitivity of the channel to
membrane tension. The effect was more pronounced after the action of
both chymotrypsin and carboxypeptidase.
Furthermore, we examined whether chymotrypsin or carboxypeptidase was
effective when applied to closed channels. After application of
increased pressure to the patch under control conditions, pressure was
released to close the channels, and the protease was superfused for
5-7 min. The patch was then washed with the control solution, and the
open probability at different pressures was determined again. As shown
in Fig. 4, D and E, both chymotrypsin and
carboxypeptidase were able to induce a shift of the curve to lower
pressure, indicating that they are active on closed channels as well.
Effects of Proteases Present in the Pipette on Purified MscL
Reconstituted in Liposomes--
Outside-out patches cannot be obtained
with giant proteoliposomes. To examine the effects of a protease on the
outside of a patch, the protease was added to the pipette solution.
After seal formation and excision of the patch, pressure was applied rapidly until activation of the channels. The pressure was kept constant, and the channel activity was monitored. Increase in channel
activity was observed when chymotrypsin (300 µg/ml) or trypsin (250 µg/ml) was present in the pipette, but an upward drift of the
baseline was observed upon application of the pressure. Reduction of
trypsin or chymotrypsin concentration to 50 and 60 µg/ml,
respectively, completely suppressed the mechanical instability of the
patches. Under these conditions, we observed either with trypsin
(n = 33) (Fig.
5A) or chymotrypsin
(n = 5) (Fig. 5B) an increase in channel
activity at constant pressure, without a change in channel unit
conductance. After 1 or 2 min, the channels became exquisitely
sensitive to pressure, and application of pressure as low as 3-5 mm Hg
was in general sufficient to activate the channels. In two cases, the
channels were even observed to gate at apparent zero applied pressure.
However, the channels remained mechanosensitive in that decrease (or
increase) of pressure always resulted in a decrease (or increase) in
open probability. We never observed, for instance, a permanently open
level, within 20 min of continuous recording. A similar activation of
the channels was also observed when Pronase (250 µg/ml), which has no
specificity, was present in the pipette (n = 4).
Importantly, when control experiments were performed with
carboxypeptidase (500 µg/ml) in the pipette, no activation of the
channels was observed (n = 9) (Fig. 5C,
compare with Fig. 3A). We next examined how the presence of
trypsin or chymotrypsin in the pipette affects the relationship between
channel open probability and applied pressure. As shown in Fig.
6A, both proteases induce a
dramatic shift of the activation curve reflected in a decrease pressure
required for half-activation and a significant increase in the
sensitivity of the channel to membrane tension. Moreover, the presence
of trypsin or chymotrypsin or Pronase in the pipette drastically
altered the channel open time. Fig. 6B, which displays
successive segments of recordings obtained after seal formation, in the
presence of trypsin in the pipette, shows how the channel open time
increased progressively with time.
Trypsin or chymotrypsin in the pipette was effective on gating channels
but not on closed channels. In a typical experiment in which trypsin
was present in the pipette, after excision of the patch, pressure was
increased progressively until, at 50 mm Hg, the channels started to
gate with a fast kinetics. Pressure was then released immediately to
close the channels. After 10 min, pressure was applied again. The
channels started to gate at 46 mm Hg. The channel kinetics was similar
to that of control experiments characterized by short openings.
Pressure was maintained, and the channel activity increased rapidly.
After 2 min, the threshold of activation dropped to 3 mm Hg, and the
channel kinetics exhibited characteristic long open times (not shown).
This type of experiment was repeated four times with trypsin and two
times with chymotrypsin with a similar result. We also incubated the
giant proteoliposomes for 20 min in a bath containing 50 µg/ml
trypsin. The liposomes were then patched with pipettes that contained
no trypsin. Under these conditions, the threshold of activation for the
channels was always relatively high (between 25 and 60 mm Hg), as in
control experiments. The long open times were not observed.
Finally we looked for the effects of proteases on both sides of the
channel. The full effect of trypsin, present in the pipette, was
observed. After that, chymotrypsin was perfused in the bath, followed
by carboxypeptidase. A further slight enhancement in the activity of
the channels was always observed after perfusion of the proteases in
the bath. However, the channels always remained mechanosensitive; a
permanently open level was never observed.
In this study we examined the effect of different proteases on the
mechanosensitivity of MscL either in the native membrane or in a
reconstituted system. Our results indicated that MscL had the same
orientation in the plasma membrane as in giant liposomes. This
conclusion is based on the following findings. First, trypsin or
chymotrypsin, when present in the pipette, activated the reconstituted channels in a different manner than when perfused in the bath. Second,
carboxypeptidase activated the reconstituted channels when it was
perfused in the bath, but not when it was present in the pipette.
Therefore, in patch-clamp experiments performed on the plasma membrane
and on giant liposomes, the amino and carboxyl termini were accessible
from the bath, whereas the periplasmic loop faced the pipette. Because,
with the reconstituted system, all experiments always yielded identical
results, we concluded that MscL was in all cases reconstituted with the
native orientation in giant liposomes.
The experiments performed by applying proteases in the bath clearly
indicate that limited proteolysis of either the NH2
terminus or the COOH terminus resulted in an increased
mechanosensitivity of the channels. This effect was characterized by
both a reduction in p1/2 and an increase in the
slope of the curve in the Boltzmann distribution. These results modify
somewhat the conclusion of previous studies, performed on mutated
channels, that most of the COOH-terminal extension plays no role in
mechanosensitivity (23, 24). The interest of the approach that has been
used here is that it allows monitoring of the activity of channels in
the same patch before and after application of proteases. The approach
that uses mutated channels necessarily implies comparison of channel
activities recorded in different patches, which may be more
problematic. A limitation of the protease approach, however, is that
the precise cleavage sites are unknown. Chymotrypsin cleavage sites are
likely to be Phe-7 and Phe-10 (Fig. 1), indicating that deletion of
part of the NH2 terminus (the first 7 or 10 amino acids of
the protein) increased the sensitivity to pressure of MscL channels.
Aminopeptidase and carboxypeptidase hydrolyze one amino acid after
another, starting from the NH2 terminus and the COOH
terminus, respectively, and stopping at an unknown distance of the
membrane determined by steric hindrance. It is therefore not possible
to know to what extent the NH2 and the COOH termini have
been deleted in these experiments.
Independently of its importance for the understanding of the channel
mechanosensitivity, the finding that a limited proteolysis of the
NH2 or COOH terminus of MscL increases its sensitivity to
membrane tension might have a physiological relevance. In particular, it is remarkable that the numerous homologs of MscL which have now been
found all have substantial cytoplasmic COOH-terminal extension. Several
residues in this extension are conserved (25). This fact is difficult
to understand, if, as suggested previously, this part of the protein
plays no part in the channel function. A suggestion raised by our
results is that the COOH-terminal extension plays a modulatory role,
perhaps through interactions with other molecules.
The most striking result of this study is that the channel remains
mechanosensitive after the external loop has been cleaved. As discussed
above, the external loop, the only non-membranous part of the channel
on the periplasmic side, is accessible from the pipette in the
reconstituted system. Therefore the change in channel kinetics and
mechanosensitivity observed when trypsin or chymotrypsin is present in
the pipette indicates that the loop was cleaved at one or two of the
trypsin- or chymotrypsin-sensitive sites present in this region of the
molecule (Fig. 1). Indeed, it is extremely unlikely that cleavage might
have occurred at sites present in the transmembrane segments because
this is never observed for membrane proteins. Given the symmetry of the
homo-oligomer, one may expect that after a steady state has been
reached, all of the subunits of the channels have been cleaved.
Importantly, the effect of trypsin or chymotrypsin was only observed
when the channel was gating, indicating that its opening results in a
conformational change of the loop which exposes its protease-sensitive
sites. Reconstitution of functional membrane proteins from proteolytic fragments or from genetically cleaved fragments has been reported for
membrane protein (for review, see Ref. 26). However, to our knowledge,
this is the first time that the activity of a membrane protein has been
monitored at the level of a single molecule after cleavage of all the
of loops of the protein. That, after this treatment, the channel
retained its main characteristic, mechanosensitivity, and that its
conductance had not been altered, attest to the importance of the
interactions between the transmembrane helices for both the stability
and activity of the molecule. We are left with the remarkable
conclusion that two unconnected segments plus a few amino acids on each
side of the membrane are sufficient for the activity of a
mechanosensitive channel. The integrity of the loop seemed not to be
essential for mechanosensitivity itself. Therefore the external loop
appears to act as a spring that resists opening of the channel and
promotes its closure when it is open. Its stiffness sets the level of
sensitivity of the channel to membrane tension.
Taken as a whole, the results presented here strongly suggest that all
of the different extramembranous parts of the channel counteract the
movement of the transmembrane helices triggered by a change in membrane
tension. The COOH termini interacting with each other by forming a
bundle (19) would resist the movement of the M2 helices. The
cytoplasmic NH2 termini may also interact with each other
and resist the movement of the M1 helices. The geometry of the external
loop counteracts the movement of the two helices relative to each
other. This leads us to propose that both M1 and M2 helices move when
the channel opens. This could be possible if membrane tension itself,
or thinning of the membrane upon stretch, modifies the orientation of
each helix in the membrane and/or specifically modifies the interaction
in the membrane between the two helices of an MscL subunit. The
movement of these helices would be at the basis of mechanosensation for
this molecule. It is not known how membrane tension or membrane
thinning can affect transmembrane helices in membrane proteins, and it
is therefore unclear what makes MscL helices specifically sensitive to
membrane tension. The importance of the transmembrane helices for
mechanosensation which is proposed here is consistent with a previous
report indicating that several residues in both helices are highly
conserved in MscL homologs (25). Moreover, a recent study has
highlighted the importance of helix M1 for mechanosensitivity. Randomly
mutagenized mscL genes were expressed in bacteria that were
screened for gain-of-function mutants with impaired growth. The most
severe mutations, which resulted in channels gating at anomalous low
tension, were found on one facet of transmembrane helix M1 (27).
In the closed conformation, as revealed by x-ray diffraction, the pore
formed by M1 helices, is a funnel whose radius varies from 18 Å on the
periplasmic side to 2 Å on the cytoplasmic side, where it is occluded
(19). Therefore, opening the channel requires a tilting of the M1
helices that are presumed to be pulled away from each other upon
application of tension to the membrane. MscL, which has a high
conductance and is able to catalyze the release of molecules such as
thioredoxin (12), has a very large pore whose diameter was estimated to
be around 40 Å by electrophysiological experiments (28). Given the
mean distance between
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Trypsin- and chymotrypsin-sensitive sites of
the extramembranous parts of the MscL subunit. The topology of the
E. coli MscL subunit in the membrane is based on PhoA fusion
experiments (16) and the structure of the M. tuberculosis
MscL (18). Trypsin-sensitive sites, lysines (K) and arginines (R)
outside the transmembrane domain M1 and M2, are shown as filled
circles. The aromatic phenylalanines (F) outside M1 which are
potential chymotrypsin sites are shown as filled
boxes.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells (14). Cells were grown in 50 ml of LB, pH
7.2, supplemented with 30 mM MgSO4 to an
OD650 of 0.12-0.15 before the addition of cephalexin (60 µg/ml). Giant round cells (5-7 µm in diameter) were harvested 3-4
h later. Cells resuspended in 10 mM Tris-HCl buffer, pH 7, 100 mM NaCl, 400 mM sucrose, were incubated at
37 °C for 30 min in the presence of lysozyme (400 µg/ml). 2 µl
of this suspension was deposited in the patch-clamp chamber and covered with 50 µl of 10 mM Tris-HCl, pH 7, 1 mM
EDTA, 100 mM KCl. 2 ml of 10 mM HEPES-KOH, pH
7.4, 100 mM KCl, 10 mM MgCl2 was
then added to the patch-clamp chamber for recording.
110-136) proteins were purified in one step,
using a glutathione S-transferase fusion system, as
described previously (16). A few microliters of purified protein in 50 mM octyl 
-D-glucoside was added to 1 ml of
10 mM HEPES-KOH, pH 7.4, 100 mM KCl, 25 mM octyl -D-glucoside containing 1 mg of
sonicated lipids (azolectin from soybean, type IV-S, Sigma) to achieve
a lipid to protein ratio of 500-2,500. After a 20-min incubation, 160 mg wet weight of SM-2 Bio-Beads (Bio-Rad) was added to the suspension
to remove the detergent (20). The suspension was agitated for 3 h,
the Bio-Beads were discarded, and the suspension was centrifuged for 30 min at 90,000 rpm using a TL100 Beckman ultracentrifuge. The pellet was
resuspended in 15 µl of 10 mM HEPES-KOH, pH 7.4. The
proteoliposomes were then fused into giant proteoliposomes amenable to
patch-clamp recording, using a cycle of dehydration-rehydration as
described previously (21). Rehydration was performed in 10 mM HEPES-KOH, pH 7.4, 100 mM KCl. 2 µl of the
giant proteoliposome suspension was deposited in the patch-clamp chamber and diluted with 2 ml of the bath solution for
electrophysiological recording.
-p-tosyl-L-lysine
chloromethyl ketone-treated), carboxypeptidase Y (from baker's yeast),
leucine aminopeptidase (from porcine kidney), and Pronase E from
Streptomyces griseus were from Sigma. Control experiments
were performed using bovine serum albumin in the bath or in the
pipette. Negative pressure (suction) in the pipette was applied by
syringe and monitored with piezo-electric pressure transducer (Bioblock
scientific). Unitary currents were recorded using a Biologic RK-300
patch-clamp amplifier with a 10-gigaohmsfeedback resistance and stored
on digital audio tape (Biologic DTR 1200 DAT recorder). Records were
subsequently filtered at 1 kHz (
3 dB point) through a four-pole
Bessel low pass filter, digitized off-line at a rate of 2 kHz, and
analyzed on a personal computer, with a program developed by G. Sadoc
(Gif sur Yvette). Data were plotted on a Hewlett-Packard laserjet
printer, using Sigmaplot software (Jandel).
where Pmax is the maximum probability of
channel being open, p is the suction,
p1/2 is the suction at which the open
probability is 0.5, and
(Eq. 1)
is the sensitivity to the applied suction.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (24K):
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Fig. 2.
Effect of trypsin superfused in the bath on
MscL channels present in the native membrane. Patch-clamp
experiments were performed on the giant protoplasts. After excision,
pressure was applied to the inside-out patch as indicated. The patch
was superfused first with the bath solution and then with the bath
solution containing 250 µg/ml trypsin as indicated. Application of
trypsin increased the activity of the channels, which, however,
remained mechanosensitive and closed upon release of pressure. Bath
medium: 100 mM KCl, 10 mM MgCl2, 10 mM HEPES-KOH, pH 7.4. Pipette medium: similar to bath
medium with, in addition, 2 mM CaCl2. The
membrane potential was +10 mV. C, closed level for all
channels in the patch. Pressure is indicated at the bottom.
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Fig. 3.
Effects of proteases superfused in the bath
on purified MscL reconstituted in liposomes. Patch-clamp
experiments were performed on giant proteoliposomes. After excision,
pressure was applied to the inside-out patch as indicated. The patch
was superfused first with the bath solution then with the bath solution
containing chymotrypsin or carboxypeptidase or aminopeptidase, as
indicated. In all cases, application of the proteases increased the
activity of the channels, which, however, remained mechanosensitive and
closed upon release of pressure. Bath medium: 100 mM KCl,
10 mM HEPES-KOH, pH 7.4. Pipette medium: similar to bath
medium with, in addition, 1 mM MgCl2, 0.1 mM CaCl2. The membrane potential was +10 mV.
Panel A, effect of carboxypeptidase (500 µg/ml). The
asterisk indicates a transient decrease in channel activity
which was observed at the onset of carboxypeptidase superfusion.
Panel B, effect of chymotrypsin (300 µg/ml). Panel
C, effect of aminopeptidase (500 µg/ml).
110-136), in which the last 27 residues had been deleted, was
still functional, whereas deletion of 6 additional residues suppressed
channel activity totally (23, 24). The mechanosensitivity of the
110-136 mutant was reported to be similar to that of the wild type.
We reconstituted the purified 110-136 mutant in giant liposomes.
The threshold of activation was not modified, but the slope of the
activation curve was enhanced considerably compared with the wild type.
Superfusion of excised patches by carboxypeptidase led nevertheless to
an increase in channel activity at constant applied pressure
(n = 3, not shown). Taken together, these results indicate that deletion of residues of the COOH terminus before and
after residue 110 modulates the mechanosensitivity of the channel.
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Fig. 4.
Pressure dependence of the MscL channel
reconstituted in liposomes before and after superfusion of the
proteases in the bath. Open probability Po
multiplied by the number N of channels in the patch
versus the applied pressure, at fixed membrane potential
(+10 mV) before (open circles) and after (full
circles) the application of proteases in the bath to an excised
inside-out patch is shown. Each point was obtained by integrating the
current through open channels in a 15-s segment of recording and by
dividing the integral by the time of recording multiplied by the
unitary current. The data were fitted to a Boltzmann distribution of
the form N·Po = N·Pmax (1 + exp (p1/2 p))1, where
N is the unknown number of channels in the patch,
Po is the open probability,
Pmax is the maximum open probability,
p is the pressure, p1/2 is the
pressure at which the open probability is 0.5, and is the
sensitivity. In panels A, B, C, and
F, after determination of open probabilities under control
conditions, the indicated protease was applied on gating channels for
5-7 min. The patch was washed, and the new open probabilities were
determined again for each value of applied pressure. In D
and E, after determination of open probabilities under
control conditions, the pressure was released to close the channels,
and the indicated protease was applied to the patch for 5-7 min. The
patch was then washed before determination of the new open
probabilities. Ionic conditions were as in Fig. 3. Panel A,
trypsin (250 µg/ml) superfused on gating channels.
p1/2 was 53 versus 44 mm Hg, and
1/ was 3.9 versus 1.8 mm Hg for the intact and
proteolyzed channels, respectively.
N·Pmax was 7. Panel B,
chymotrypsin (300 µg/ml) superfused on gating channels.
p1/2 was 38 versus 20 mm Hg, and
1/ was 4 versus 1.3 mm Hg for the intact and proteolyzed
channels, respectively. N·Pmax was
65. Panel C, carboxypeptidase (500 µg/ml) superfused on
gating channels. p1/2 was 62 versus
28 mm Hg, and 1/ was 5 versus 3.4 mm Hg for the intact
and proteolyzed channels, respectively.
N·Pmax was 30. Panel D,
chymotrypsin (300 µg/ml) superfused on closed channels.
p1/2 was 74.5 versus 42 mm Hg, and
1/ was 7 versus 3 mm Hg for the intact and proteolyzed
channels, respectively. N·Pmax was
80. Panel E, carboxypeptidase (500 µg/ml) superfused on
gating channels. p1/2 was 52 versus
36.4 mm Hg, and 1/ was 3.7 versus 1.3 mm Hg for the
intact and proteolyzed channels, respectively.
N·Pmax was 5. Panel F,
chymotrypsin (300 µg/ml) was first superfused on gating channels
followed by carboxypeptidase (500 µg/ml). p1/2
was 67 versus 11.3 mm Hg, and 1/ was 5.9 versus 1.02 mm Hg for the intact and proteolyzed channels,
respectively. N·Pmax was 20.
View larger version (39K):
[in a new window]
Fig. 5.
Effects of proteases present in the pipette
on liposome patches. After excision of the patch, the activity of
the channels was monitored at constant applied pressure. Panel
A, the presence of trypsin (50 µg/ml) in the pipette resulted in
a rapid increase, at constant pressure, of the activity of the
channels, which remained mechanosensitive and could be closed upon
release of pressure. Panel B, a similar activation was
observed when chymotrypsin (60 µg/ml) was present in the pipette.
Panel C, in contrast, the presence of carboxypeptidase (500 µg/ml) in the pipette did not produce any variation in channel
activity. The membrane potential was +10 mV. The ionic conditions were
as in Fig. 3.
View larger version (22K):
[in a new window]
Fig. 6.
Proteolysis of the external part of the
channel alters its pressure dependence and drastically modifies its
kinetics. Panel A, open probability
Po versus the applied pressure, at
fixed membrane potential (+10 mV), of the MscL channel after activation
by trypsin (50 µg/ml) (circles) or chymotrypsin (60 µg/ml) (squares) present in the pipette. After seal
formation, pressure was applied, and the activation of the channels was
monitored for 10 min until a steady state was reached. The open
probability at each applied pressure was then determined. Each point
was obtained by integrating the current through open channels in a 15-s
segment of recording and by dividing the integral by the time of
recording multiplied by the unitary current and the total number of
channels in the patch. The data were fitted to a Boltzmann distribution
as defined in Fig. 5. After the action of trypsin,
p1/2 was 5 mm Hg, and 1/ was 0.4 mm Hg. After
the action of chymotrypsin, p1/2 was 7.3 mm Hg,
and 1/ was 0.4 mm Hg. The broken line represents the
Boltzmann distribution obtained by averaging the parameters of all of
the control curves (in the absence of proteases) displayed in Fig. 5
(p1/2 = 67 mm Hg; 1/ = 4.85 mm Hg).
Panel B, trypsin in the pipette altered the channel
kinetics, increasing the open time. Successive segments of recordings
were obtained at different times, as indicated, following seal
formation and excision of the patch, with 50 µg/ml trypsin in the
pipette. For each segment, only the amount of pressure necessary to
activate one open channel level was applied.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices in membrane proteins, it is doubtful
that only M1 helices are sufficient to make such a large pore, and it
is therefore probable that M2 helices also participate in the formation
of the open pore (28). These considerations lead to the putative model
of Fig. 7 which enables us to interpret
the whole of the data presented here. An increase in membrane tension
modifies the interactions between M1 and M2 helices or between these
helices and the core of the membrane causing both helices to tilt, with
the M2 helices intercalating between the M1 helices. These movements
are opposed by the non-membranous domains, thus setting the level of
mechanosensitivity of the channel. On each -helix, the amino acids
immediately adjacent to the membrane on the cytoplasmic side certainly
play an important role because their deletion results in an inactive
channel (23, 24). This could not be seen in our experiments, probably
because these residues are too close to the membrane to be accessible to proteases, but it is of interest to note that trypsin or
chymotrypsin, perfused in the bath, totally abolished the activity of
another mechanosensitive channel, MscS, in patch-clamp experiments
performed on the native membrane (29). It is possible that these
cytoplasmic residues are important to keep the helices in an
appropriate orientation relative to the plane of the membrane.
View larger version (22K):
[in a new window]
Fig. 7.
A model of the molecular mechanism of
MscL. For the sake of simplicity only two subunits of the channel
are shown, and their different elements are represented in the same
plane. The channel is closed by the M1 helices oriented at an oblique
angle with respect to the plane of the membrane. An increase in
membrane tension modifies the interactions between M1 and M2 helices or
between these helices and the core of the membrane causing both helices
to tilt, with the M2 helices intercalating between the M1 helices. The
cytoplasmic end-terminal extensions resist the movement of M1 and M2
helices. Similarly, the external connecting loops counteract the
movement of helices M1 and M2, relative to each other. Proteolysis of
all of these parts of the molecule facilitates the movement of the
helices, thus enhancing the sensitivity of the channel to membrane
tension.
Are the results described here relevant for the understanding of the
molecular mechanisms of mechanosensitive channels outside the bacterial
domain? Although bacterial mechanosensitive channels have been shown to
gate in response to bilayer tension alone, it is usually considered
that the tethered model is more likely to describe the functioning of
eukaryotic mechanosensitive channels. In this model, a direct
connection is hypothesized between the gate of the channel and proteins
located in the extracellular matrix and/or the cytoskeleton (30). A
displacement of the channel relative to the extracellular matrix or the
cytoskeleton will cause the channel to open or close. In the case of
mechanotransduction in Caenorhabditis elegans, for instance,
several non-membranous proteins are believed to interact with the
mechanosensitive channel (31, 32). The MEC-4 and MEC-10 proteins, which
belong to the DEG/ENaC superfamily, are likely to encode subunits of
this mechanosensitive channel. These proteins have a similar predicted
topology that includes two transmembrane domains, an extracellular
loop, and internal COOH and NH2 termini (31, 32). It is
clear, however, that modifications of the cytoskeleton might alter the
mechanical properties of the membrane. Furthermore, the results
presented here indicate that interactions with the end terminals and/or the loop of such proteins might greatly alter their mechanosensitivity, even if the channels are gated by membrane tension alone. Although no
sequence similarity exists between MscL and the members of the DEG/ENaC
superfamily, the overall structural similarity is striking. It is
therefore possible that some of these channels share a common
mechanism, for which the scheme outlined above could provide a working model.
| |
FOOTNOTES |
|---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: URA CNRS 1218, Faculté de Pharmacie, Université Paris-Sud, 5 rue Jean-Baptiste Clément, 92296 Châtenay-Malabry Cedex, France.
To whom correspondence should be addressed. Tel.:
33-1-6915-7194; Fax: 33-1-6985-3715; E-mail:
alexandre.ghazi@biomemb.u-psud.fr.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: Msc, mechanosensitive channel(s); MscM, mini; MscS, small; MscL, large channel.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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