From the Department of Cell Biology, Nencki Institute of Experimental Biology; 3, Pasteur Street, 02-093 Warsaw, Poland
Received for publication, November 26, 2002, and in revised form, January 23, 2003
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ABSTRACT |
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Heptameric YggB is a mechanosensitive ion channel
(MscS) from the inner membrane of Escherichia coli. We
demonstrate, using the patch clamp technique, that cross-linking of the
YggB C termini led to irreversible inhibition of the channel
activities. Application of Ni2+ to the
YggB-His6 channels with the hexahistidine tags added to the
ends of their C termini also resulted in a marked but reversible decrease of activities. Western blot revealed that
YggB-His6 oligomers are more stable in the presence of
Ni2+, providing evidence that Ni2+ is
coordinated between C termini from different subunits of the channel.
Intersubunit coordination of Ni2+ affecting channel
activities occurred in the channel closed conformation and not in the
open state. This may suggest that the C termini move apart upon channel
opening and are involved in the channel activation. We propose that the
as yet undefined C-terminal region may form a cytoplasmic gate of the
channel. The results are discussed and interpreted based on the
recently released quaternary structure of the channel.
Mechanosensitive (MS)1
ion channels open upon membrane tension, and therefore they represent
the simplest mechanosensors. MS channels have been implicated in many
physiological processes from growth and cell volume regulation to
hearing, blood pressure regulation, and pain sensation (reviewed in
Ref. 1). Bacterial MS channels protect these cells against hypoosmotic
shock. Two types of MS channels from the cytoplasmic membrane of
Escherichia coli, MscL and MscS, play an essential role in
the physiology of this bacterium, allowing the efflux of solutes from
the cytoplasm when osmolarity of the external medium decreases (2-4).
MscL, the large conductance MS channel, has been cloned (5), and a
quaternary structure of its closed conformation has been determined (6). Based on this structure and the analysis of the channel gating,
the open conformation has been predicted (7, 8) and experimentally
confirmed (9, 10). Functional homologues of this channel have been
found in other bacteria (11) and Archea (12), and structurally related
protein from Neurospora has been also reported (13). The
functional channel is a pentamer, and each subunit consists of two
The activity of MscS, the E. coli MS channel of a smaller
conductance (16), consists of activities of two separate ion channels of very similar conductance encoded by yggB and
kefA (3). KefA is a large multidomain protein (1120 amino
acids), spanning the inner membrane and possibly having a link to the
outer membrane (17), whereas YggB is a small protein of 286 amino acids
residing in the inner membrane. Both proteins show amino acid sequence homology in the region corresponding to the entire YggB sequence (3)
presented in Fig. 1A. The
activities of the KefA and YggB channels recorded directly from the
E. coli membrane are kinetically distinct; YggB shows
inactivation during sustained pressure, i.e. adaptation
(18), whereas KefA does not adapt to pressure (3, 16). The YggB
channels are more abundant than those of KefA, and their activities
have been recorded after reconstitution of a purified protein in planar
lipid bilayers (19, 20), indicating that, similar to MscL, YggB senses
membrane stress directly.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical membrane-spanning domains TM1 and TM2 with both the C and N
termini located in the cytoplasm (6). TM1s line the pore, and their
hydrophobic residues form the primary, transmembrane gate (6, 14). It
is postulated that there are two gates involved in the opening of the
channel: the transmembrane and the cytoplasmic gates (7, 8) acting in
accordance (15). The transmembrane gate is proposed to act as a
pressure sensor, and upon application of pressure, this gate permits
initial expansion of the channel without its full opening (7, 8, 10).
It is proposed that the other, cytoplasmic gate, which allows full
activation of the channel, is composed of five
-helical S1 segments
of the cytoplasmic N termini being connected with TM1s via flexible
linkers. According to the model, the applied pressure is transmitted to
the S1 segments through the flexible linkers and pulls them apart. The
channel may fully open when the interactions between the five S1
segments of the cytoplasmic gate break down (7, 8, 10).
View larger version (67K):
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Fig. 1.
Amino acid sequence of YggB and its
quaternary structure. A, YggB has three
membrane-spanning helices, indicated above the sequence as gray
cylinders, with its N terminus in the periplasm and its C terminus
located in the cytoplasm. The positions of lysines from the cytoplasmic
regions are indicated with black boxes. The upward
arrow indicates the position where YggB was truncated (see
"Discussion"). B, the middle panel shows a
side view of the quaternary structure of the YggB heptamer with one
subunit marked in dark gray. Right side, the top
view of the heptamer is presented as seen from the periplasmic side.
Left side, the structure of one subunit with transmembrane
helices indicated as TM1, TM2, and TM3 and with three cytoplasmic
C-terminal regions indicated as Middle domain,
Lower
/
domain, and
-barrel
strand.
The quaternary structure of the YggB channel was recently published
(21). The structure reveals that the functional channel is a heptamer
and has three transmembrane domains, TM1, TM2, and TM3, in each of
seven subunit (Fig. 1B). The TM3s line the channel pore. The
cytoplasmic domains are composed mostly of -sheets and surround the
large water-filled chamber with a diameter of ~40 Å. Each subunit of
the assembly consists of a middle
domain and a lower
/
domain
(Fig. 1B, left view), and all seven subunits are
linked together by a
barrel composed of seven strands, which are
located at the very ends of the C termini (Fig. 1B,
middle view).
In this paper we have studied a possible role for C termini in
functioning of the YggB channels. We demonstrate that cross-linking of
the YggB C termini yields inactive channels. We also show that intersubunit coordination of Ni2+ in the
YggB-His6 channels prevents the channels from opening. The
Ni2+ coordination leading to the inhibition of activities
occurs in the channel closed state, and we did not observe it in the
channel open conformation. This may suggest that the C termini move
apart upon channel opening and are involved in the process of the
channel gating.
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EXPERIMENTAL PROCEDURES |
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Chemicals--
Nonpolymerized, microscopy grade glutaraldehyde
(GDH) (Grade I) was purchased from Sigma and stored at 80 °C in
small aliquots. Bis(sulfosuccinimidyl)suberate (BS3)
(Sigma) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
hydrochloride (EDC) (Sigma) were dissolved in the bath solution shortly
before use. SDS and
isopropyl-1-thio-
-D-galactopyranoside were from Biomol.
Bacterial Strains, Constructs, and Overexpression--
All of
the electrophysiological experiments were performed on E. coli strains: MJF 379, Frag1
kefA::kan; MJF429, Frag1
kefA::kan yggB. Frag1 is a wild-type strain, a
derivative of E. coli K-12. All of the strains were kindly
provided by I. R. Booth (University of Aberdeen, Aberdeen, UK).
The construct pYggB-His6 with a histidine tag added to the
end of its C terminus was obtained as follows. The yggB open
reading frame was amplified from genomic DNA of E. coli
wild-type DH5 strain with proofreading Pfu polymerase
(Promega) using the following primers: upper primer,
5'-TAGCCCATGGAAGATTTGAATGTTGTC-3' (underlined bases represent the NcoI restriction
endonuclease recognition site, and the sequence of yggb open
reading frame is in italics), and lower primer,
5'-GGTACCTTAGTGATGGTGATGGTGATGCGCAGCTTTGTCTTCTTT-3' (underlined bases represent KpnI restriction endonuclease
recognition site; the sequence in italics represents the
yggB open reading frame, and the bold sequence codes the
six-histidine tag followed by a stop codon). The resulting PCR product
was digested with NcoI and KpnI (Promega),
purified using QIAex II gel purification kit (Qiagen), and ligated into
fragment of pTrc99A vector (Amersham Biosciences) digested with
NcoI and KpnI. The resulting plasmid is carrying
YggB-His6 under control of
isopropyl-1-thio-
-D-galactopyranoside-inducible trc (trp/lac) promoter. The
pYggBHis6 construct was verified by restriction digestion
and sequencing of the insert.
The pYggBHis6 construct was expressed in the MJF429 strain.
The bacteria were grown at 37 °C overnight in liquid LB broth containing 10 g/liter bacto-tryptone, 5 g/liter yeast extract, 5 g/liter NaCl, and supplemented with ampicillin (100 µg/ml). The
overnight culture was diluted to A600 = 0.2, and the bacteria were next grown to A600 = 0.6. Expression was induced by adding 1 mM
isopropyl-1-thio--D-galactopyranoside for 3 h. The
cells were spun down and frozen in liquid nitrogen. Lysate was obtained by treatment of cells with lyzozyme (25 µg/ml) (Sigma) in the buffer
C containing 50 mM HEPES, 100 mM NaCl, pH 7.4, in the presence of 1 mM phenylmethylsulfonyl fluoride
(Merck). The lysate was treated with DNase (5 µg/ml) (Sigma) and spun
down in Sorvall SS-34 rotor at 10 000 rpm for 10 min.
Western Blotting-- An insoluble fraction of E. coli cells containing membranes was resuspended in buffer C and divided equally into Eppendorf tubes. NiCl2 or MgCl2 (as indicated) was added to the final concentration of 50 mM, and the samples were incubated for 30 min at room temperature. The membrane fraction was spun down in a microcentrifuge at 14,000 rpm and washed once with buffer C. The pellet was solubilized in 0.5× Laemmli sample buffer and incubated for 5 min at the indicated temperature.
For Western blot, the proteins were separated in 9% SDS-PAGE mini-gels and electroblotted on a polyvinylidene difluoride membrane using semi-dry transfer apparatus (Bio-Rad). The membrane was blocked by overnight incubation in 2% bovine serum albumin (SERVA) in TBST buffer (25 mM Tris, 100 mM NaCl, 0.05% Tween 20, pH 7.4). The YggB-His6 protein was detected by monoclonal anti-His C-terminal antibodies (Invitrogen) in 1:7500 dilution in TBST. The antibodies did not react with any protein from a lysate of a wild-type E. coli cells (MJF429) not carrying pYggB-His6 (not shown). As secondary antibodies, the anti-mouse IgG conjugated to alkaline phosphatase antibodies (Promega) in dilution 1:5000 were used. For detection of alkaline phosphatase, 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium color substrate (Promega) was used according to manufacturer's instructions. The protein masses and the band intensities were estimated according to prestained protein standards (Bio-Rad) and measured from Western blot scans using Image Quant software (Molecular Dynamics).
Electrophysiological Recordings and Data Analysis--
All of
the experiments were performed on E. coli giant protoplasts
prepared as described previously (22). Single channel recordings were
obtained from inside-out excised membrane patches, and the experimental
procedure, including the equipment used and the application and
measuring of suction, was the same as described earlier (18). Bath
solution was 150 mM KCl, 400 mM sorbitol, 4 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, pH 7.2, whereas the pipette solution was the same
except that the sorbitol concentration was 300 mM. 60-s
intervals or longer were maintained between applications of suction.
The data were acquired (with a sampling rate of 2.5 kHz), filtered at 1 kHz, and analyzed using pCLAMP6 software. The mean single-channel open
probability, Po during the pressure pulse (not
shorter than 12 s) was calculated by integrating the current
passing through all active channels I during the pulse and
dividing this integral by the current through a single open channel
i and the number of active channels N according
to the formula Po = I/Ni.
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RESULTS |
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Conservation of YggB Domains among Its Bacterial
Homologues--
Homologues of the E. coli YggB can be found
in a wide range of bacteria and archea (3). In Fig.
2 we compared 36 amino acid sequences of
bacterial YggB homologues. In this figure each bar
represents a single residue in the YggB sequence and its length is
proportional to the identity of amino acids estimated for this residue.
Among three membrane domains TM3 shows the highest level of homology as
a whole (44% identity comparing to 30 and 23% estimated for TM1 and
TM2, respectively), but there are also numerous short spans of C
terminus whose identities are higher then 50%, implicating the
importance of that region.
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Cross-linkers Inhibit YggB Activities in Membrane Patches--
The
model of the MscL gating postulates that the cytoplasmic gate composed
of five N termini occludes the pore of the channel until a mechanical
force pulls them apart and the channel opens (7, 8). If a cytoplasmic
gate exists in the YggB channel, it should be composed of its C
termini. In the amino acid sequence of the cytoplasmic region of YggB
all lysines but one (Lys60) are situated in the C terminus
(Fig. 1A). We assumed that cross-linking of lysines from
different C termini of the channel would hamper or prevent the channel
opening providing its C termini are being pulled apart during opening.
We therefore applied lysine-specific reagents to the cytoplasmic side
of the membrane patches containing the YggB channels. Fig.
3 shows effects of GDH, EDC, and highly lysine-specific BS3 on the channels. Each reagent, at
concentration 2 mM, was applied to the closed channels, and
the recordings were obtained at positive pipette voltages (+15 mV).
Nonpolymerized GDH of undefined cross-linking distance and
BS3 with a spacer arm of 11.4 Å inhibited the YggB
activity irreversibly, whereas EDC with a spacer arm of 0 did not
affect it. Each set of traces in Fig. 3 represents a single experiment,
but similar effects of GDH, BS3, and EDC were observed in
at least three other experiments in which these cross-linkers were
used. The difference in control traces arises from a different number
of channels in each patch and also from the different pressure applied
in each case. The rate of adaptation is inversely proportional to the
suction (18), and accordingly, the highest rate of adaptation is
observed in the control trace of experiment with the lowest suction
applied (Fig. 3B).
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Inhibition of YggB-His6 Channel Activities by Ni2+ Coordination-- The previous set of experiments showed that cross-linking of lysines by BS3 on the cytoplasmic side of the closed YggB channel reduces the probability of the channel being open upon application of pressure. An important question, however, arises: which lysines were those involved in the cross-linking preventing the channel from opening. BS3 could bind lysines situated within a single C terminus or lysines from C termini of different subunits. Alternatively, Lys60 located in the linker between TM1 and TM2 and situated in the cytoplasm (Fig. 1) could be also involved. Cross-linking studies performed with disuccinimidyl suberate showed that lysine-specific cross-linkers react with groups from different subunits (19). We also confirmed this observation using BS3 (a water-soluble analogue of disuccinimidyl suberate).2 Therefore, we assumed that cross-linking of lysines from different subunits of the YggB closed channel irreversibly prevented the opening of the channel. However, we wanted to verify this assumption with a different experimental procedure. We noticed that three of all eight lysines of the C terminus are situated within the stretch of the last nine amino acids (Fig. 1A) (the entire sequence of the C terminus consists of ~160 amino acids), and accordingly the highest probability of cross-linking occurs in this region. Therefore, we added a His6 tag to the end of the C terminus and studied the effect of Ni2+ binding to their imidazole groups. It was shown previously that the addition of His5 tag to the hemolysin channel renders a channel that could be inhibited by Ni2+ (23). It is worthwhile to note at this point that there are no other histidines in the entire amino acid sequence of YggB. We reasoned that, if in the closed state of the YggB-His6 channel its tagged C termini are close enough to coordinate Ni2+, the gating should be altered. This indeed proved to be the case.
The response to pressure of the YggB channels was essentially the same
whether or not they had the attached C-terminal histidine tag (not
shown). Fig. 4 shows the effect of
exposure of the YggB-His6 channels to 0.5 and 5 mM NiCl2 at negative pipette voltage (15 mV).
Addition of 0.5 mM Ni2+ reduced the YggB
activity (measured as an open probability (Po)) to 89.1% (second trace) and after application of 5 mM Ni2+ the activity decreased to 32.7%
(third trace) of that observed in the control (top
trace). After removal of Ni2+ with 2 mM
EDTA (bottom trace), the YggB activity returned almost to
the control level (93% of control). Similar effect was observed in
eight other patches, and one of these experiments is presented in Fig.
5 (middle trace). In this
experiment six concentrations of Ni2+ were
used: 0.25, 0.5, 1, 2, 5, and 10 mM. Each point represents the channel open probability Po at various
Ni2+ concentrations normalized to the open probability
Poc in control. As shown in this figure, at
least five recordings of the channel activity were taken at each
Ni2+ concentration, and each recording was performed within
1 min after the end of the preceding pressure pulse. The most severe reduction in activity was observed at 1 mM
Ni2+, and the lowest Po was at 5 and
10 mM Ni2+. In three experiments of similar
procedure but at positive pipette voltage (+15 mV), we did not observe
this effect, and the data from one of these experiments are presented
in Fig. 5 (top trace). Control experiments of identical
procedure were performed on the channels without the His6
tag (three cases). The activities of these channels were not affected
by the addition of Ni2+, and one of the control experiments
is shown in Fig. 5 (bottom trace).
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We applied the antiHis6 antibodies to three membrane patches containing YggB-His6 channels. We did not observe any inhibiting effect of the antibodies on the channel activities (not shown).
Ni2+ Coordination in YggB-His6 in Vitro-- Inhibition of channel activities of YggB-His6 in the presence of Ni2+ suggested that histidines from different subunits are involved in coordinating Ni2+. We looked, therefore, for confirmation of this hypothesis in vitro.
Western blot of YggB-His6 (Fig.
6) in the absence of Ni2+ and
at 25 °C revealed a ladder pattern of six bands (left
panel, first lane) indicated by numbers.
Under our conditions of electrophoresis, the lowest band migrated close
to the front of the gel. It represents the monomeric polypeptide of 28 kDa. The dimer, trimer, tetramer, pentamer and hexamer are present at
47, 93, 150, 190, and 210 kDa, respectively. The oligomers were stable
in SDS buffer at 25 °C. At higher temperatures they dissociated; at
55 °C the bands of monomers, dimers, and trimers are visible, and at
75 °C only the bands of monomers and dimers are visible. Nickel ions
bound to YggB-His6 oligomers and the electrophoretic
mobility of the resulting complexes was lower than the mobility of the
YggB-His6 oligomers present in the absence of
Ni2+. At 25 °C all of the bands up to hexamer are well
resolved, and the binding seems to be stable from trimer to hexamer.
The patterns yielded at 55 °C and at 75 °C are very similar to
that observed at 25 °C, indicating that the Ni2+-bound
oligomers are stable at higher temperatures. This is in a marked
contrast to the YggB oligomers existing in the absence of
Ni2+. We used Mg2+ as a control for the effect
of Ni2+, and the ladders obtained in the presence of
Mg2+ (third lane in each panel) were
very similar to the control ones (first lane in each
panel) at all of the temperatures tested.
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The amount of fraction applied to each lane was the same; however, in control at room temperature the overall intensity of the bands representing dimers and higher oligomers is higher than in the other lanes. The overall intensity of the bands in the lanes with Ni2+ (normalized to the intensity at room temperature) increases with an increase of temperature (1.0, 2.5, and 3.1), indicating that more protein could enter the gel at higher temperatures as dimers and higher oligomers. In contrast, the higher the temperature the lower the intensity of the bands in the control lanes (1.0, 0.13, and 0.10) and in the lanes with Mg2+ (1.0, 0.38, and 0.47), suggesting that more protein can exist in a monomeric state. The monomeric band migrates at the front of the gel, and an estimation of its intensity is difficult. This explanation applies as well to the apparent difference in intensity between the bands in the presence and in the absence of Ni2+ at room temperature (Fig. 6, first panel, second and first lanes, respectively).
The observed stability of the Ni2+-bound oligomers allows
us to conclude that histidines from different subunits of
YggB-His6 channel participate in Ni2+
coordination. An additional confirmation of this conclusion comes from
the YggB crystal structure, which revealed that the strands located
at the ends of C termini form a parallel
-barrel (Fig. 1B). The ends of the strands from the
-barrel are in a
close proximity, and after addition of His6 epitopes,
Ni2+ coordination by histidines from different subunits is
very likely to occur.
Ni2+ Coordination in the Open State of the YggB
Channels--
We exposed the C termini of the open
YggB-His6 channels to Ni2+, assuming that the
Ni2+ coordination between the His6-tagged C
termini might not occur. The control experiment was basically similar
to that in which we had investigated Ni2+ coordination in
the closed YggB-His6 channels, but we introduced a time
scale. In this experiment we measured channel activity by a short
pressure pulse, and then 5 mM NiCl2 was applied
to the closed channels (Fig. 7). After
exposure to Ni2+ for 2 min, a second pulse of identical
pressure was applied. A change in the channel activities in both pulses
was measured as a ratio:
Imax2/Imax1, where
Imax1 and Imax2 represent
peak currents (corresponding to the number of active channels) during the first and the second pulse, respectively. Fewer channels open in
response to the second pulse because of the Ni2+
coordination and the ratio
Imax2/Imax1 = 0.46 ± 0.01 (n = 3). After washout of the chamber with the
bath solution devoid of NiCl2 and with 2 mM
EDTA similar two-pulse procedure was performed. The response to both
pulses was very similar,
Imax2/Imax1 = 0.99 ± 0.02 (n = 3), and it returned to its initial level.
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A procedure of similar time scale was used to the open
YggB-His6 channels. In this experiment, the first pressure
pulse was not released, and 5 mM NiCl2 was
applied to the open channels. After ~3 min the pressure was released,
and the second short pulse was applied. The activities during the
second pulse were reduced (Imax2/Imax1 = 0.67 ± 0.13s, n = 5) (Fig. 8,
middle trace); however, the degree of the decrease was
similar to the control experiment (Fig. 8, top trace), in
which no nickel ions were added and
Imax2/Imax1 = 0.67 ± 0.08 (n = 4). This suggests that the decline of
activities was due to the lack of recovery of the adapted channels from
their inactive state. We found previously that the recovery rate from an inactive state of the YggB channels was 72 and 95% following intervals of 60 and 120 s, respectively (18). In the experiment presented in Fig. 8, the bottom recording was obtained 2 min
after the middle recording, and all of the channels were
able to recover from their inactive state. Over this time
Ni2+ was present in the chamber; however, nickel ions were
not coordinated by the closed channels as seen from
Imax1 of the lower recording (Imax1 from the bottom
recording/Imax1 from the middle
recording = 1.074). We applied additional 5 mM
NiCl2; so over the next 2 min the closed channels were
exposed to 10 mM NiCl2, and still we did not
observe any effect of coordinating Ni2+ by histidines. As
shown in Fig. 5, the most severe reduction of the YggB-His6
activity occurred at 1 mM Ni2+, suggesting that
the large number of nickel ions must be coordinated by many histidines
from different subunits to have an effect on the channel activity.
Single histidine coordinates nickel ion with a low affinity (millimolar
range) (24); however, nickel ion is coordinated within a stretch of
histidines with a high affinity (micromolar range) (25). This implies
that in the experiment presented in Fig. 8, nickel ions bound to
histidines when the channels were open and probably saturated the
imidazole groups within a single histidine tag (with a high affinity),
preventing the coordination between subunits (with a low affinity) even
after exposing the channels to the high Ni2+
concentration.
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The set of the two experiments described above shows that the closed
channels were able to coordinate Ni2+ between their
subunits, and it resulted in a reduction of the number of active
channels. In the open channels, although they were exposed over similar
time period to the same concentration of Ni2+, intersubunit
binding of Ni2+ did not occur because there was no
inhibition of the channel activities.
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DISCUSSION |
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The existing model predicts that MscL, the large conductance MS channel from E. coli may be gated in a two-step manner (7, 8, 10). In this model there are two gates involved in the channel opening: the transmembrane gate that consists of the intramembraneous channel domains and the cytoplasmic one that is composed of the parts of the N termini. MscS, the small conductance MS channel from E. coli, is different from MscL in its amino acid sequence, membrane topology, and channel activities. The most striking difference is that MscL functions as pentamer and has two membrane-spanning domains, TM1 and TM2, whereas functional YggB is a heptamer and has three membrane domains, TM1, TM2, and TM3 (21). The YggB TM3 domains line the channel pore and, similar to the TM1 domains of MscL, form the transmembrane gate of the channel (21). It has been proposed that both channels, although different, might have a common evolutionary origin (12). The hypothesis was based on a high sequence identity between the MscL TM1 and the YggB TM3 helices. The YggB TM3 helix shows the highest level of conservation among all three membrane domains in various YggB homologues in bacteria (3) (Fig. 2) and eukaryota (13). There are also short spans of C terminus that are highly conserved among YggB bacterial (Fig. 2), archean (12), and eukaryotic homologues (13).
In the model of the MscL functioning, the role of its C terminus is not
clear. The large portion of it (the last 27 amino acids of the total
41) can be deleted, and the truncated channel still remains active;
however, greater deletions result in an inactivation of the channel
(26, 27). Within the portion of C terminus that can be deleted without
affecting channel activities there is an -helical S3 domain. It has
been found recently that S3 helices form a pentameric coiled-coil
structure and separate upon channel opening. It is suggested that they
may not be directly involved in the channel gating (28). The role of
the conserved portion of C terminus adjacent to TM2 and indispensable
for the channel proper function has yet to be established.
The quaternary structure of YggB reveals that extramembranous
C-terminal domains are composed mostly of -sheets and surround a
large water-filled chamber with eight openings with diameters of
~8-14 Å (Fig. 1B, middle panel). The YggB
structure may reflect an open configuration of the channel, and it is
suggested that the movement of solutes to the water chamber occurs via
the openings. The entire cytoplasmic domain would serve as a screening
filter against large molecules (21). At the end opposite to the
membrane of this assembly, there is a parallel
-barrel structure
composed of seven strands, which, in our experiments, were
tagged with the hexahistidine tags.
From the experimental results presented in this study we can conclude
that the C termini of the YggB channel move apart upon channel opening.
The intersubunit Ni2+ coordination between the histidine
tags added to the ends of the -barrel strands leads to inhibition of
the channel activities, and it occurs only when the channel is closed.
This result suggests that these parts are close to each other in the
channel closed state, and they are more distant to each other when the
channel is open. It is likely that they move apart during opening of
the channel when mechanical force is applied. We have also shown that YggB channels become inactive when their cytoplasmic parts were exposed
to BS3 and the nonpolymerized GDH in the closed state of
the channel. We failed to observe this phenomenon when EDC was used.
Our interpretation of these data is that after the cross-linking of
lysines from the YggB C termini, the cytoplasmic domain becomes more
rigid, and therefore the closed conformation of the channel is stabilized.
These results suggest that the entire cytoplasmic domain of the channel is a dynamic structure. It is likely that the movement of the ends of C termini may occur during activation of the channel, and this is due to conformational changes within each subunit and/or due to a change of interactions between subunits. There are several examples in the literature showing that large cytoplasmic domains directly or indirectly participate in the channel gating. Activation of the voltage-gated K+ channel Kv is coupled to the conformational changes in its cytoplasmic domains T1 (29, 30), and the conformational changes induced by Ca2+ binding to the cytoplasmic domains of the bacterial calcium-activated K+ channel MthK are linked to the membrane domains, resulting in the channel activation (31). Cytoplasmic C termini of KcsA, pH-activated bacterial K+ channel, do not directly participate in gating; however, they contribute to modulating of the pH gating and stabilize the closed conformation of the channel (32).
The Ni2+ coordination between YggB subunits occurred with a
Kd in a millimolar range. This affinity is similar
to that estimated for a single imidazole group in solution (24).
Coordination of Ni2+ between multiple histidines on
adjacent subunits of the cyclic nucleotide gating channel was reported
to occur in the range of micromolar concentrations of Ni2+
(33); however, histidines from the center of -hemolysin polypeptide from Staphylococcus aureus were coordinated by
Zn2+ at a concentration of 0.1-0.6 mM (23).
Even higher concentrations of Ni2+ (0.1-50 mM)
were reported to inhibit epithelial sodium channel by coordination of
histidines from two extracellular subunits
and
(34). In our
case a histidine tag was added to the end of a very long C terminus
(~160 amino acids) whose mobility is not known. High concentration of
Ni2+ yielding the inhibiting effect on the channel
activities is probably due to the requirement of coordination of many
nickel ions by multiple subunits. This suggestion is also supported by
another result from this report: a lack of the channel inhibition in
the presence of antiHis6 antibodies. Specific antibodies
can block ion channels providing the targets are located on external
parts of the channels (35). Based on the channel crystal structure, we
assume that the histidine tags added to the ends of the
-barrel strands (Fig. 1B) are easily accessible to the antibodies.
However, they may not bind a sufficient number of the antibody
molecules to block the channel. The internal diameter of the
-barrel
is estimated to be 8 Å (20), and the external one is ~20 Å (our estimation). Because of the flexible hinge present in the antibodies, these molecules bivalently bind epitopes separated by 40-100 Å (36,
37). Using the crystal structure of the Fab antibody fragment with six
histidines bound, we estimated the minimal distance for bivalent
binding to be ~37 Å. Because the external diameter of the
-barrel
is ~20 Å, seven histidine tags can bind monovalently only one
anti-His6 molecule. One monovalently bound antibody
molecule does not suffice to inhibit the channel.
The inhibition of YggB-His6 activities in the presence of
Ni2+ was voltage-dependent. The open
probability of MscS activities are modulated by voltage (16), and it is
likely that voltage induces conformational changes of the channel.
Based on the crystal structure of YggB, such changes within the
transmembrane regions are suggested (21, 38), and they might be
transmitted to the cytoplasmic domains of YggB similarly to how it was
shown on the Kv channel and its T1 domains (31). Even small changes in
the position of the -barrel strands may affect Ni2+
binding to the histidine tags.
We demonstrated previously that exposure of the cytoplasmic side of the MscS channels to 1 mg/ml Pronase almost entirely abolished their activities (18). We concluded that Pronase removed responsiveness of the channels to the membrane tension by disrupting the tension transmission mechanism responsible for the opening of the channel. Now, knowing the amino acid sequence of YggB, the topology in the membrane, and its quaternary structure, we assume that Pronase could digest the C termini and/or the cytoplasmic linker between the TM1 and TM2 domains (Fig. 1A). In the case of the latter, the entire structure of the channel would be destroyed, and we could not expect the channel to be active. In the case of the C terminus digestion, a question arises as to which part of it was removed because this region was crucial for the proper channel function. In our preliminary studies leading to localization of this region, we wanted to delete a part of C terminus and obtain an inactive channel. We removed the last 89 amino acids from the C terminus (the last amino acid in the remaining portion was Asp197, indicated in Fig. 1A) and examined membrane patches of the mutant. We did not detect any YggB activity in any of the examined patches (two preparations, 20 patches of each).2 At present we cannot entirely rule out a possibility that the truncated C termini make the YggB channels unable to assembly in the membrane. However, if taken together with our former observation regarding the effect of Pronase on the assembled channels, the lack of activity in the mutant suggests strongly that the C termini may indeed be involved in activation of the YggB channel.
Based on the results summarized and discussed above, we propose that
the YggB C termini are involved in the gating of YggB. It means that in
addition to the channel transmembrane gate composed of its TM3s, the
other cytoplasmic gate may be formed by as yet undefined fragments of C
termini. As calculated, the heptameric symmetric
transmembrane gate of YggB cannot make a tight constriction resulting
in the closed conformation of the channel because the helices cannot
come closer than at a distance of ~9 Å (38). It is suggested that to
close the conducting pathway, the symmetry of the gate might be broken
or that the periplasmic linker between the TM2 and TM3 helix should
become a part of the gate (38). Alternatively, a part of the C terminus
may contribute to gating by composing a part of the transmembrane gate
or by forming a separate gate. Which fragments of C termini might be
involved in gating? We suggest that the gate may exist within the
removed stretch of 89 amino acids. The fragment proved to be crucial
for gating, and there are several highly conserved regions within it.
More deletion mutations are needed to localize the region of the
hypothetical gate.
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ACKNOWLEDGEMENT |
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We thank Dr. I. R. Booth from the University of Aberdeen for providing the E. coli strains used in this study.
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FOOTNOTES |
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* This work was supported by Grant KBN 6P04C 002 20 from the State Committee for Scientific Research and funding from the Nencki Institute of Experimental Biology.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.
Fellow of the Foundation for Polish Science.
§ To whom correspondence should be addressed: Dept. Cell Biology, Nencki Institute of Experimental Biology, 3, Pasteur St., 02-093 Warsaw, Poland. Tel.: 4422-659-8571; Fax: 4422-822-5342; E-mail: kubalski@nencki.gov.pl.
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M212073200
2 P. Koprowski and A. Kubalski, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: MS, mechanosensitive; TM, transmembrane domain; GDH, glutaraldehyde; BS3, bis(sulfosuccinimidyl)suberate; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride.
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