Báo cáo khoa học: The outer membrane component of the multidrug efflux pump from Pseudomonas aeruginosa may be a gated channel pptx

The outer membrane component of the multidrug efflux pump
from
Pseudomonas aeruginosa
may be a gated channel
Eisaku Yoshihara, Hideaki Maseda and Kohjiro Saito
Department of Molecular Life Science, School of Medicine Tokai University, Isehara, Japan
OprM, the outer membrane component of the MexAB-
OprM multidrug efflux pump of Pseudomonas aeruginosa,
has been assumed to facilitate the export of antibiotics across
the outer membrane of this organism. Here we purified to
homogeneity the OprM protein, reconstituted it into lipo-
some membranes, and tested its channel activity by using the
liposome swelling assay. It was demonstrated that OprM is a
channel-forming protein and exhibits the channel property
that amino acids diffuse more efficiently than saccharides.
However, antibiotics showed no significant diffusion
through the OprM channel in the liposome membrane,
suggesting that OprM functions as a gated channel. We
reasoned that the protease treatment may cause the distur-
bance of the gate structure of OprM. Hence, we treated
OprM reconstituted in the membranes with a-chymotrypsin
and examined its solute permeability. The results demon-
strated that the protease treatment caused the opening of an
OprM channel through which antibiotics were able to dif-
fuse. To elucidate which cleavage is intimately related to the
opening, we constructed mutant OprM proteins where the
amino acid at the cleavage site was replaced with another
amino acid. By examining the channel activity of these
mutant proteins, it was shown that the proteolysis at tyrosine
185 and tyrosine 196 of OprM caused the channel opening.
Furthermore, these residues were shown to face into the

periplasmic space and interact with other component(s). We
considered the possible opening mechanism of the OprM
channel based on the structure of TolC, a homologue of
OprM.
Keywords: multidrug efflux pump; OprM; channel; gate;
P. aeruginosa.
Pseudomonas aeruginosa causes opportunistic infections in
immunocompromised patients and exhibits natural and
acquired resistance to structurally and functionally diverse
antibiotics [1]. The broad specific antibiotic resistance of
P. aeruginosa is mainly attributable to the synergy of a
tight outer membrane barrier and expression of multidrug
efflux pumps with a low specificity for antibiotics [2–7]. In
P. aeruginosa, four operons encoding multidrug efflux
pumps have been reported. Among these, the mexAB-
oprM operon is expressed constitutively in the wild-type
strain and overexpressed in the nalB-type mutant that
exhibits an elevated level of resistance to fluoroquinolones,
tetracycline, chloramphenicol, macrolides, trimethoprim,
and b-lactams [8–13]. The MexCD-OprJ and MexEF-
OprN pumps that are homologous to MexAB-OprM, are
highly expressed in the nfxB-andnfxC-type mutants,
respectively, and each contributes to multidrug resistance
[11–15]. MexXY has been shown to act in conjunction
with OprM and also confers multidrug resistance on
P. aeruginosa [16–18].
These efflux pumps of P. aeruginosa consist of two inner
membrane-associated components, MexB (MexD, MexF,
and MexY) and MexA (MexC, MexE, and MexX), and an
outer membrane component, OprM (OprJ, and OprN)

[2–4,10,11,15,18]. MexB is a member of the resistance-
nodulation-cell division (RND) family and has been
presumed to act as a drug-proton antiporter. MexA is a
periplasmic fusion protein and is assumed to link the inner
and outer membranes. It has been predicted that OprM
forms a channel allowing the antibiotics to pass through the
outer membrane [2,3,19–21].
Although three components of the multidrug efflux pump
are essential to confer multidrug resistance on P. aeruginosa
[17,22,23], the biochemical and biophysical studies of the
pump components are limited. Recently, Zgurskaya and
Nikaido [24] reported a reconstituted system to investigate
the function of AcrB, a member of the RND family and an
inner membrane component of the multidrug efflux pump
of E. coli. They reconstituted the purified AcrB into
liposome membranes and measured the efflux activity.
AcrB was shown to possess catalytic activity in extrusion of
fluorescent phospholipids from the liposome membrane in
the presence of DpH, and its activity was competitively
inhibited by the substrates of the AcrAB-TolC pump. This
reconstituted system does not directly measure the extrusion
of its substrates, but appears to be applicable to other
members of the RND family such as MexB.
AcrA of E. coli is a member of the membrane fusion
protein (MFP) family and is assumed to act as a linker
between the inner membrane transporter and the outer
membrane channel. Zgurskaya and Nikaido [25] demon-
strated that AcrA exists as a highly asymmetric monomeric
molecule in an elongated form that is sufficient to span the
periplasmic space.

Correspondence to E. Yoshihara, Department of Molecular Life
Science, School of Medicine Tokai University, Isehara, 259-1193,
Japan Fax: + 81 463 93 5437, Tel.: + 81 463 93 5436.
E-mail:
Abbreviations: RND, resistance-nodulation-cell division; MFP,
membrane fusion protein.
Enzymes: a-chymotrypsin (EC 3.4.21.1)
(Received 16 April 2002, revised 6 July 2002, accepted 24 July 2002)
Eur. J. Biochem. 269, 4738–4745 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03134.x
OprM, OprJ and OprN are mutually homologous and
also homologous to TolC, an outer membrane protein of
E. coli. TolC is a versatile protein with multiple roles in
extruding the antibacterial drugs or exporting toxins by
forming complexes with different counterparts [26,27].
Bentz et al. [28] demonstrated that TolC forms a channel
in planar lipid bilayer membranes and that the pore size of
the TolC channel appeared to be insufficient for the
exportation of drugs or toxins. Indeed, Koronakis et al.
[29] recently revealed the crystal structure of TolC and
showed that it forms a b-barrel channel in the outer
membrane.
We tried elucidating the function of the outer membrane
component of the multidrug efflux pump of P. aeruginosa
and showed that OprM forms a channel with some
specificity for amino acids and peptides. Furthermore, we
present data indicating that OprM functions as a gated
channel allowing antibiotics to diffuse through the outer
membrane of this organism.
MATERIALS AND METHODS
Purification of the outer membrane components

of multidrug efflux pumps
The oprM protein was isolated from P. aeruginosa
TNP058 (nalB-type mutant). One litre of fully grown cells
was diluted with 10 L of LB-medium (10 g of Bacto-
tryptone,5gofyeastextractand10gofNaClperlitre,
pH 7.3) and the flask was rotated at 200 r.p.m. at 37 °C
for 4 h. The harvested cells were suspended in 50 mL of
20 m
M
sodium phosphate buffer, pH 7.6, and disrupted
by passing through a French pressure cell three times at
1200 kg cm
)2
. After removing unbroken cells by centri-
fugation at 7000 g for 10 min, the supernatant was
centrifuged at 100 000 g for 60 min at 15 °C. The pellet
was suspended in 50 mL of 20 m
M
sodium phosphate
buffer, pH 7.6, containing 0.8% of sodium lauroylsarco-
sinate and incubated at 30 °C for 30 min to solubilize the
inner membrane proteins. After centrifugation at
100 000 g for 60 min at 15 °C, the pellet was solubilized
in 50 mL of 10 m
M
sodium phosphate buffer, pH 8.0,
containing 2.5% of b-octylglucoside and centrifuged at
100 000 g for 60 min at 15 °C to remove the insoluble
materials. The supernatant was subjected to DEAE-high
performance liquid chromatography equilibrated in

10 m
M
sodium phosphate buffer, pH 8.0, containing 1%
b-octylglucoside and the column was eluted with a
0–500 m
M
linear gradient of NaCl. Fractions containing
OprM were pooled and subjected again to the DEAE-
chromatography [30,31]. The purified protein was subjec-
tedtoSDS/PAGEtoestimatethehomogeneityofthe
purified protein.
To confirm that the purified protein is OprM, we
determined the N-terminal amino acid sequences of the
products generated by protease treatment. The purified
sample was mixed with a-chymotrypsin (10 lgÆmL
)1
)in
10 m
M
sodium phosphate buffer, pH 8.0, containing 1%
b-octylglucoside, and incubated at 37 °C for 5 h. Samples
were subjected to SDS/PAGE (12% gel) and blotted
electrically onto a poly(vinylidene difluoride) membrane.
Protein bands were excised from the membrane and
subjected to Edman degradation using a protein sequencer.
Measurement of the permeability of OprM
Proteoliposomes were prepared as described previously with
minor modifications [30]. Briefly, an appropriate amount of
OprM was mixed with 1 lmole of the lipids phosphatidyl-
choline (PtdCho) and dicetylphosphate at the molar ratio of

97 : 3 in 250 lLof2%b-octylglucoside, and then dialysed
against a large excess of deionized water at 4 °Covernight.
The liposome suspension was transferred to a glass tube,
dried under an N
2
gas stream, and retained in an evacuated
desiccator for 30 min The lipid film was suspended in
133 lLof40mosMstachyosein1m
M
Mops buffer,
pH 7.2 and then vortexed vigorously for 20 s. Diffusion
rates of the test solutes were determined by measuring the
time course of the D
450nm
change [30,32].
The effect of the protease digestion of OprM on the
channel activity was examined as follows. The liposome
membranes with or without OprM were suspended in 20
mosM stachyose or 6% Dextran T-10 in 1 m
M
Mops
buffer, pH 7.2, containing a-chymotrypsin (final concen-
tration of 100 lgÆmL
)1
), and incubated at 37 °Cfor3h.
Then the diffusion rates were measured by the liposome
swelling assay.
Construction of the mutant OprM proteins
To introduce the mutation in the oprM gene, we used a
site-directed mutagenesis system kit (Takara Shuzo). Oligo-

nucleotide primers, Y24S (5¢-GTTCTGCCCGGAGGCC
TGCC) and Y196S (5¢-GCCGACGTCGGAGCTGC
GCT) were employed to substitute Tyr-24 or Tyr-196 to
Ser, respectively. To construct the oprM gene coding the
OprM with two mutations at tyrosine 185 and tyrosine 196,
we used the pKF18 bearing the gene coding the mutant
OprM (Y196C) and the oligonucleotides, Y185C (5¢-ACT
CTTCTGGCAGGTGCCCAG) as the template and the
primer, respectively. The nucleotide sequences of these
mutant genes were determined using an ABI model 373A
sequencer to confirm the introduction of the mutations.
Proteolysis of OprM in the cells by a-chymotrypsin
P. aeruginosa PAO1 cells were grown in 4 mL L broth at
37 °C for 4 h. Harvested cells were suspended in NaCl/P
i
and mixed with a-chymotrypsin (final concentration of
100 lgÆmL
)1
) in the absence or presence of 1 m
M
EDTA.
The cells were incubated at 37 °Cfor60minandthen
0.5 m
M
p-toluenesulfonyl fluoride was added. The cells were
disrupted by sonication and the membrane fraction was
solubilized in the sample buffer. These samples were
subjected to SDS/PAGE and the proteins were blotted on
a poly(vinylidene difluoride) membrane. OprM and its
fragments were visualized by immunostaining with poly-

clonal antibodies directed against OprM [33].
Chemicals
EggyolkPtdCho(typeV-E)anda-chymotrypsin were from
Sigma; N-lauroylsarcosinate and dicetylphosphate were
obtained from Nacalai Tesque Inc., Kyoto. Bacto-tryptone
and yeast extracts were purchased from Difco. b-octylgluco-
side was from Alexis Corp., Switzerland. All other chemicals
used were of the highest purity available commercially.
Ó FEBS 2002 Gating of the OprM channel of MexAB-OprM pump in P. aeruginosa (Eur. J. Biochem. 269) 4739
RESULTS
Purification and identification of OprM
OprM was isolated from P. aeruginosa nalB-type mutant
overexpressing the MexAB-OprM pump and purified by
ion-exchange HPLC in the presence of b-octylglucoside as
described in Materials and methods. SDS/PAGE showed
that the purified protein appeared to be homogeneous
(Fig. 1, lane 2). However, in order to confirm that the
purified protein is OprM, we determined the N-terminal
amino acid sequences of fragment 1, 2, 3 and 4 that were
produced by a-chymotrypsin treatment (Fig. 1, lane 3). The
N-terminal amino acid sequences of fragment 1, 2 and 4 were
determined to be GQNTGAAAVP, DVGVASALDL and
GQNTGAAAVP, respectively, which matched completely
the sequences from glycine 25 to proline 34, from aspartate
197 to leucine 206, and from glycine 25 to proline 34 of
OprM [9]. The results demonstrate that the protein purified
was OprM. On the other hand, the N-terminal sequence of
fragment 3 could not be determined, suggesting that this
fragment contains the N-terminus of the OprM protein since
OprM has been demonstrated to be acylated [34].

Characterization of OprM
The outer membrane components of the multidrug efflux
pumps in P. aeruginosa havebeenassumedtoforma
channel through which antibiotics pass to cross the outer
membrane. Then, to demonstrate the channel activity and
properties of OprM, we examined the permeability of
various solutes through the OprM protein reconstituted
into liposome membranes by using the liposome swelling
assay [30]. First, we tested the glycine permeability, and
found that Gly did not diffuse through the liposomes
without any protein, but diffused through the liposome
containing the OprM protein. The diffusion rates of Gly
increased linearly depending on the amount of OprM
reconstituted into the liposome membranes (Fig. 2 inset).
These results clearly demonstrate that OprM forms the
channel through which Gly can diffuse. Next we exam-
ined the OprM channel properties by measuring the
diffusion of various solutes such as amino acids, peptides
and saccharides. As shown in Fig. 2, it was demonstrated
that the OprM channel possesses a pore allowing the
diffusion of these solutes so that amino acids and
peptides (except the hydrophobic amino acids such as
leucine and phenylalanine) diffused more efficiently than
saccharides. This specific diffusion of the OprM channel
may be unique among the outer membrane channels so
far reported.
Next, we tested the diffusion of antibiotics such as
cephalexin (M
r
374) and cephaloridine (M

r
415). We chose
these drugs as test solutes since they are zwitterionic
b-lactams extruded by the MexAB-OprM pump (data not
shown). The liposome swelling method used here cannot be
applied to negatively or positively charged drugs. However,
it was found that these antibiotics did not diffuse through
the OprM channel, suggesting that OprM may function as a
gated channel.
Opening of the OprM channel
Next we designed an experiment to demonstrate the gating
function of the OprM channel. In general the gate domain
of channel proteins seems to have a flexible region, and the
flexible regions are prone to digestion by proteases.
Accordingly, we expected that the protease treatment might
affect the gating of the OprM channel.
We chose a-chymotrypsin to digest OprM because this
enzyme was shown to digest only at two sites in OprM
(Fig. 1). Even when OprM exists in the liposome mem-
branes, the same sites were digested by a-chymotrypsin
(data not shown). Therefore we treated the OprM
containing liposomes and measured the diffusion of
various solutes including antibiotics through these lipo-
somes. As shown in Fig. 3, it was clearly demonstrated
that the protease treatment caused a noticeable increase in
the diffusion rates of amino acids, peptides and saccha-
rides, and surprisingly allowed cephalexin and cephalo-
ridine to permeate through the OprM channel. These
results suggest that the protease treatment rendered the
channel open probably by affecting the gate structure of

OprM. One may argue that the activation of the OprM
channel by the protease treatment is caused by the gross
destruction of the OprM protein. However, this possibility
seems very unlikely since the protease treatment caused no
significant change in the fluorescence spectrum of OprM,
indicating that no gross structural alteration of the OprM
protein occurred by the protease treatment (data not
shown).
Fig. 1. SDS/PAGE of purified OprM. OprM was purified as described
in Materials and methods and was subjected to SDS polyacrylamide
gel electrophoresis after heating at 100 °C for 5 min. Lane 1, molecular
mass markers: myoglobin (17 kDa), carbonic anhydrase (30 kDa),
aldolase (42 kDa), albumin (66 kDa), a-galactosidase (116 kDa) and
myosin(200 kDa);Lane2,purifiedOprM;Lane3,OprMtreatedwith
a-chymotrypsin. Fragments are indicated by F1, F2, F3 and F4
according to the molecular masses.
4740 E. Yoshihara et al. (Eur. J. Biochem. 269) Ó FEBS 2002
The region involved in the opening of the OprM
channel
Next we investigated how the protease treatment caused the
opening of the OprM channel. As OprM was digested at
tyrosine 24 and tyrosine 196 by a-chymotrypsin, we
examined which proteolysis is responsible for the channel
opening. To answer this issue, we constructed mutant OprM
proteins, that are resistant to the a-chymotrypsin digestion,
by replacing the amino acid at position 24 or 196 with
another one. The mutant OprM (Y24S) and OprM (Y196S)
genes were constructed by site-directed mutagenesis as
described in Materials and methods, and these mutant
proteins were purified by the method described above.

First, we checked whether these mutant proteins were
resistant to a-chymotrypsin. Upon digestion of the mutant
OprM (Y24S) by a-chymotrypsin, it was shown that
fragment 2 and 3 alone were generated (Fig. 4B), the result
indicating that there is no cleavage at the amino acid 24 as
expected. On the contrary, the a-chymotrypsin digestion of
OprM (Y196S) generated all fragments and caused the open
state of channel through which antibiotics permeate (data
not shown), suggesting that another site(s) near tyrosine 196
may be digested by a-chymotrypsin. Since tyrosine 185
resides near tyrosine 196, we considered the possibility that
tyrosine 185 may be digested by a-chymotrypsin. Then,
tyrosine 185 of OprM (Y196S) was substituted with cysteine
and the OprM (Y185C/Y196S) protein was purified des-
cribed above. The digestion of OprM (Y185C/Y196S) by
a-chymotrypsin produced only fragment 1 (Fig. 4A), indi-
cating that the digestion occurred at tyrosine 24 but not at the
positions 185 and 196 of OprM (Y185C/Y196S) as expected.
To examine the effect of these mutations on the OprM
channel activity, we measured the diffusion rates of various
solutes through these mutant proteins. As depicted in
Fig. 5(A), OprM (Y185C/Y196S) exhibited permeability
Fig. 2. Permeability of OprM reconstituted into the liposome membrane. Proteoliposomes were prepared from 1 lmol of lipids (PtdCho and
dicetylphosphate in a 97 : 3 molar ratio) and 5 lg of the purified OprM, and the diffusion rates of amino acids, peptides and saccharides were
examined by the liposome swelling assay as described previously [30]. Solutes used here were 1, glycine; 2, alanine; 3, serine; 4, proline; 5, threonine;
6, glycylglycine; 7, leucine; 8, methionine; 9; phenylalanine; 10, glycylglycylglycine; 11, ribose; 12; arabinose; 13, galactose; 14; glucose and 15,
a-methylglucoside. Inset: effect of the amount of OprM on the diffusion rates of glycine. The proteoliposomes were prepared from the appropriate
amount of OprM and the diffusion rates of glycine through these liposomes were determined.
Fig. 3. Permeability of the OprM channel treated with a-chymotrypsin.
Proteoliposomes were prepared according to the procedure as des-

cribed in the legend of Fig. 2 except that the liposome was suspended in
Dextran T-10 solution containing a-chymotrypsin (100 lgÆmL
)1
). The
proteoliposomes were incubated at 37 °C for 2 h and then subjected to
the liposome swelling assay. The data for the treated and untreated
OprM with protease are represented by closed and open circles,
respectively. Solutes used here were 1, glycine; 2, proline; 3, threonine;
4, glycylglycine; 5, arabinose; 6, ribose; 7, glucose; 8, glycylglycylgly-
cine; 9, a-methylglucoside; 10, cephalexin; and 11, cephaloridine.
Ó FEBS 2002 Gating of the OprM channel of MexAB-OprM pump in P. aeruginosa (Eur. J. Biochem. 269) 4741
almost the same as that of the wild-type OprM (open
circles), showing that the mutation appears not to affect the
channel activity. On the contrary, OprM (Y24S) was shown
to have slightly less permeability towards amino acids and
peptides compared with the wild-type OprM (Fig. 5B, open
circles), which diminished the specificity of the OprM
channel for amino acids and peptides. As it was shown that
these mutations produced no, or a less significant, effect on
the channel activity of OprM, we then investigated the effect
of protease digestion on the channel activity of these mutant
proteins. When OprM (Y185C/Y196S) was digested by
a-chymotrypsin and subjected to the liposome swelling
assay, it was shown that the protease treatment caused a
slight effect on the saccharide diffusion but no significant
effect on the diffusion of amino acids and peptides
(Fig. 5A). Furthermore, the treatment caused no effect on
the permeability of antibiotics through the OprM channel.
These results indicate that the cleavage at tyrosine 24 is
unrelated to the channel opening.

Next, we examined the effect of protease digestion on the
channel activity of OprM (Y24S). As show in Fig. 5B, it was
clearly demonstrated that the protease treatment enhanced
noticeably the permeability of amino acids, peptides and
saccharides. More importantly, the protease treatment
allowed the diffusion of cephalexin and cephaloridine
through the OprM channel.
All these data indicate that the digestion at tyrosine 185
and tyrosine 196 but not at tyrosine 24 is responsible for
opening of the OprM channel.
Tyrosine-185 and tyrosine 196 face into the periplasm
As it was shown that the tyrosine 185 and tyrosine 196 were
directly involved in the opening of the OprM channel, the
next issue to address was whether tyrosine 185 and tyrosine
196 faced into the periplasmic space or the extracellular
medium. To clarify this, we examined the accessibility of
a-chymotrypsin to tyrosine 185 and 196 of OprM existing in
the cell. Hence, P. aeruginosa cells were suspended in NaCl/
P
i
and digested by a-chymotrypsin as described in the legend
of Fig. 6. The digestion of OprM was assessed by an
immuno Western blot using a polyclonal anti-OprM
antibody. As shown in Fig. 6, the protease treatment
caused no digestion of the OprM protein. Next, we
suspended the cells in the presence of EDTA to permeabilize
the outer membrane, and then added a-chymotrypsin to the
Fig. 4. SDS/PAGE of the mutant OprM (Y185C/Y196S) or OprM
(Y24S) treated with a-chymotrypsin. (A) The mutant OprM (Y185C/
Y196S) was treated with a-chymotrypsin (100 lgÆmL

)1
)at37°Cfor
2 h and subjected to SDS/PAGE: Lane 1, mol markers; lane 2,
OprM (Y185C/Y196S) without the protease treatment; lane 3, OprM
(Y185C/Y196S) treated with a-chymotrypsin. (B) Lane 1, OprM
(Y24S) without protease teatment; lane 2, OprM (Y24S) treated with
a-chymotrypsin (100 lgÆmL
)1
)at37°Cfor2h.
Fig. 5. Permeability of the mutant OprM (Y185C/Y196S) channel (A) and OprM (Y24S) (B) with or without the protease treatment. The mutant
OprM (Y185C/Y196S) or OprM (Y24S) channel was reconstituted in liposome membranes with or without a-chymotrypsin in Dextran T-10,
incubated at 37 °C for 2 h and then subjected to the liposome swelling assay. Open and closed circles represent the protease-untreated and treated
data, respectively. Solutes used here were 1, glycine; 2, alanine; 3, serine; 4, threonine; 5, glycylglycine; 6, methionine; 7, phenylalanine; 8, ribose; 9,
glucose; 10, a-methylglucoside; 11, cephalexin; and 12, cephaloridine.
4742 E. Yoshihara et al. (Eur. J. Biochem. 269) Ó FEBS 2002
cell suspension. It was demonstrated that only fragment 1
was produced (Fig. 6, lane 3), suggesting that a-chymo-
trypsin can access tyrosine 24 under these conditions. It
seems unlikely that the EDTA treatment may alter the
accessibility of the surface region of OprM since if so, both
sites of OprM should be digested. These results suggest that
tyrosine 24 faces into the periplasmic space, and that
tyrosine 185 and 195 may be protected from the access of
a-chymotrypsin. As it was considered that this protected
state is produced by the interaction between OprM and
other pump component(s), we reconstituted the purified
OprM protein into liposomes followed by a-chymotrypsin
digestion. When the digestion products were examined by
SDS/PAGE (Fig. 7), it was found that all fragments were
produced under these conditions. Since fragment 4 is

produced when a-chymotrypsin digested at both sites of
OprM, these results indicate that tyrosine 185 and 195, and
tyrosine 24 face into the same side of the membrane, that is,
tyrosine 185 and 195 of OprM reside in the periplasmic
space.
DISCUSSION
In order to investigate the assumed channel activity of the
outer membrane component of the MexAB-OprM pump,
we purified the OprM protein, reconstituted it into liposome
membranes and examined the permeability through these
proteoliposomes by using the liposome swelling assay [30].
When glycine was used as a test solution, it was clearly
demonstrated that glycine could not diffuse through the
liposome without OprM but diffused through those
containing OprM. Its diffusion rates increased linearly to
the amount of OprM reconstituted into the liposome
membranes (Fig. 2). These data indicate unequivocally that
OprM is a channel-forming protein, and are consistent with
the data presented by Wong et al. [35]. They reported that
OprM possesses channel activity with an average single-
channel conductance of about 80 pS in 1
M
KCl.
Next, in order to reveal the characteristics of the OprM
protein, we measured the diffusion rates of various solutes
such as amino acids, peptides and saccharides through the
OprM channel. Consequently it was demonstrated that all
the amino acids, peptides and saccharides so far tested
diffused through the OprM channel, but the permeability of
the amino acids and peptides appeared to be superior to that

of the saccharides (Fig. 2). To our knowledge, this kind of
specificity seems to be unique among the outer membrane
channels so far reported. Interestingly this specificity of the
OprM channel disappeared by the substitution of tyrosine
24 to serine (Fig. 5B). Furthermore, OprJ and OprN, being
the outer membrane components of MexCD-OprJ and
MexEF-OprN pumps, were shown to exhibit similar
specificity (unpublished data)
1
. Therefore, this kind of
specificity may contribute to the function of the efflux pump.
Since it was demonstrated that OprM is a channel protein
but antibiotics could not diffuse through the purified OprM
protein, we considered the possibility that OprM may be a
gated channel. To date, many gated channels have been
reported and the calcium-activated potassium channel from
rat adrenal chromaffin cells is one of them. Solaro et al. [36]
have shown that the activation of this channel induced by
Fig. 6. Proteolysis of OprM in the cells with or without EDTA treat-
ment. The experiments were carried out according to the method
described in Materials and methods. Lane 1 represents the cells with-
out the a-chymotrypsin treatment. Lanes 2 and 3 represent the cells
treated with a-chymotrypsin in the absence or presence of 1 m
M
EDTA, respectively.
Fig. 7. Proteolysis of OprM in the liposome membranes. The liposomes
with OprM were prepared as described in materials and methods
except that the liposomes were dialyzed against 20 m
M
sodium phos-

phate buffer, pH 8.0. The liposome suspension was mixed with
a-chymotrypsin (final concentration of 100 lgÆmL
)1
) and incubated at
37 °C for 2 h. Then the mixture was added to the sample buffer and
subjected to SDS/PAGE. Proteins were stained by Coomassie blue
dye. Lane 1, molecular mass markers; lane 2, proteoliposmes without
the protease treatment; lane 3, proteoliposomes with the protease
treatment.
Ó FEBS 2002 Gating of the OprM channel of MexAB-OprM pump in P. aeruginosa (Eur. J. Biochem. 269) 4743
applying a voltage was followed by an inactivation process,
and the tryrpsin treatment of this channel diminished the
inactivation process. These results indicate that the inacti-
vation process is carried out by plunging the inactivation
domain into the open channel; the inactivation domain lost
this gating function by the protease treatment. Accordingly,
we thought that protease treatment might be applicable to
show the gating function of the OprM channel. When
OprM reconstituted into the liposome membranes was
digested by a-chymotrypsin and the permeability of the
treated OprM examined, it was demonstrated that this
treatment enabled antibiotics to permeate through the
OprM channel, probably by inducing the opening of it
(Fig. 3).
Next, in order to investigate how the OprM channel is
opened by the protease treatment, we determined which
digestion is responsible for the channel opening. Conse-
quently it was demonstrated that the digestion at tyrosine
185 and tyrosine 196 but not at tyrosine 24 is responsible for
the channel opening. The N-terminal inactivation domain

of the potassium channel has been demonstrated to
participate in the inactivation process [37,38]. However,
this gating mechanism is unlikely to hold for the OprM
channel because the digestion at tyrosine 24 located near the
N-terminus of OprM is unrelated to the channel opening.
Recently Li and Poole [21] reported the mutational
analysis of OprM of P. aeruginosa. They showed that two of
several deletions which were stably expressed, spanning
residues G199 to A209 and A278 to N286 were unable to
restore the antibiotic resistance of the cell, indicating that
these regions are essential to the proper function of OprM.
Since tyrosine 196 is very near from glycine 199, it is
conceivable that the region from tyrosine 185 to alanine 209
might take an important role in the OprM function, e.g. the
channel gating.
OprM is a homologue of TolC of E. coli whose crystal
structure has been solved recently by Koronakis et al.[29].
TolC is assembled into a trimer and forms a unique
structure called a Ôchannel-tunnelÕ. TolC traverses the
outer membrane by forming a b-barrel, whereas a-helical
bundles form a long tunnel with a tapered proximal end.
They propose that this channel may transport its
substrates by opening the proximal end of the tunnel.
We compared the amino acid sequences between TolC
and OprM to consider the gating of the OprM channel.
Based on the alignment of OprM and TolC presented by
Li and Poole [21], it was found that tyrosine 185 and
tyrosine 196 of OprM correspond to isoleucine 133 and
phenylalanine 144 of TolC, respectively. The crystal
structure of TolC shows that these residues are located

at the C-terminal side of the helix 3 of TolC and this
portion participates in forming the tapered proximal end
of TolC. From these results we propose the possible
opening mechanism that tyrosine 185 and tyrosine 196 are
located at the tapered end of OprM and the digestion at
these residues dilates this portion. However, what triggers
the opening of the OprM channel during the MexAB-
OprM pump transporting drugs into the extracellular
environment? The digestion profile of OprM in the cellls
or the liposome membranes by a-chymtrypsin showed
that tyrosine 185 and 196 face into the periplasm and
probably interact with other component(s). Furthermore,
MexA is thought to link the outer channel with an inner
membrane component. Accordingly, it is considered that
the interaction of MexA with this portion of OprM may
trigger the opening of this channel.
However, we cannot exclude the possibility of MexB as a
partner of OprM because this protein possesses two large
loops extruding into the periplasm and so can contact with
OprM [39].
On the contrary, Zhao et al. [40] reported that a TonB
homologue in P. aeruginosa influenced multidrug resist-
ance, suggesting interaction between the Ton B homologue
and OprM channel. Therefore, the Ton B homologue may
contribute to the triggering of the channel opening.
To the best of our knowledge, this is the first biochemical
study demonstrating that the outer membrane component
of the multidrug efflux pumps works as a gated channel.
Our study reported here may help to understand the
molecular function of the multidrug efflux pumps. How-

ever, many issues still remain to be clarified, including how
protein-to-protein interaction triggers the opening of the
OprM channel.
ACKNOWLEDGEMENTS
This work was supported by a Grant-in-Aid for Scientific Research
from the Ministry of Education, Science, Sports, and Culture, the
Ministry of Health, Labour and Welfare for Antibiotic Resistance
Research Project, the Japan Association for the Promotion of Science,
and Tokai University School of Medicine, Project Research.
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Ó FEBS 2002 Gating of the OprM channel of MexAB-OprM pump in P. aeruginosa (Eur. J. Biochem. 269) 4745