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INTRODUCTION |
Vibrio cholerae is a Gram-negative bacterium causing
cholera, a several diarrheal disease in humans. After ingestion and
passage through the gastric acid barrier, this bacterium colonizes the small intestine, where it produces virulence factors such as cholera toxin. There is much evidence that the production of virulence factors
is under a complex array of environmental stimuli that influence the
activity of regulators, such as the transmembrane protein ToxR (1).
Examples of factors that modulate expression of the ToxR regulon
include pH, salt, amino acids, and bile. As bacteria progress through
different microenvironments during the infection process, it is likely
that they sense and respond to these, and maybe some yet unknown,
stimuli to maximize survival, colonization, and virulence. Little is
known about the importance of the outer membrane composition in these
processes. Because the expression of outer membrane proteins is itself
regulated by many of the environmental conditions that also influence
virulence factor production (1-4), it is clear that there is a need
for a thorough characterization of the function of outer membrane proteins to elucidate the reason for this parallel regulation. In
particular, much would be learned from investigating the role that
outer membrane proteins may play in allowing specific chemical environmental signals to reach ToxR and other cytoplasmic membrane regulators, at the appropriate time and in the appropriate circumstances.
Six major outer membrane proteins have been reported in V. cholerae (5). The 45-kDa protein OmpS is a maltoporin induced upon growth on maltose and similar to the LamB porin of
Escherichia coli (6). OmpV (25 kDa) is a heat-induced,
highly immunogenic protein associated with peptidoglycan (7). The
35-kDa outer membrane protein (OmpA) is heat modifiable and reminiscent
of OmpA of E. coli (8). The OmpX protein (27 kDa), although
osmoregulated and trypsin-resistant, does not have pore-forming
properties and is not classified as a porin (5). Finally, OmpU (38 kDa)
and OmpT (40 kDa) have been shown to allow the transport of hydrophilic solutes in liposome swelling assays (5) and are considered general
diffusion porins. Similar proteins have also been described in other
Vibrio species and shown to have expression patterns that
are sensitive to medium composition or external conditions (4,
9-11).
The expression of the ompU and ompT porin genes
is regulated by ToxR in opposing ways; ToxR activates ompU
transcription but represses ompT transcription (2, 12, 13).
Levels of OmpU and OmpT porins are also affected by changes in
osmolarity and by other environmental signals (2, 5). Of particular
interest is the stimulation by bile of the ToxR-dependent
transcription of ompU (4), because it has also been shown
that cells expressing only OmpU porin are more resistant to bile than
cells expressing solely OmpT (14). These types of OmpU-expressing cells
also have a greater ability to colonize the small intestine and to express virulence factors than cells expressing solely OmpT (14). Interestingly, the OmpU-mediated flux of
-lactam antibiotics is also
impervious to the presence of bile in the external solution, whereas
permeability through OmpT is reduced by bile in a
concentration-dependent manner (15).
With the long-term goal of identifying the molecular basis for this
difference in bile sensitivity between the two porins and in the spirit
of establishing correlations between porin function and the ability of
external stimuli to activate cytoplasmic membrane sensors, we have
undertaken a detailed biophysical study of the pore properties of the
OmpU and OmpT porins by electrophysiology. Comparative studies between
these two porins have been so far limited to a description of their
sugar and antibiotic permeability (5, 15). This type of data only
provides some very general characterization of pore properties.
Electrophysiological techniques can provide more precise and more
extensive knowledge on parameters such as selectivity, modulation,
probability of being in the open state, etc. A previous
electrophysiological analysis was performed on OmpU reconstituted in
planar lipid bilayers, but a comparison with OmpT was not provided
(16). The 40-kDa MOMP (major outer membrane protein) of
Vibrio anguillarum was investigated with a similar
technique (17). The work presented here provides the first detailed
characterization of purified OmpU and OmpT by two complementary
electrophysiological techniques, namely patch clamp and planar lipid
bilayers. The former approach allows the detection of single channels
and provides high resolution analysis of channel behavior (18); the
latter allows the investigation of population of channels and is
particularly useful in the study of voltage dependence (19). Together,
these two techniques have allowed us to highlight a number of
distinctive functional features of the OmpU and OmpT porins, which may
shed some light on the impact of regulation of outer membrane
composition on cell survival and pathogenesis.
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EXPERIMENTAL PROCEDURES |
Strains, Chemicals, and Media--
The strains used were
V. cholerae KKV1107 and KKV1108, expressing only
ompU or ompT, respectively (15). The cells were
grown in LB broth (1% tryptone, 0.5% NaCl, and 0.5% yeast extract)
with 100 µg/ml streptomycin. Tryptone and yeast extract were from
Difco Laboratories. N-Octyl-oligo-oxyethylene
(Octyl-POE)1 was purchased
from Alexis Biochemicals. Other chemicals were from Sigma or Fisher.
Protein Purification--
The cells were grown in LB broth to an
OD600 of ~0.8, harvested by centrifugation at
6,000 × g, washed with cold 10 mM Hepes (pH 7.6), and resuspended at a concentration of 20 g of wet
weight/100 ml of 10 mM Hepes (pH 7.6) containing 0.4 mg/ml
DNase. The cells were subsequently broken with a French press at 16,000 p.s.i. The unbroken cells were removed by centrifugation at 8,000 × g for 45 min at 4 °C, and a total membrane fraction
was obtained after ultracentrifugation of the supernatant at
350,000 × g for 15 min at 4 °C. The membrane pellet
was resuspended with a glass-Teflon homogenizer in 20 mM
sodium phosphate buffer (pH 7.6) containing 10 mM NaCl and
1% Octyl-POE and stirred for 30 min at 4 °C (first extraction). The
suspension was then ultracentrifuged at 350,000 × g
for 15 min at 4 °C. Another extraction at 1% Octyl-POE, followed by
two extractions at 3% Octyl-POE, was performed on the samples. OmpU
and OmpT porins were specifically extracted at 3% Octyl-POE. The 3%
extracts were subjected to dialysis (Spectra/Por 7, VWR) against 10 mM sodium phosphate buffer (pH 7.6), 50 mM
NaCl, 0.5% Octyl-POE, and the proteins were quantified with the
bicinchoninic acid assay (Pierce). Five mg of proteins were applied to
an anion exchange column (Mono Q HR5/5; Pharmacia Corp.) equilibrated
with 10 mM sodium phosphate buffer (pH 7.6), 50 mM NaCl, 0.5% Octyl-POE. The proteins were eluted with a
salt gradient (final concentration, 1 M NaCl). In some
cases, fractions containing the protein of interest were concentrated
on a Centriprep or Microcon (Amicon) unit (10,000 molecular weight
cut-off), and subsequently purified by size exclusion chromatography on
a Superdex 75 HR 10/30 column (Pharmacia Corp.). Protein visualization
and purity were assessed by silver staining after SDS-PAGE. The samples
were either left at room temperature or heated at 96 °C for 10 min
prior to electrophoresis. The OmpU or OmpT bands were identified by
Western blot using rabbit antibodies. For N-terminal sequencing, the
proteins were transferred to a polyvinylidene difluoride
membrane, and sequenced at the Protein Chemistry Core Laboratory of
Baylor College of Medicine (Houston, TX).
Reconstitution in Planar Lipid Bilayers--
Planar lipid
bilayers were formed with azolectin, a lipid preparation containing
essentially phosphatidylcholine (Sigma), as described (20).
Reconstitution of channels was performed by adding 1-2 µg of pure
porin into ~4 ml of 1 M KCl, 10 mM Hepes (pH
7.4) in each compartment. Voltage ramps were applied with an Agilent
function generator at a rate of 1.6 mV/s. The critical voltage for
voltage dependence (Vc) was defined as the
highest voltage at which the slope of the curve tangent reverses sign.
Reconstitution in Liposomes--
Patch clamp experiments were
performed on blisters induced from giant liposomes containing the
reconstituted pure porin (21). Reconstitution into azolectin
multi-lamellar liposomes was performed by a 1-h incubation of the
lipids and pure protein at room temperature followed by a 3-h
incubation of this mixture in the presence of 40 mg/ml BioBeads
(Bio-Rad) (22). Protein:lipids ratios of 1:6,000 to 1:14,000 (w:w) were
typically used. Following this step, the beads were removed by
sedimentation, and blister formation was obtained according to
previously published methods (21). The patches were obtained as
described with 10-megohm pipettes (21). Patch clamp experiments were
performed with the following buffers in the pipette and/or the bath, as
dictated by the experimental protocol: Buffer A (150 mM
KCl, 5 mM Hepes, 0.1 mM K-EDTA, 0.01 mM CaCl2, pH 7.2) or Buffer C (50 mM KCl, 5 mM Hepes, 0.1 mM K-EDTA, 0.01 mM CaCl2, pH 7.2).
Data Recording and Analysis--
The currents were recorded with
an Axopatch-1D amplifier (Axon Instruments), using the CV-4 headstage
for patch clamp experiments and the CV-4B headstage for bilayer
experiments. The current was first filtered at 1 KHz. Continuous
recordings were digitized (VR-100, Instrutech), and data acquisition
was done with the Acquire program (Bruxton) at 84.75-µs sampling
intervals for patch clamp experiments or at 1.356-ms sampling intervals
for bilayer experiments. Analysis was done with a Windows-based program
developed in the laboratory. For OmpT, single channel currents were
measured from the Gaussian fit of amplitude histograms (Igor,
Wavemetrics). The peaks were well defined and gave current values that
were identical to those measured by fitting lines directly on the
computer screen through the open and closed current levels of
individual events. Because the gating transitions are too infrequent
and short-lived in OmpU to give reliable amplitude histograms, single channel currents were obtained from individual events and averaged. Measurements of average closed times or average time at the base line
and calculation of closing probability were obtained from kinetic
analysis of the closures (23). Closing probability was defined as the
ratio of the total time spent at any closed level to the total duration
of the analyzed trace.
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RESULTS |
Purification of OmpU and OmpT--
The solubilization of OmpU and
OmpT from V. cholerae outer membranes was performed by using
Octyl-POE, a detergent successfully used for the crystallization and
functional studies on E. coli and V. anguillarum porins (17, 24). After solubilization and dialysis
(see "Experimental Procedures"), a single anion exchange chromatography step was sufficient to obtain OmpU in a pure form. Although the protein elutes over a wide range of salt concentrations (from 150 to 230 mM NaCl), it was typically found as a
single protein species in two or three fractions eluting at 150 mM NaCl in the elution buffer. These fractions were pooled
and analyzed by SDS-PAGE as shown in Fig.
1. The sample heated at 96 °C ran as a
single band in a gel containing 0.1% SDS (Fig. 1A,
lane 96), as revealed by silver staining. This single band
migrates at a molecular mass of 38 kDa and represents the
denatured monomer of OmpU. If not heated, the same sample shows the
expected trimeric form, running as a ladder of bands at 97 kDa and
above (labeled trimer in Fig. 1A, lane
RT). The ladder is due to the association of the protein with
various amounts of tightly bound lipopolysaccharide molecules, as seen
for OmpF (25). Surprisingly, two other bands of lower molecular masses
also appear at 66 and 31 kDa. Because the same heated sample contains
no other protein except OmpU, the 66- and 31-kDa bands represent other
forms of OmpU, namely the dimer and a monomeric form with a distinct
migration pattern relative to the denatured monomer (labeled
folded monomer in Fig. 1A, because it is likely
to have retained some secondary structure at room temperature). In
addition, these bands were also revealed in a Western blot with an
anti-OmpU antibody (not shown). The existence of a folded monomer has
been seen in MOMP, a porin from Campylobacter jejuni, and
demonstrated to originate from an SDS sensitivity of the trimer even at
room temperature (26). As done for MOMP, we have repeated the SDS-PAGE
procedure, but this time with 10 times less SDS (Fig. 1B).
When the sample is not heated, the protein migrates only as a trimer,
and no dimer or folded monomers are observed, confirming that the
migration pattern observed in 0.1% SDS is due to a loss of tertiary
structure of OmpU. This behavior is unusual for most porins, which are
characterized by their resistance to SDS at room temperature (27).

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Fig. 1.
Protein profiles of purified OmpU and OmpT
samples. A and B, SDS-PAGE analysis of
purified OmpU, with SDS concentrations in the gels as indicated. The
positions of trimers, denatured monomers, and folded monomers marked to
the right of B apply to both panels.
C, protein profiles of three OmpT samples. TQS3 was obtained
from a 3% extract purified with anion exchange chromatography followed
by size exclusion chromatography (70-kDa cut-off). OmpT eluted in the
void volume together with OmpV and OmpA (identified by N-terminal
sequencing). Two consecutive anion exchange chromatographies yield a
pure sample of OmpT (TQQ3). OmpT extracted at 1% Octyl-POE
is fully purified with a single anion exchange chromatography
(TQ1). For all three panels, M indicates the
molecular mass marker, and 96 or RT indicates the
temperature (96 °C or room temperature, respectively) at which
proteins in sample buffer were treated prior to electrophoresis. All of
the gels are silver-stained.
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A similar anion exchange protocol was applied to the purification of
OmpT, extracted with 3% Octyl-POE, and subsequently diluted to 0.5%
Octyl-POE. The protein eluted at NaCl concentrations ranging from 180 to 210 mM but was never found in a pure form. Fractions containing OmpT associated with the least number of contaminants were
pooled and subjected to size exclusion chromatography (70 kDa cut-off).
The void volume was collected (sample TQS3) and analyzed by SDS-PAGE
(Fig. 1C). The nonheated sample (Fig. 1C, lane RT) shows some high molecular mass bands that disappear
upon denaturation at 96 °C, revealing the presence of trimeric and dimeric forms of OmpT. The heated sample (lane 96) contains
three polypeptides identified by N-terminal sequencing as OmpT, OmpV, and the heat-modifiable OmpA, indicating that these proteins migrated as a complex on the size exclusion column. Nevertheless, we were able
to obtain pure samples of OmpT, either by performing two consecutive
anion exchange chromatography steps (Fig. 1C, sample TQQ3) or by extraction of OmpT with 1% Octyl-POE followed by
single anion exchange chromatography (Fig. 1C, sample
TQ1). Samples TQQ3 and TQ1 were used for electrophysiology.
Single Channel Recordings--
For patch clamp experiments, the
purified porins in 0.5% Octyl-POE were reconstituted into liposomes.
Control experiments were performed where the same volume of a
protein-free 0.5% Octyl-POE solution was added to liposomes, and no
channel activity was detected. In the presence of OmpU or OmpT, current
fluctuations are readily observed. The electrophysiological signatures
of OmpU and OmpT are quite different. Both display a typical porin
pattern, i.e. a predominance of channels in the open state,
but vary in the frequency and average dwell time of the closing
transitions. The traces of OmpU activity shown in Fig.
2 are similar to typical OmpF or OmpC
traces (23, 28, 29). The current level labeled BL (base
line) corresponds to the total current flowing through all the channels
of the patch (macroscopic current). Departures from the base line, seen
as downward or upward deflections at positive or negative pipette
potentials, respectively, represent transient closing events. The
closing events typically average a duration in the range of 0.5-1.0
ms, irrespective of the membrane voltage. A slight increase in the
frequency of these transitions is observed at higher potentials,
leading to a decrease in the time spent at the base-line level (more on
this below). For the patch shown in Fig. 2, for example, the average
time spent at the base-line level in between two consecutive closing
events went from 52 ms at
30 mV to 36 ms at
80 mV. This type of
activity has been seen reproducibly in 27 individual patches.

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Fig. 2.
Electrophysiological signature of OmpU.
Representative current traces obtained at the indicated pipette
voltages in buffer A. The preferred current level, denoted base line
(BL) corresponds to the total current flowing through all
open porins of the patch. Transient closures are represented by
downward or upward deflections from the base line at positive and
negative pipette voltages, respectively.
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OmpT is characterized by a much more active gating pattern (Fig.
3). At small potentials, the predominant
current level is also that corresponding to the current flowing through
all the channels, but the average duration at the base line is much
shorter than for OmpU (for example, ~16 ms at
30 mV for the
illustrated patch, which has a comparable number of channels to the
OmpU patch above). Notably, the average duration of the closing events
ranges from 2 to 30 ms, depending on the voltage used. These values are much larger than the 0.5-1.0-ms average closing duration found for
OmpU. As seen in Fig. 3, OmpT displays frequent prolonged closures,
which are never seen with OmpU. The combination of more frequent and
more prolonged closures makes OmpT a more active and less frequently
open channel than OmpU. This gating pattern has been consistently
observed in 31 patches. Occasionally (in <10% of the traces), the
channel temporarily switches to an even more active gating pattern
characterized by an intense flickering activity (not shown).

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Fig. 3.
Electrophysiological signature of OmpT.
Representative current traces obtained at the indicated pipette
voltages in buffer A. The base line (BL) corresponds to the
total current flowing through all open porins of the patch and is
highlighted by a dotted line. Transient closures are
represented by downward or upward deflections from the base line at
positive and negative pipette voltages, respectively.
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A close inspection of the gating patterns also reveals differences
between the two porins. Representative traces are shown on an expanded
time scale in Fig. 4 to highlight the
details of the closing events. The most frequently observed events in
OmpU patches are closures of small amplitudes, corresponding to a
conductance of 51 pS. On occasion, such events are interrupted by
closures of larger amplitude that are usually five or six times the
small event amplitude. Examples of such transitions are shown in Fig. 4A, where the dashed lines mark the small event
current level and its multiples. It is unclear whether the 51-pS
conductance represents the monomeric conductance or that of a
substate.

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Fig. 4.
Detailed gating patterns of OmpU and
OmpT. Representative current traces are shown on an expanded time
scale to highlight the differences in OmpU and OmpT kinetics. The base
line (BL) corresponds to the total current flowing through
all open porins of the patch. To help comparing traces, all of the
traces are displayed with transient closures shown as upward
transitions, irrespective of the recording voltage. The dashed
lines represent the current levels that are multiples of the
predominant conductance for OmpU (51 pS) or multiples of the monomeric
conductance for OmpT (120 pS). The asterisks below
traces C and D mark the 50-80-pS subconductance
state of OmpT. Note that the OmpT traces are interrupted in the middle,
because specific segments were chosen to highlight certain features.
The pipette voltages are 40 mV (trace A), 50 mV
(traces B and C), and +60 mV (trace
D). All of the traces originate from different patches.
Note the different scale bars.
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For OmpT, the most frequent transition, as clearly seen in the rapid
and prolonged closures of the traces in Fig. 3, has a conductance of
354 pS. Events of conductances that are 1/3 or 2/3 of
this value are also often seen (although not consistently in all
patches, or even at all voltages in the same patch). Such events are
displayed in Fig. 4B. Because the 354-pS conductance is
consistently found at all voltages and in all patches, it may represent
the conductance of the trimer. The dashed lines in Fig. 4
(B-D) have been drawn at levels that represent the
monomeric conductance of ~120 pS and its multiples. Occasionally, a
conductance level of 50-80 pS appears mixed in with the other levels.
This presumed substate is marked by asterisks in the
traces of Fig. 4 (C and D), where it is seen to
be of smaller size than the multiples of the monomer conductance marked
by the dashed lines.
Current-Voltage Plots and Selectivity--
The selectivity of the
OmpU and OmpT channels was investigated by measuring current amplitudes
at various voltages in symmetric and asymmetric ionic conditions. The
slope of the obtained current-voltage plot yields the conductance,
whereas the x axis intercept represents the potential at
which there is not net current (reversal potential, Erev). In symmetric conditions (same ionic
composition of the buffer in the pipette and the bath, here 150 mM KCl), Erev is 0 mV, as expected
in the absence of any driving force for ion movement. In asymmetric
conditions (150 mM KCl in the pipette buffer; 50 mM KCl in the bath buffer), the current is null at a
potential that precisely counterbalances the driving force originating from the concentration gradient. In this condition,
Erev depends on the relative permeabilities of
the channel for anions and cations. Such permeability ratio can be
calculated from Erev by using the Goldman-Hodgkin-Katz equation (30).
As observed for OmpF and OmpC, the frequency and duration of closing
events being so low, it is not possible to obtain amplitude histograms
for OmpU. Thus, we measured the amplitude of individual events for
three separate patches. The data points plotted in Fig.
5A are the averages of current
amplitudes obtained for the most frequent transition (the small
conductance seen in Fig. 4A). The slope conductances
obtained from the linear regressions were identical in symmetric
(closed symbols) and asymmetric (open symbols) conditions (51 pS). The reversal potential was calculated to be
23.7
mV, indicating a preference for cations over anions, with a relative
permeability ratio PK/PCl
of 13.8. In the case of OmpT, amplitude histograms are easily obtained
because the closures are frequent and long-lived. The peaks of the
amplitudes were fitted with Gaussian curves, and the current values
obtained from such fits were averaged across four individual
experiments and plotted in Fig. 5B. The linear regressions
produced slope conductances of 354 and 248 pS in symmetric
(closed symbols) and asymmetric (open symbols)
conditions, respectively. The nonparallel shift of the current-voltage
plots indicates an effect of ionic conditions on the conductance, a
hint that the ionic occupancy of single channels has not yet reached
saturation in 50 mM KCl. The calculated reversal potential
was
15 mV. Thus, OmpT is much less cation-selective channel than
OmpU, with a relative permeability ratio
PK/PCl of 3.6.

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Fig. 5.
Selectivity of OmpU and OmpT. The
current values were obtained for the predominant conductance of each
channel, either by measurement of individual events from three separate
patches (OmpU, A) or from amplitude histograms from four
individual experiments (OmpT, B). The ionic conditions were:
150 mM KCl in both pipette and bath solutions ( ) and 150 mM KCl in the pipette solution and 50 mM KCl in
the bath solution ( ). The other buffer components are given under
"Experimental Procedures." The data were fitted with regression
lines, using SigmaPlot (Jandel). The reversal potentials correspond to
the x axis intercepts.
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Voltage Dependence--
Although the voltage-induced inactivation
of channels has not been demonstrated in vivo and may not
have a physiological relevance (31), it is a well known
electrophysiological property of porins that can be used as a
comparative tool (19, 29, 32). We have used the two complementary
approaches of patch clamp and planar lipid bilayers to investigate this
phenomenon with the V. cholerae porins. By performing
voltage ramps on planar lipid bilayers containing a large number of
reconstituted porins, it is possible to easily measure the so-called
critical voltage (Vc), i.e. the
threshold voltage at which channel inactivation is detected. Typical
figure 8-shaped hysteresis loops were found for both OmpU and OmpT in
planar lipid bilayers where purified proteins had been added to both
sides of the membrane (Fig. 6). The
graphs clearly show that Vc is much larger for
OmpU than for OmpT, and thus that OmpT is more voltage-sensitive. The
values of Vc were found to be 163 ± 9 mV
and
170 ± 10 mV for OmpU (n = 10) and 92 ± 11 mV and
92 ± 11 mV for OmpT (n = 11), for
the positive and negative voltage ranges, respectively. Fig.
6B was obtained from a bilayer with a large macroscopic
current, and thus, the curve is rather smooth. The inset
illustrates an experiment where the macroscopic current was comparable
with that of the experiment in Fig. 6A and shows that in
this case, as in Fig. 6A, it is also possible to observe
sharp deflections in the current because of the abrupt closing of one
or a few channels.

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Fig. 6.
Critical voltage for OmpU and OmpT
inactivation. Triangular voltage ramps were applied at a rate of
1.6 mV/sec to planar lipid bilayers containing purified OmpU
(A) or OmpT (B) channels. Each plot shows the
current obtained in response to the applied voltage for a single cycle
of the following sequence: 0 mV maximum positive voltage 0 mV
maximum negative voltage 0 mV (sequence shown by small
arrows near the OmpU trace). The maximum voltages were ±200 mV
for OmpU and ±130 mV for OmpT. The inset in
B is obtained from an OmpT bilayer with comparable
macroscopic current to the OmpU bilayer of A. For the
inset, only the 0 mV +130 mV and the 0 mV 130 mV
ramps are shown for sake of clarity. See "Experimental Procedures"
for definition of the critical voltage.
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Patch clamp experiments allowed us to observe the detailed behavior of
individual channels. For OmpU, increasing the membrane voltage causes a
small increase in the frequency of closing transitions and has no
effect on the average closed time. However, because the effect is mild
and the closures are still extremely short-lived, there is no
appreciable increase in overall closing probability (Fig.
7) in the voltage range of
110 to +110
mV (it is hard to obtain stable recordings at voltages greater than
this). A similar analysis was performed with OmpT channels. As
indicated above, OmpT patches can show some variability in the extent
of gating to conductance levels that are fractions of the trimeric
conductance. To ensure consistency, we have restricted our kinetic
analysis to OmpT patches that mostly display closing transitions to the trimeric conductance level over the whole voltage range investigated and have a limited gating to lower conductance levels. Fig. 7 shows the
averaged results obtained from seven such patches. As seen in planar
bilayer experiments, OmpT reveals a more voltage-sensitive behavior
than OmpU, with increased closing probability at voltages greater than
±50 mV. There is a hint of some asymmetry in this behavior, because
negative pipette voltages tend to be more effective than positive one.
As pointed out earlier, the increase in closing probability originates
from a combination of increased closing frequency, more prolonged
closed times, and gating to current levels that correspond to the
simultaneous closure of multiple trimers.

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Fig. 7.
Voltage dependence of channel gating.
The closing probability was obtained in patch clamp experiments as
described under "Experimental Procedures" and averaged across three
OmpU patches ( ) and seven OmpT patches ( ) at the indicated
voltages, in symmetric buffer A. The errors bars are S.D.
and may lie within the thickness of the symbols for some
points.
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DISCUSSION |
The outer membrane of V. cholerae contains six major
proteins, of which only OmpT and OmpU appear to have properties that are similar to those of the E. coli general diffusion porins
OmpF and OmpC (5). Much work has been performed on the purification and
biochemical analysis of the OmpU porin. However, functional characterization of OmpU and OmpT has been limited. The experiments presented here provide a detailed and more complete comparative study
of these major outer membrane proteins.
Different procedures have been documented in the literature for the
purification of major outer membrane proteins from various Vibrio species. Detergents such as Triton X-100 (5), sodium deoxycholate (10), Genapol (16), and Octyl-POE (17) have been used. The
use of Triton X-100 was justified by Chakrabarti et al. (5)
on the basis of the irreversible denaturation of OmpU by SDS, although
no data on this phenomenon were presented. Here, we have confirmed that
the presence of SDS in electrophoresis gels is sufficient to promote a
breakdown of OmpU tertiary structure, a behavior that is not seen with
E. coli porins (27) but has been documented for porins of
other organisms (26), including other Vibrios (10). It is
likely that the 31-kDa band that we detect in nonheated samples
corresponds to the 32-kDa form reported for OmpU when heated at
50 °C (33, 34). Our initial purification of OmpT following an
extraction with 3% Octyl-POE yielded a complex of OmpT with OmpA and
OmpV, as evidenced by the co-migration of these polypeptides on a size
exclusion column. This complex was not documented by Chakrabarti
et al. (5) in their OmpT purification, but it is likely that
this is due to their use of Triton X-100 (and not Octyl-POE) for
solubilization. Indeed, we also found that the complex could not be
isolated from samples first solubilized with 1% Octyl-POE, and thus
the ability of the three proteins to remain in close association is,
not surprisingly, dependent on solubilization conditions. Whether the
existence of the complex has functional implications remains to be shown.
The electrophysiological demonstration of pore activity has been
documented for V. cholerae OmpU (16) and for a major outer membrane protein MOMP of V. anguillarum (17). The porins
described in these reports have similarly functional properties as
those presented here, such as large conductance, cation selectivity, and oscillations between open and closed states. Only liposome swelling
and antibiotic flux assays were performed with OmpT (5, 15). The patch
clamp analysis presented here allows a detailed comparison of the
activity of these two pore-forming proteins. The salient
distinctive features that this work has revealed can be summarized as
follows: 1) OmpT exhibits more frequent and more prolonged closures
than OmpU and thus appears to be a more dynamic channel; 2) OmpT
is much less specific for cations than OmpU; and 3) OmpT is more
voltage-sensitive than OmpU. Much of the previous literature on these
proteins attempts to draw analogies between these proteins and the
E. coli porins OmpF and OmpC. We believe that some caution
has to be exercised in making such comparisons. For example, OmpT has
more resemblance to OmpF than to OmpC on the basis of poor cation
selectivity and the pattern of environment-regulated gene expression
(27). However, its pore properties based on permeability to sugars (5)
put it closer to the smaller OmpC than to OmpF. Despite this, the most
common gating steps of OmpT correspond to a conductance that is greater
than that of OmpU, even if we take into consideration the conductance
of the less frequent, albeit large, transitions of OmpU. This mixture
of features is reminiscent of V. anguillarum 40-kDa MOMP,
which shares some aspects (but not all) of expression regulation by the
environment with OmpC but whose pore properties suggest a functional
similarity to OmpF (10). Thus, it seems prudent to accept that OmpU and OmpT have their own distinctive features, some of them shared with OmpC
and others with OmpF. OmpT also displays unique characteristics, such
as a much lower threshold voltage for inactivation and a higher
propensity for gating than typically documented for porins. This
uniqueness may correlate with a somewhat different structure, because
no significant homology is found between OmpT and OmpF or OmpC, whereas
OmpU shares ~40% homology with OmpF or OmpC.
It is anticipated that differences in the functional properties of OmpU
and OmpT will provide insights into the physiological significance of
the complex regulation of the expression of these proteins by
environmental conditions. The expression of the ompT and
ompU genes is under the control of the transmembrane
regulator ToxR, whose activity is influenced by environmental stimuli
(1). In particular, ToxR mediates the stimulation of ompU
transcription by external bile or deoxycholate and thus appears
sensitive to these agents (4). The increased level of OmpU in the outer membrane, although not a prerequisite, appears to favor virulence factor expression and intestinal colonization in vivo. A
previous report has documented that toxR
strains, which
express ompT exclusively, are more bile-sensitive (35). In
addition, the mere swapping of porin expression (OmpU instead of OmpT,
and vice versa) in the absence of ToxR has profound effects on
pathogenic properties, with the presence of OmpU positively correlating
with increased bile resistance, expression of cholera toxin and
co-regulated pilus, and intestinal colonization (14). Some results
presented here provide a possible explanation for the increased bile
resistance of OmpU-expressing strains relative to those expressing
solely OmpT. Because OmpU is much more cation-selective than OmpT, and bile salts are negatively charged, it is anticipated that the flux of
such salts through OmpU will be lower than through OmpT. Because bile
is essentially deleterious to cells because of the detergent-induced
damage caused to the inner membrane, the reduced accessibility of the
inner membrane to bile in OmpU-containing strains will provide
protection and thus will impart such strains with the ability to better
survive bile-containing environments. This relatively simple
explanation needs experimental testing with mutants impaired in their
selectivity properties, which is underway. It remains possible that the
selectivity of the porins plays only a part in conferring bile
resistance, because it has been shown that OmpU and OmpT also have
different sensitivities to functional modulation by bile (15). The
molecular relationship between channel selectivity and inhibition by
bile has yet to be examined.
Interestingly, the nonselective nature of OmpT would also provide bile
with the ability to penetrate inside the periplasm, come into contact
with the cytoplasmic membrane-bound ToxR, and thus activate this
protein. A possible scenario can thus be imagined where initial
ingestion of contaminated water or food supply would infect the host
with V. cholerae cells that essentially express OmpT. Upon
reaching the intestine, this nonselective porin would allow some bile
to enter the periplasm and activate ToxR, thus promoting the switch
from an OmpT-containing outer membrane to an OmpU-containing outer
membrane, in addition to expression of virulence factors. The presence
of OmpU would then impart the surviving cells with the ability to
withstand the bile-containing environment and to establish
colonization. Whether some of the other distinct properties of OmpU and
OmpT that we have reported here play a role in this scenario can also
be examined with the help of specific mutants.