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J. Biol. Chem., Vol. 277, Issue 42, 39880-39886, October 18, 2002
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From the
Received for publication, July 2, 2002, and in revised form, August 14, 2002
The influenza virus M2
proton-selective ion channel is known to be essential for acidifying
the interior of virions during virus uncoating in the lumen of
endosomes. The M2 protein is a homotetramer that contains
four 19-residue transmembrane (TM) domains. These TM domains are
multifunctional, because they contain the channel pore and also anchor
the protein in membranes. The M2 protein is gated by pH,
and thus we have measured pH-gated currents, the accessibility of the
pore to Cu2+, and the effect of a protein-modifying reagent
for a series of TM domain mutant M2 proteins. The results
indicate that gating of the M2 ion channel is governed by a
single side chain at residue 41 of the TM domain and that this property
is mediated by an indole moiety. Unlike many ion channels where the
gate is formed by a whole segment of a protein, our data suggest a
model of striking simplicity for the M2 ion channel
protein, with the side chain of Trp41 blocking the pore of
the M2 channel when pHout is high and with this
side chain leaving the pore when pHout is low. Thus,
the Trp41 side chain acts as the gate that opens and closes
the pore.
The prediction that the influenza A virus M2 protein
has a proton-selective ion channel activity (Refs. 1 and 2 and reviewed
in Ref. 3) arose from a coupling of various observations on the life
cycle of influenza virus. The M2 protein is an integral membrane protein that is expressed at the plasma membrane of influenza virus-infected cells and is incorporated in small amounts into budding
virions (4, 5). Studies on the mechanism of action of the anti-viral
drug, amantadine (1-aminoadamantine hydrochloride), indicated that
viral escape mutants resistant to the drug mapped to the transmembrane
(TM)1 domain of the
M2 protein (6) and that amantadine affected two steps in
the life cycle, virus uncoating and virus maturation. The effect
of amantadine on inhibition of uncoating is general to all strains of
influenza A virus (7, 8) (reviewed in Refs. 3, 9-11). When a virion
has entered the cell by receptor-mediated endocytosis and the virus
particle is in the acidic environment of the endosomal lumen, the
M2 ion channel is activated and conducts protons across the
viral membrane. The lowered internal virion pH is thought to weaken
protein-protein interactions between the viral matrix protein (M1) and
the ribonucleoprotein (RNP) core (7, 12-15) (reviewed in Ref. 16). In
the presence of amantadine, influenza virus uncoating is incomplete,
because the M1 protein is not released from the RNPs and the RNPs fail
to enter the nucleus. Normally, influenza virus RNPs are transcribed
and replicated in the nucleus (reviewed in Ref. 10). For some influenza
virus subtypes, amantadine inhibits a "late" step in virus
replication. The M2 ion channel activity is activated
during transport of the M2 protein through the exocytic
pathway; this ion channel activity raises the lumenal pH of the
trans Golgi network (TGN), equilibrating pH with that of the
cytoplasm (1, 17-22). Thus, the intralumenal pH of the TGN is kept
above the threshold at which the hemagglutinin (HA) conformational
change to the low pH-induced form occurs, therefore preventing HA
aggregation, which blocks virus release from cells.
Direct evidence that the M2 protein has an ion channel
activity was obtained by using electrophysiological techniques and oocytes of Xenopus laevis (23-29) or mammalian
cells (30-33) that expressed the M2 protein. It was
found that the M2 channel is blocked specifically by
amantadine, is highly proton-selective, and is opened (activated) when
the N-terminal ectodomain is exposed to a low pH environment (25, 30,
31, 34). Furthermore, when mutations in the M2 protein TM
domain that confer resistance to amantadine were introduced into the
M2 protein and the mutants expressed in oocytes, the ion
channel activity was found to be insensitive to amantadine (25). In
addition, when either peptides corresponding to the M2 TM
domain or purified M2 protein were incorporated into planar
bilayers, an amantadine-sensitive current was measured (34-37).
The M2 ion channel protein is a homotetrameric integral
membrane protein with each chain of the mature protein containing 96 amino acid residues (2, 4, 5, 38-43). The coding regions for the
M2 protein have been conserved in all known strains of avian, swine, equine, and human influenza A viruses, and the amino acid
sequence of the M2 protein TM domain has been conserved to a greater extent than the remainder of the protein (44). The TM domain
consists of 19 residues, and a considerable body of experimental
evidence indicates that the M2 protein TM domain constitutes the proteinaceous core (the channel pore) that allows a
flux of protons across the membrane. M2 protein TM domain
histidine 37 is essential for both ion selectivity and activation by a
low pH environment at the M2 N-terminal ectodomain that
resides external to the virion (pHout) (25, 28).
Activation of most ligand- and voltage-gated ion channels is a process
that requires detection of the activating signal and coupling to a
"gate," the portion of the protein that prevents conduction while
in the unactivated state (45). For many ion channel proteins, the
portion of the protein that detects the activating signal (46-50) or
acts as the activation gate (51-54) may be as large as an entire TM
domain consisting of several turns of an Site-directed Mutagenesis, Protein Expression, and Measurement of
Electrical Activity--
The cDNA encoding the influenza virus
A/Udorn/72 M2 protein was subjected to site-directed
mutagenesis using four-primer PCR. The nucleotide sequence of the
entire coding region of the altered cDNAs was confirmed by
nucleotide sequencing.
Culture and Microinjection of Oocytes--
Oocytes were removed
from female X. laevis (Nasco, Fort Atkinson, WI),
defolliculated by treatment with collagenase B (2 mg/ml; Roche
Molecular Biochemicals), and incubated in ND96 (96 mM NaCl, 2 mM KCl, 3.6 mM CaCl2, 1 mM MgCl2, 2.5 mM pyruvic acid, 5 mg/ml gentamicin, 5 mM HEPES, pH 7.6, osmolality ~210
mOs/kg) at 19 °C. Oocytes at stage V were microinjected with 50 nl
of mRNA (1 ng/nl) on the day after defolliculation, incubated for
24 h in ND96 (pH 7.6), and finally incubated for 24 h in ND96
(pH 8.5) at 19 °C before use (25).
Measurement of Membrane Current and Intracellular Injections
during Recording from Oocytes--
Whole-cell currents were measured
using a two-electrode voltage clamp. Electrodes were filled with 3 M KCl, and the oocytes were bathed in either Barth's
solution (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.3 mM
NaNO3, 0.71 mM CaCl2, 0.82 mM MgSO4, 15 mM HEPES, pH 7.5, osmolality ~210 mOs/kg) or a modified solution during the recording.
Continuous current-voltage relationships were measured with ramps of
membrane voltage, because the M2 channel shows no rapid
voltage or time-dependent gating. These ramps typically spanned a range of 120 mV in 2 s. Oocyte holding potential was To identify the region of the M2 ion channel protein
that serves as its activation gate and to assess the extent to which the M2 ion channel gate was open, oocytes of X. laevis expressing the M2 protein were used.
Because the M2 ion channel is both activated by elevated
[H+]out and conducts H+ (31), the
absence of inward H+ currents in the presence of low
[H+]out does not permit the determination
that the channel is closed. One effective way to show that the channel
is closed is to show that in the presence of a deliberately induced
high [H+] inside the cell there is no outward current
while membrane voltage is held at a constant value with high driving
force for H+. Thus, we assessed gating by measuring the
presence of outward currents using two different
acidification/alkalinization protocols while membrane potential was
clamped to a constant value. Both these procedures showed the gate of
the wt M2 ion channel to be closed in high pH medium (Fig.
1, A and B). First,
the oocytes were bathed in acidic (pH 5.9) medium, imposing an inward
current sufficient to acidify the ooplasm by about one pH unit (31), and then the medium was switched rapidly to an alkaline medium (pH 8.5;
Fig. 1A). The outward current expected if the gate were open
in the alkaline medium was not observed (Fig. 1A). Secondly, HCl was injected into oocytes that were bathed in alkaline medium (Fig.
1B), and again outward currents were not observed. In
earlier experiments we had observed that the currents of oocytes
expressing the M2-W41C mutant protein were larger than
those for wt M2 protein (62), leading us to suspect that
the large indole group of the highly conserved Trp41
residue might limit current amplitude. Thus, to test the
possibility that Trp41 acts as the channel gate, mutant
proteins containing residues at position 41 with less bulky side chains
were examined for outward current. When Ala, Cys, or Phe were
substituted for tryptophan, outward currents were able to flow from
acidified oocytes (Fig. 1A, arrow). Two findings
supported the conclusion that this difference between mutant and wt
M2 proteins was due to gating by pHout and not
due to a general inability of the wt M2 protein to conduct outward current (rectification). First, the membrane conductance of oocytes expressing the wt protein, measured upon reintroduction of
bathing solution of pH 8.5 following bathing in low pH solution, returned to nearly the low value measured at pH 8.5 prior to
acidification. Secondly, the wt M2 channel protein has been
demonstrated to conduct outward current when pHout is low
and the membrane voltage is sufficiently positive (26, 31). Outward
currents were also observed for oocytes expressing M2-W41C
(7 oocytes) and M2-W41F (8 oocytes) mutant proteins when
HCl was injected intracellularly. In contrast to oocytes expressing the
wt protein, the conductance of oocytes expressing each of these three
mutant proteins, measured upon reintroduction of bathing solution of pH
8.5 following bathing in low pH solution, remained at the elevated
value measured during the acidification in low pH solution. To better
understand the gating of the M2 proton channel, we compared
the data obtained from M2 protein with data obtained from a
H+-transporting compound that does not have the ability to
be gated, the protonophore FCCP. In contrast to the findings for
wt M2 protein, current did flow from acidified oocytes
treated with FCCP when pHout was increased (Fig.
1A; see also Ref. 31). This result is similar to that
obtained when the indole side chain of Trp41 of the
M2 protein was replaced with Ala, Cys, or Phe. We also measured currents for acidified oocytes expressing the
M2-W41Y mutant protein and found that outward
H+ current did not flow after the transition from low to
high pHout (see Fig. 4). We ascertained that the outward
currents of oocytes expressing the M2 protein were specific
to the M2 protein by applying the specific M2
ion channel inhibitor, amantadine (100 µM) (25, 29). The
outward currents from oocytes expressing the mutant M2
proteins (Fig. 2A), but not
the currents from oocytes treated with FCCP (31), were inhibited by
amantadine. Thus, these data indicate the gating properties of the
M2 ion channel protein are determined by the side chain of
the residue at position 41 of the TM domain and can be influenced by a
single hydroxyl moiety.
The amplitude of the charge carried by the outward current (Fig.
2B) and the extent of acidification of the oocyte while it was bathed in low pH medium were quantified for each of several values
of acidification. The acidification was measured from the change in
reversal voltage, Vrev, of the amantadine-sensitive current
(31). It was found that the charge was proportional to the extent of
the acidification that occurred while the oocyte was bathed in solution
of low pH (Fig. 2C). This relationship was very useful
because it allowed the comparison of the outward currents from
different oocytes that have different levels of expression of the
M2 protein.
Given that Trp41 functions as a gate for outward current,
it was important to determine whether this residue also influences the
openness of the channel at high values of pHout. Thus the relationship between conductance and pHout of the wt
channel protein with that of the M2-W41A, -C, -F, and -Y
mutant proteins was compared. If the gate of the M2 ion
channel protein is removed by a mutation that does not remove its
selectivity filter, then the relationship between conductance and
pHout ought to show a greater degree of openness at high
pHout for the residue 41 mutant channels than for the wt
M2 channel. As shown in Fig.
3, the relationship between conductance
and pHout was shifted to higher pH values for all the
M2 mutant ion channels, indicating a greater
degree of openness for the mutant channels at high pHout
(see below for discussion of M2-W41Y mutant). The
conductance measured at pH 8.2, normalized to the value at pH 4.5, was
consistently higher for oocytes expressing the mutant proteins than for
oocytes expressing the wt protein. We were unable to measure this small
conductance with sufficient precision to tell whether there was a
biologically significant difference among the mutant proteins in their
minimum conductance. We also compared the Vrev of the
currents of oocytes expressing these four mutant proteins with that of
the wt protein. The measurement was made 10 s after switching
from bathing medium of pH 8.5 to bathing medium of pH 5.9, at which
time some acidification of the oocyte had already begun (31). The
values for Vrev were in the range of 35-42 mV and did not
differ between wt and the various M2-W41 mutants tested
(ANOVA p = 0.28, 28 oocytes), indicating that the ion
selectivity of these four mutant proteins does not differ significantly
from that of the wt protein. Thus, the observed shift to higher values
of pHout (Fig. 3) is consistent with the mutant
M2 proteins lacking a region that blocks the pore at high pHout.
The Gate of the Influenza Virus M2 Proton Channel Is
Formed by a Single Tryptophan Residue*
,,
, and**
Department of Neurobiology and Physiology,
the § Department of Biochemistry, Molecular Biology, and
Cell Biology, and the ¶ Howard Hughes Medical Institute,
Northwestern University, Evanston, Illinois 60208-3500
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix (55, 56). The
gate of Shaker-type channels has been studied by comparing atomic
structures of the closed state of the KcsA channel with the open state
of the calcium-activated MthK channel (57, 58). The gate is postulated
to be formed by the cytoplasmic residues of the "inner"
transmembrane helix. In the closed state the cytoplasmic residues of
this helix appose one another closely at a conserved Ala residue, and
opening results from a splaying apart of the cytoplasmic residues
starting from a conserved Gly residue that serves as a "hinge."
This postulated splaying open of the helices in the open state is
consistent with the increased accessibility of the internal residues to
organic reagents that occurs when the Shaker family channels are
activated (52, 59). The influenza virus M2 protein is a
model of minimalism because it has only a single multifunctional TM
domain, which contains the pore of the channel (25), acts both to
target the protein initially to the membrane of the rough endoplasmic
reticulum (ER) and anchor the protein in the ER membrane (61), and, as we show here, contains the activation gate.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 mV unless stated otherwise. Voltage clamping was achieved with a
two-electrode voltage clamp apparatus and the PCLAMP7 program. Intracellular injections of CuCl2, hydroxybenzyl
methanethiosulfonate, and their water controls were confirmed by
inclusion of a small quantity of 6-carboxyfluorescein. The quantity of
fluorescent dye present in the oocyte lysates was measured by fluorometry.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (21K):
[in a new window]
Fig. 1.
The conducting pore of the influenza virus wt
M2 protein, but not the M2-W41F, -C, or -A
mutant proteins, is opened by low pHout and closed by high
pHout(gated). A, oocytes expressing the
M2 protein were bathed in acidic media, resulting in an
inward H+ current (downward deflection) and acidification.
Upon return to alkaline solution, the oocytes expressing the
M2-W41A, -C, and -F mutant proteins conducted outward
H+ currents (arrow), but those expressing the wt
M2 protein did not (data not shown for M2-W41C,
-A). No currents were seen for the mutant channels prior to
acidification because at pHout = 8.5 the concentration of
conducting H+ is very low. Uninjected oocytes
treated with the protonophore FCCP did not display gating; they
conducted inward H+ current when bathed in acidic solutions
and also conducted outward H+ current upon return to an
alkaline bathing solution (arrow). B, oocytes
expressing the wt M2 protein bathed in alkaline solution
did not display outward currents when injected with HCl. Those
expressing the M2-W41C and -F (see arrow) mutant
proteins did (ANOVA p <0.01; injections confirmed with
fluorescent indicator, ANOVA p = 0.25;
M2-W41C not shown), showing that the mutant channel
proteins were open at high pH values, whereas under these conditions
the wt M2 channel protein was closed.
View larger version (14K):
[in a new window]
Fig. 2.
Outward current of M2-W41F mutant
protein is amantadine-sensitive, and its amplitude is proportional to
prior acidification. A, amantadine sensitivity of the
outward H+ currents was evaluated by bathing an oocyte that
had an outward current of at least 0.5 µA (such as shown in Fig.
1A) in solution of pH 5.9 with amantadine for 1 min prior to
return to high pHout or by returning the oocyte immediately
from pH 5.9 solution without amantadine to pH 8.5 solution containing
amantadine. Outward H+ current was not detectable in the
former case and was attenuated by 90% in the latter case, consistent
with the known forward reaction rate constant for amantadine (29).
B, oocytes were bathed in solutions of pH 5.9 for various
times (Vhold = 20 mV), and the charge carried by the
outward H+ currents that flowed upon return to high
pHout was measured (area above interrupted
line). C, the charge carried by outward H+
current was proportional to oocyte acidification as judged by the shift
in Vrev of this highly H+- selective channel.
The finding that the amplitude of the outward charge and current was
proportional to prior acidification made it possible to compare the
outward currents of different cells expressing different proteins in
later experiments. To allow comparison between individual cells
(see "Results"), the amplitude of the outward current was divided
by the change in Vrev to yield a normalized outward
current.
View larger version (17K):
[in a new window]
Fig. 3.
Relationship between membrane conductance and
pHout for oocytes expressing the wt M2 ion
channel protein and the M2-W41A, -C, -F, and -Y mutant
proteins. Conductance was normalized to the value obtained at pH
4.5. Note the shift to higher pH values for the mutants (ANOVA
p <0.01).
Although these results confirmed our suspicion that the bulky indole
side chain of Trp41 is the gate of the channel, it is
possible nevertheless that other amino acids in the inner TM-spanning
region of the protein, but not the wider amantadine-accessible outer
region of the TM domain (29, 63), also participate in the gating
process. Therefore, we performed cysteine-scanning mutagenesis for the
residues that form the cytoplasmic-proximal -helical turns of the TM
domain (42, 62). We used the presence of outward currents into alkaline solutions as an indication that the gate was open. It was found that
replacement by cysteine at only one position, Trp41,
produced a protein that allowed significant outward currents to flow
(Fig. 4).
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To determine whether mutation to cysteine at residue 41 causes large
scale changes in the conformation of the protein, a cysteine-specific reagent was injected into oocytes expressing the M2-W41C
mutant protein, and the gating properties were examined to see whether they were altered in a manner consistent with the known biology. Ideally, one would prefer to restore the function of the wt
M2 channel, but the reagent necessary to produce a Trp-like
side chain, 7-indole methanthiosulfonate, was not commercially
available. We thus resorted to restoring the function of the
M2-W41Y mutant channel from the M2-W41C mutant
channel, because the M2-W41Y mutant channel resembled the
wt M2 channel by not permitting outward currents to flow
from acidified oocytes into solutions of high pHout. The
reagent chosen to do this, hydroxybenzyl methanethiosulfonate, was
expected to produce an altered channel that mimicked the
M2-W41Y mutant channel by forming a Tyr-like side chain
consisting of a phenol adduct with cysteine. It was considered likely
that this adduct would alter the function of the M2-W41C
mutant channel protein, causing it to mimic the M2-W41Y
mutant channel protein if the conformation of the M2-W41C
mutant protein permits the reagent to react with its cysteine side
chain. Amantadine-sensitive currents were recorded 30 min after
injection of 50 nl of 40 mM reagent during a 2-min period
in pH 6.2; during the 30-min period, oocytes were bathed in medium of
pH 7.5. Intracellular injection of this reagent significantly decreased
the amplitude of outward currents from acidified oocytes (see Fig.
5, 30% reduction; ANOVA p
<0.05), consistent with the data obtained for the M2-W41Y
mutant protein. The currents of control oocytes expressing
M2-W41F mutant protein were not affected by intracellular
injection of hydroxybenzyl methanethiosulfonate (ANOVA
p = 0.48, n = 6; injections confirmed by co-injection of fluorescent tracer). This result confirms the notion
that the nature of the side chain at M2 residue 41 determines the gating properties of the M2 ion channel.
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To add support to the notion that the indole side chain of
M2-Trp41 is capable of preventing current flow
through the conducting pore of the M2 protein, we
investigated whether this side chain prevented a reagent applied
intracellularly from accessing the cytoplasmic portion of the channel
pore. Advantage was taken of our finding (64) that Cu2+,
but not Cu1+, applied extracellularly, is capable of
inhibiting the M2 ion channel. The mechanism of inhibition
is by coordination with the His37 selectivity filter,
located closer to the lipid interior of the membrane than
M2-Trp41. The accessibility of intracellularly
applied Cu2+ to the His37 residue in the wt
M2 and M2-W41A mutant proteins was tested. The
mutant M2-W41A was chosen for study because alanine has the smallest side chain among the residue 41 mutant proteins that were
constructed, and indeed M2-W41A has larger currents than the wt M2 protein. Oocytes expressing the wt M2
channel and the M2-W41A mutant were bathed in low pH
solution to activate the M2 ion channel, and the
conductance of the oocytes was measured every minute (Fig.
6). After the first conductance
measurement, Cu2+ was injected intracellularly, and the
effect of Cu2+ injection on oocyte conductance was
measured. For control oocytes expressing either the wt M2
protein or the M2-W41A mutant protein that were not
injected with Cu2+, conductance increased with time while
they were bathed in solutions of low pH because the cytoplasm of the
oocytes became acidified and the concentration of conducting ions near
the membrane increased. The currents for the M2-W41A mutant
channel increased more than for the wt M2 channel, because
the mutant channel allowed more protons to pass. For oocytes expressing
the wt M2 protein, the conductance continued to increase
even after the injection of Cu2+ (injections confirmed by
co-injection of fluorescent tracer). However, for oocytes expressing
the M2-W41A mutant protein, the conductance decreased after
intracellular injection of Cu2+ (two-way ANOVA p
<0.01). These results indicate that accessibility of the
His37 selectivity filter to intracellularly injected
Cu2+ is limited by the bulky indole side chain of
M2-Trp41.
|
. 500 µM, confirmed by
co-injection of fluorescent indicator, p = 0.46), and
the effect on conductance was measured. The conductance of oocytes
expressing either the wt M2 ion channel protein (
) or
the M2-W41A (
) mutant protein that were not injected
with Cu2+ increased monotonically as they became acidified
in the low pH solution (the M2-W41A mutant increased faster
because its currents are larger). The conductance for
M2-W41A mutant channel decreased after Cu2+
injection (
), but the conductance for the wt M2 channel
did not (
), demonstrating the ability of the indole side chain of
Trp41 to limit accessibility to the His37
selectivity filter.
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DISCUSSION |
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Taken together, these data suggest a model for the activation of
the M2 ion channel (Fig. 7).
In the presence of high pHout the channel pore is
obstructed by the indole side chain of Trp41. When
pHout is lowered, the His37 H+
selectivity filter becomes protonated; as a result, the indole of
Trp41 rotates to permit H+ to flow. This
movement may be accomplished by cation-pi interactions (65, 66). After
returning to high pHout, outward current will not flow
because the deprotonation of the His37 selectivity filter
in high pH medium causes the indole of Trp41 to return to
its pore-blocking position. If Trp41 is mutated to have a
smaller side chain, pore blockage cannot occur. Thus, His37
acts as the detector of low pHout, and Trp41
acts as the gate. These data are consistent with the notion that the
Trp41 region of the M2 ion channel undergoes
pH-dependent conformational changes as deduced from
cysteine-scanning mutagenesis experiments. It was observed that
residues 40-43 showed a pH-dependent propensity to form
inter-subunit disulfide bonds on oxidation (67). Our data do not allow
us to determine the contribution made to the gating process by the
positively charged nitrogen atom of the imidazole side chain of
His37. Furthermore, we cannot distinguish which of the four
His residues of each tetramer participate in transport of
H+ from those that participate in interactions with Trp.
Our observation that the M2-W41Y mutant channel is more
similar to the wt M2 channel in its gating behavior than
the M2-W41F mutant channel is consistent with our proposed
mechanism of His-Trp interactions mediating channel gating. For the
small ribonuclease, barnase, interactions among amino acids have been
studied by measuring the stability of the protein. The
pH- dependent interaction between His18 and
Tyr94 was observed to be stronger than the interaction in a
barnase mutant between His18 and Phe94 (68).
This difference between Phe and Tyr is also consistent with the
increase in electronegativity of the aromatic ring of phenol that
results from interaction of the phenolic hydroxyl with a hydrogen bond
acceptor (69), an interaction that is possible for Tyr but not Phe.
However, we do not completely understand the behavior of the
M2-W41Y mutant protein because it requires greater
reductions of pH than the M2-W41C mutant protein to be activated (Fig. 3) but does not permit the passage of outward current
into solutions of high pH.
|
Our data are also consistent with a model in which
M2-Trp41 does not physically block the pore but
instead chemically interacts with His37 in such a fashion
as to prevent His37 from binding either H+ or
Cu2+ presented from the cytoplasmic end of the pore. Thus,
our model is consistent with a change in conformation of the side chain of a single amino acid altering the activation state of the channel. This is simpler than the changes in backbone conformation that are
required for either the acetlycholine receptor (53) or K+
channels (51, 52, 54, 57-59, 60, 70). This proposed mechanism for
influenza virus M2 ion channel gating has the following advantages for the virus: 1) The functions of selectivity and activation are built into only two residues of this small viral protein. 2) Transient exposure to low pHout will result in
lasting acidification of the virion because protons are retained by the tryptophan gate, increasing the effectiveness of the small number of
M2 molecules found in the virion. This mechanism for gating of the M2 ion channel protein may also serve as a model to
help understand the gating interactions that occur in more complicated ion channels.
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ACKNOWLEDGEMENTS |
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We thank Drs. T. Cross, D. Dougherty, and J. Lear for reading the manuscript and Yuan Lin for constructing the mutants.
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FOOTNOTES |
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* This work was supported by Research Grants R37 AI-20201 (to R. A. L.) and R01 AI-31882 (to L. H. P.) from NIAID, National Institutes of Health.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.
An Investigator of the Howard Hughes Medical Institute.
** To whom correspondence should be addressed: Dept. of Neurobiology and Physiology, Hogan Hall, 2153 N. Campus Dr., Northwestern University, Evanston, IL 60208-3500. Tel.: 847-491-7915; Fax: 847-491-211; E-mail: larry-pinto@northwestern.edu.
Published, JBC Papers in Press, August 14, 2002, DOI 10.1074/jbc.M206582200
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ABBREVIATIONS |
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The abbreviations used are: TM, transmembrane; RNP, ribonucleoprotein; ER, endoplasmic reticulum; wt, wild type; ANOVA, analysis of variance; Vrev, reversal voltage; FCCP, mesoxalonitrile4-trifluoromethoxyphenylhydrazone.
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DISCUSSION
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