Cu(II) Inhibition of the Proton Translocation Machinery of the
Influenza A Virus M2 Protein*
Chris S.
Gandhi
,
Kevin
Shuck§,
James D.
Lear¶,
Gregg R.
Dieckmann¶,
William F.
DeGrado¶,
Robert A.
Lamb§
, and
Lawrence H.
Pinto
**
From the
Department of Neurobiology and Physiology,
Northwestern University, Evanston, Illinois 60208-3520, the
§ Howard Hughes Medical Institute and Department of
Biochemistry, Molecular Biology and Cell Biology, Northwestern
University, Evanston, Illinois 60208-3500, and the ¶ Department
of Biochemistry and Biophysics, The Johnson Foundation, School of
Medicine, University of Pennsylvania,
Philadelphia, Pennsylvania 19104-6059
 |
ABSTRACT |
The homotetrameric
M2 integral membrane protein of influenza virus forms
a proton-selective ion channel. An essential histidine residue (His-37)
in the M2 transmembrane domain is believed to play an
important role in the conduction mechanism of this channel. Also, this
residue is believed to form hydrogen-bonded interactions with the
ammonium group of the anti-viral compound, amantadine. A molecular
model of this channel suggests that the imidazole side chains of His-37
from symmetry-related monomers of the homotetrameric pore converge to
form a coordination site for transition metals. Thus, membrane currents
of oocytes of Xenopus laevis expressing the M2
protein were recorded when the solution bathing the oocytes contained
various transition metals. Membrane currents were strongly and
reversibly inhibited by Cu2+ with biphasic reaction
kinetics. The biphasic inhibition curves may be explained by a two-site
model involving a fast-binding peripheral site with low specificity for
divalent metal ions, as well as a high affinity site
(Kdiss ~2 µM) that lies deep
within the pore and shows rather slow-binding kinetics
(kon = 18.6 ± 0.9 M
1 s
1). The pH dependence of
the interaction with the high affinity Cu2+-binding site
parallels the pH dependence of inhibition by amantadine, which has
previously been ascribed to protonation of His-37. The voltage
dependence of the inhibition at the high affinity site indicates that
the binding site lies within the transmembrane region of the pore.
Furthermore, the inhibition by Cu2+ could be prevented by
prior application of the reversible blocker of M2 channel
activity, BL-1743, providing further support for the location of the
site within the pore region of M2. Finally, substitutions
of His-37 by alanine or glycine eliminated the high affinity site and
resulted in membrane currents that were only partially inhibited at
millimolar concentrations of Cu2+. Binding of
Cu2+ to the high affinity site resulted in an approximately
equal inhibition of both inward and outward currents. The wild-type protein showed very high specificity for Cu2+ and was only
partially inhibited by 1 mM Ni2+,
Pt2+, and Zn2+. These data are discussed in
terms of the functional role of His-37 in the mechanism of proton
translocation through the channel.
 |
INTRODUCTION |
The M2 protein of influenza A virus is thought to
function as an ion channel that permits protons to enter virus
particles during uncoating of virions in endosomes. In addition, in
influenza virus-infected cells the M2 protein causes the
equilibration of pH between the acidic lumen of the trans-Golgi network
and the cytoplasm (reviewed in Refs. 1 and 2). The M2
protein contains a 24-residue N-terminal extracellular domain, a single
internal hydrophobic domain of 19 residues which acts as a
transmembrane domain and forms the pore of the channel, and a
54-residue cytoplasmic tail (3). Chemical cross-linking studies showed
the M2 protein to be minimally a homotetramer (4-6), and
statistical analysis of the ion channel activity of mixed oligomers
indicated that the minimal active oligomer is a homotetramer (7).
Despite the small size of the active M2 oligomer, several
pieces of evidence indicate that the ion channel activity is intrinsic
to the M2 protein. First, ion channel activity has also
been observed in two expression systems in addition to oocytes,
mammalian cells (8) and yeast (9). Second, the activity has also been
reconstituted in artificial lipid bilayers (10) from purified
M2 protein. The currents associated with the M2
ion channel are inhibited by amantadine, its methyl derivative
rimantadine, and the spirene-containing compound BL-1743
(Structure 1). Mutant viruses resistant
to amantadine or BL-1743 have been isolated, and most have been found
to map to the extracellular half of the transmembrane domain of the
M2 protein (11-13). These mutant M2 proteins
have ion channel activity that is insensitive to the compound used to
generate the resistant mutant virus (8, 12, 14-16). Inhibition by
BL-1743 also has the useful property that, unlike inhibition by
amantadine, it is reversible on the time scale of physiological
experiments (15).
The ion selectivity of the M2 ion channel has been examined
with ion substitution and reversal voltage measurements. The channel possesses at least 105-fold selectivity for H+
(8, 17). This finding has been confirmed with intracellular pH
measurements, but the former experiments indicate that other monovalent
cations such as Na+ also permeate (17).
Recently, we proposed a model for the three-dimensional structure of
the transmembrane region of M2, based on a mathematical analysis of the functional properties of a series of mutants (18). The
predicted structure consists of four
-helices arranged with approximate 4-fold symmetry about a central channel, which spans the
transmembrane region of the protein. This model is in very good
agreement with spectroscopic data (19-21) and with an independently proposed structure based on molecular dynamics calculations (22). Our
model provides a rationale for the proton selectivity of the channel;
the tetramer defines a continuous, water-filled pore, which is
interrupted at only one position, His-37 (Fig.
1A) which may act as a proton
shuttle similar to that of carbonic anhydrase (23, 24). A considerable
body of evidence shows that His-37 is indeed essential for the activity
of M2, and mutation of this residue leads to a channel that
conducts ions in a pH-independent manner (25).

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Fig. 1.
Location of histidine residues and possible
mode of interaction of the M2 transmembrane region with
amantadine. A, the previously described model of the
transmembrane region of the M2 protein (18). The fourth
helix of the tetramer has been removed from the front of the structure
to reveal the pore interior. A large cavity can be seen at Gly-34,
followed by an occlusion formed by the His-37 side chains. The backbone
atoms and histidine side chains are shown as stick and
ball-and-stick representations, respectively. A surface
generated using a 1.4-Å radius probe (32) is shown for each helix. The
histidine side chains from each of the helices pack together in an
arrangement that may greatly retard the flow of most ions through the
channel. However, by alternately protonating/deprotonating the
N and N atoms of
the histidine residues, it may be possible to shuttle protons through
the channel in a mechanism that resembles the proton shuttle of
carbonic anhydrase (23, 24). The central pore in our model reaches its
widest diameter (~4 Å) near the center of the bilayer, just above
His-37. The widening of the aqueous pore may be important for
minimizing the dehydration energy of protons as they pass through the
channel; charged species diffusing through an aqueous pore in an
otherwise low dielectric environment encounter an unfavorable
dehydration energy, which reaches a maximum near the center of the
bilayer. This energy barrier may be lowered by widening the pore near
the center of the bilayer (26). B, a modified model
including amantadine within the pore showing possible interaction with
the side chains of His-37. Amantadine is shown in stick
representation with the amine group protonated. The model was generated
by placing one molecule of amantadine within the M2 pore,
centered at the largest opening at Gly-34, doing one round of energy
refinement (500 steps steepest descent/5000 steps conjugate gradient)
with fixed M2 atom positions, followed by a second round of
energy minimization with no atoms fixed. The proposed cavity in
M2 is also believed to comprise the binding site for
amantadine and BL-1743. Amantadine-resistant forms of the virus show
mutations at sites that line the proposed central pore of the channel.
In our model, the hydrophobic adamantyl group interacts with the lining
of the pore, which is composed primarily of hydrophobic groups. Also,
the ammonium group forms stabilizing H-bonded interactions with the
electron lone pairs on the imidazole nitrogen atoms of His-37 (shown in
A). Minimizations were performed in the absence of water
using Discover (Biosym/Molecular Simulations Inc., San Diego, CA). The
figure was generated using Insight95 (Biosym/MSI).
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Amantadine inhibits M2 with a dissociation constant of
approximately 0.2 µM and a relatively slow rate of
association (16). This drug binds to the channel with a half-time on
the order of 10-20 min when the drug is present at low micromolar
concentrations (the second order rate constant ranges from 150 to 1000 M
1 s
1 for M2 from
different amantadine-sensitive subtypes of virus). Interestingly,
another positively charged, hydrophobic drug, BL-1743, is known to bind
M2 at a similar rate, although its rate of dissociation varies widely; the half-time for dissociation of BL-1743 from M2 is approximately 3 min (15), while amantadine
dissociates at a rate too slow to be experimentally measured. The
relatively slow rates at which these drugs associate with the channel
may relate to the mechanism by which they enter the pore. Both drugs are relatively bulky and may experience some steric hindrance as they
enter the channel. In addition, the partial or full dehydration of
positively charged groups as the drugs enter the channel may be
energetically difficult, leading to a slow rate of penetration. We
suggest that these drugs interact with the lining of the pore, which is
composed primarily of hydrophobic groups that form a widening near the
center of the bilayer similar to that found in the K+
channel (26).
To test further the role of His-37 in the function and inhibition of
M2, we have examined the ability of Cu(II) to inhibit the
channel. Examination of the model (Fig. 1) indicated that this ion
could interact with the four histidine imidazole groups similarly to
the role proposed for histidine as the chelating ligand for Cu(II)
binding to prion protein (27). We demonstrate that Cu(II) indeed binds
to a high affinity site within the protein in a slow,
time-dependent process.
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EXPERIMENTAL PROCEDURES |
Site-specific mutagenesis of M2 cDNA was
performed as described previously (15, 19). In vitro
synthesis of mRNA was performed using the mMessage mMachine T7
Transcription Kit (Ambion, Austin, TX).
Microinjection and Culture of Oocytes--
Ovarian lobules from
individually identified Xenopus laevis females (Nasco, Fort
Atkinson, WI) were surgically removed and treated with collagenase B (2 mg/ml; Boehringer Mannheim) in Ca2+-free OR-2 solution
(82.5 mM NaCl, 2 mM KCl, 1 mM
MgCl2, 5 mM HEPES-NaOH, pH 7.5) at 24 °C for
30-45 min to liberate oocytes from follicle cells. Defolliculated
oocytes were washed in OR-2 and maintained in ND-96 solution (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 2.5 mM sodium pyruvate, 5 mM HEPES-NaOH, pH 7.5)
until injection of ~40 ng of mRNA. After this time they were maintained in ND-96, pH 8.5, in order to minimize the proton flux due
to the action of the M2 ion channel. All solutions were
equilibrated with room air except for solutions containing Cu(I), which
were equilibrated with N2. In control experiments we found
that the currents of oocytes expressing the M2 protein
incubated in Barth's solution equilibrated with N2 (pH 7.5 or pH 6.2) for up to 20 min were indistinguishable from those of
oocytes incubated at the same pH in Barth's solution equilibrated with
room air. Metabolic labeling of oocytes and analysis of proteins by
SDS-polyacrylamide gel electrophoresis was carried out as described
previously (15).
Electrophysiological Recordings--
24-48 h after RNA
injection, whole-cell currents were recorded with a two-electrode
voltage-clamp apparatus consisting of a differential preamplifier
(Nihon Kohden MEZ-7101, Tokyo, Japan) that recorded the voltage
difference between a pipette (filled with 3 M KCl) located
in the cell and another in the surrounding bath. A voltage-clamp
amplifier (Nihon Kohden CEZ-1100) provided feedback current to the
oocyte through a second intracellular pipette. Oocyte currents were
recorded in standard Barth's solution (0.3 mM
NaNO3, 0.71 mM CaCl2, 0.82 mM MgSO4, 1.0 mM KCl, 2.4 mM NaHCO3, 88 mM NaCl, 15.0 mM MES,1 pH 6.2, or 15.0 mM HEPES, pH 7.5) or a modified Barth's solution as indicated. Amantadine hydrochloride (Sigma) (10 mM stock
in Barth's solution), CuCl2, and BL-1743 (kindly provided
by Dr. Mark Krystal, Squibb, Wallingford, CT) were diluted as
indicated. To check that the oocytes did not develop nonspecific
leakage currents during the recordings, we applied amantadine
hydrochloride (100 µM) for 2-5 min at the end of the
measurements from each oocyte. Data from an oocyte were only used if
the current in the presence of amantadine was less than 10% of the
initial current.
Reagents--
CdCl2, MgCl2,
PtCl2(NH4)2, and ZnCl2
were purchased from Sigma; CuCl2 and Cu(I)
tetrakis(acetonitrile)hexafluorophosphate were purchased from Aldrich;
MnCl2 was purchased from J. T. Baker Inc., and
AgNO3 was purchased from Fisher.
 |
RESULTS |
Inhibition of Currents by Cu2+--
Cu2+
is a potent, time-dependent
inhibitor of the channel activity of oocytes that express the
M2 protein (Fig. 2 and Table I). In the absence of M2 protein, Xenopus
oocytes have very small currents in the range of pH 4.0-pH 9.0. Oocytes
that express M2 protein have currents that are dependent on
pH of the bathing medium (14, 25). For pH 7.5 the current is double the
background current found in control oocytes. However, at pH 6.2 the
current is about 8-fold higher than the current at pH 7.5 (14, 25). Bathing oocytes that express wild-type M2 protein in a
solution that contains 100 µM amantadine causes the
currents to decrease to background levels within 2-3 min (14, 16).
Prolonged exposure of oocytes to Cu2+ can be toxic, so we
took a number of precautions to ensure that the observed inhibition was
specific to the M2 ion channel. Before applying media
containing transition metals, we first confirmed that the whole-cell
current of each oocyte was activated by low pHout. We also
tested each cell for the inhibition of current by amantadine by
applying the drug after washing out the transition metal. The data
reported here are from oocytes whose currents were increased 4-6-fold
by decreasing pHout from pH 7.5 to pH 6.2 and whose
currents were reduced to 100-200 nA by amantadine (100 µM for 2 min). These values for activation by low pH and inhibition by amantadine are within the range for
M2-expressing oocytes in the absence of transition metals
and thus provide assurance that the inhibition reported is specific to
the M2 ion channel and is not confounded with nonspecific
leakage currents.

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Fig. 2.
Time course of inhibition of the wild-type
M2 ion channel protein by Cu2+. The
symbols in this and the following figures show the recorded
currents (mean ± S.E. for 4 cells), and the lines show
the currents fit with a double exponential function. See Equation 1.
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(Eq. 1)
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See Table I for parameters that were fitted to these
data.
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Table I
Parameters fitted to the time course of inhibition of the wild-type
M2 ion channel protein by Cu2+ (see Fig. 2)
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Addition of Cu2+ to the bathing solution (pHout = 6.2) gives rise to a time-dependent decrease in the
amantadine-sensitive channel activity, approaching full inhibition at
long times (Fig. 2 and Table I). The time course of inhibition differs
from that for amantadine or other hydrophobic drugs, which generally
show simple, pseudo-order first order decay kinetics under conditions
where the drugs are in large excess. Instead, the data for inhibition by Cu2+ is biphasic, and the data can be fit by a sum of
two exponentials (Fig. 2 and Table I), representing a fast and a slow
process with both relaxation times linearly proportional to the
concentration of Cu2+ between 100 and 1000 µM. Indeed, we show below that the initial rapid rate
(93 ± 5 M
1 s
1) is
associated with a nonspecific, partial block of the channel that is not
dependent on His-37, whereas the slower process (18.6 ± 0.9 M
1 s
1) requires the presence of
histidine at position 37. The second order rate constant associated
with this latter process is nearly 2 orders of magnitude slower than
the value observed for inhibition of the M2 channel
activity (A/Udorn subtype) by
amantadine.

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Fig. 3.
Time course of washout after Cu2+
exposures of Fig. 1. Note the apparently paradoxical faster
recovery rate for the 1 mM concentration experiment. The
lines are fits to the double exponential function as shown
in Equation 2.
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(Eq. 2)
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See Table II for parameters that were fitted to these
data.
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Fig. 3 and Table II illustrate the
recovery of the amantadine-sensitive channel activity following removal
of Cu2+. Again, the data can be fit by two exponentials.
Interestingly, the curves show a small initial burst, possibly
reflecting dissociation from the low affinity site, followed by a slow
recovery of channel activity representing dissociation from the high
affinity site. Indeed, the fraction of the fast component of the
recovery kinetics decreased with time of preincubation with
Cu2+ and was essentially unmeasurable after the long (over
20 min) incubation period with 100 µM Cu2+
(see Fig. 3 legend and Table II). The slow dissociation rate constant
(1.8 × 10
4 s
1) corresponds to a
half-life of approximately 2 h for copper bound to the high
affinity site. The dissociation constant for binding of
Cu2+ to the low affinity site of M2 can be
estimated to be approximately 100 µM from the ratio of
the fast process on and off rate constants, assuming that these are
kinetically independent (mechanistic analysis from Appendix A yields
400 µM or 250 µM, depending on the
mechanism chosen). The corresponding dissociation constant for the high affinity site (using the ratio of slow off to fast on rate constants) would be about 2 µM (Appendix A yields 1.9 or 1.6 µM), significantly tighter.
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Table II
Parameters fitted to the time course after exposure to Cu2+
(see Fig. 3)
Values represent means ± S.D.; in three cases the standard
deviation could not be calculated.
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pH Dependence of the Cu2+-binding Site--
To help
identify the nature of the binding sites, we first determined if they
could be titrated within the range of pH we were able to test. This
titration was done by measuring the time course of inhibition and
recovery from inhibition in solutions of various pH values. These
experiments paralleled previous experiments in which we showed that
amantadine binds less strongly at low pH, and we showed that the pH
dependence of the interaction required the presence of His-37.
Similarly, the time course of inhibition was slower at acidic pH (Fig.
4), and the recovery was faster for the
lower values of pH. We were able to test only the effects of pH in the
range pH 5.2-6.2. Below pH 5.2 irreversible effects due to large
proton currents flowing for many seconds were seen, and above pH 6.2 the current amplitude was too small to measure inhibition directly.

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Fig. 4.
pH dependence of onset of inhibition and
recovery from inhibition by 0.25 mM Cu2+.
, pH 6.2; , pH 5.7; , pH 5.2. The arrow indicates
the time at which washout began, and the data points are connected by
the lines.
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Voltage Dependence of Cu2+ Binding--
If the
Cu2+-binding sites lie within the transmembrane region,
then the rate of association of the positively charged copper ion may
be accelerated at negative applied potentials. To test this possibility
we measured the time course of inhibition by 250 µM
Cu2+ at various holding voltages. We found that the rate of
onset of inhibition was greater for more
negative holding voltages (Fig. 5 and
Table III), consistent with the presence
of at least one Cu2+-binding site that is located at least
partially inside the electric field of the membrane.

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Fig. 5.
Voltage dependence of onset of inhibition and
recovery from inhibition by 0.25 mM Cu2+.
, 40 mV; , 20 mV; , 0 mV; , +20 mV; , +40 mV. The
arrow indicates the time at which washout began.
Lines are fits to double exponential functions as yielding
the below tabulated fast and slow forward (kff and
kfs) and backward (kbf and
kbs) rate constants. See Table III for parameters
that were fitted to these data.
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Table III
Parameters fitted to the time course of onset of inhibition and
recovery from inhibition by 0.25 mM Cu2+ for
membrane voltages between 40 and +40 mV (see Fig. 5)
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Directionality of Cu2+ Inhibition--
Cationic,
open-pore blocker molecules usually attenuate only the current
originating from the side of the membrane to which they are applied. An
example of this is the block of the outward, but not inward,
K+ current of the squid axon by internally applied
tetraethylammonium+ cation (28). We tested the
directionality of the inhibition by Cu2+ by measuring the
current-voltage relationship of the oocyte (pHout = 6.2)
with slowly varying ramps of current before and at various times after
the application of Cu2+. For these experiments, the leakage
current in the presence of amantadine was subtracted in order to obtain
an accurate estimate of the reversal potential, and the membrane
voltage was restricted to +50 mV to avoid activating endogenous
channels. We found (in six cells) that inward and outward currents were
attenuated equally (see Fig. 6) and that
the reversal voltage remained constant with increasing percentage
inhibition by Cu2+. Thus, inhibition by Cu2+,
like inhibition by amantadine (see Fig. 6, inset), is
bi-directional.

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Fig. 6.
Cu2+ inhibits both inward and
outward amantadine-sensitive currents. The current-voltage
relationship of an oocyte expressing the wild-type M2 ion
channel was measured at the times shown after application of 0.1 mM Cu2+ to the bathing medium. The
inset shows the inhibition after addition of 10 µM amantadine to the bathing medium. Note that both
inward and outward currents were inhibited by both compounds.
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Binding of BL-1743 Prevents Binding of Cu2+--
If
Cu2+ binds to an internal site, it should be possible to
block binding by prior application of a compound that either competes for the same site or occupies the outer regions of the pore. The compound BL-1743 has been shown to be a reversible inhibitor of the
currents of the M2 ion channel (15). Mutations conferring resistance to BL-1743 map to the pore region immediately above His-37
(as viewed in Fig. 1), suggesting that BL-1743 penetrates deeply into
the pore.
We tested the ability of BL-1743 to prevent inhibition by
Cu2+. The test depended on the fact that recovery from
inhibition by BL-1743 (Fig. 7 and Ref.
15) is considerably faster than that from 0.5 mM
Cu2+ (4 min versus 4 h, respectively). To
determine whether BL-1743 prevents inhibition by Cu2+, we
performed the following steps: (i) inhibited the channel completely
with BL-1743; (ii) while maintaining the concentration of BL-1743
constant, added 0.5 mM Cu2+ to the solution for
5 min (such that the Cu2+ would have completely inhibited
the currents had it been applied alone); (iii) washed out free
Cu2+ from the recording chamber briefly (2 min) while
BL-1743 was still maintained in the solution; (iv) washed out BL-1743
and measured the time course of recovery. Our earlier results
demonstrated that the time course of recovery from inhibition by
Cu2+ alone is much slower than that from BL-1743. Thus, if
Cu2+ had gained access to an internal binding site in the
presence of BL-1743, then during washout of BL-1743 the recovery would have been slow and incomplete. However, we found that the time course
of the recovery during washout of BL-1743 did not differ from the time
course that would have been measured had Cu2+ not been
applied (Fig. 7). Thus, BL-1743 prevented the binding of
Cu2+ to a presumably internal site.

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Fig. 7.
Prior exposure to BL-1743 prevents inhibition
by Cu2+. The oocyte currents were measured at pH 6.2 to determine the maximal current. The current was then completely
inhibited by BL-1743 (0.1 mM). While inhibited, 0.5 mM Cu2+ was applied to the oocyte for 5 min, a
long enough time to achieve full inhibition from which recovery would
have been very slow had the Cu2+ been applied alone (see
Fig. 2). After 2 min washout of Cu2+, the BL-1743 was
finally washed out, and the time course of the fraction of the recovery
was measured ( ). This recovery was compared with the recovery from
0.1 mM BL-1743 alone ( ) and the recovery from 0.5 mM Cu2+ alone ( ). Note that the time course
of recovery of current of oocytes treated with BL-1743 before
Cu2+ ( ) was not distinguishable from that of oocytes
inhibited by BL-1743 alone ( ; data from Ref. 15).
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Mutation of His-37 Eliminates the High Affinity
Cu2+-binding Site--
The above results strongly suggest
that the high affinity Cu2+-binding site lies within the
transmembrane pore of M2. To confirm these observations, we
examined the ability of Cu2+ to inhibit a number of
variants of M2 in which potential chelating groups were
altered. The amino acid sequence of the transmembrane region of
M2 (residues 24-43) is:
Asp-Pro-Leu-Val-Val-Ala-Ala-Ser-Ile-Ile-Gly-Ile-Leu-His-Leu-Ile-Leu-Trp-Ile-Leu. The only titratable residue in the transmembrane region is
His-37, and this residue has been shown to be important for amantadine inhibition. Thus, we compared the ability of Cu2+ to
inhibit the mutant proteins M2-H37A and M2-H37G
with its ability to inhibit the wild-type M2 protein. In
addition, we examined the role of the Asp-24, which lies near the N
terminus of the
-helix, and Ser-31, which lines a portion of the
predicted pore. Finally, wild-type M2 protein has two
cysteine residues at positions 17 and 19 in the extracellular domain.
It appeared unlikely that they would be responsible for binding
Cu2+ because these residues participate in disulfide
bonding to stabilize the homotetramer (4-6). Nevertheless, we tested
for this possibility by using a previously characterized
"cysteineless" mutant in which each of the three cysteine residues
of the M2 protein, found at positions 17, 19, and 50, were
mutated to serine.
We examined the time course of inhibition and recovery from inhibition
of the currents of the M2-H37A and M2-H37G
mutant proteins by 0.1 and 1.0 mM Cu2+. The
limiting fractional inhibition was less than for the wild-type protein,
and the rates of inhibition and recovery
for both of these mutant proteins were faster than for the wild-type
M2 protein (Fig. 8 and Table
IV). Unlike the data for the wild-type protein, that for both mutants
could be well fit by single exponentials. The rate of inhibition was
proportional too, and the rate of recovery from inhibition was
independent of the Cu2+ concentration. Both rates were also
independent of membrane voltage. Taken together with the above kinetic
data for the wild-type protein, these data strongly suggest that
mutation of His-37 results in the removal of the high affinity site,
while maintaining the integrity of the peripheral binding site.

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Fig. 8.
Inhibition of the current of the
M2H37-G mutant protein by 0.1 mM
Cu2+ is rapid, incomplete, and reversible. Inhibition
is also relatively independent of membrane voltage. , 40 mV; ,
20 mV; , 0 mV; , +20 mV. See Table IV for parameters that were
fitted to these data. The lines show single exponential fits
with the following rate constants: inset, the time course of
inhibition by 0.1 mM Cu2+ of the
M2-H37A mutant protein (average of four cells) is compared
with that of the wild-type protein (data from Fig. 1 and Fig. 2); note
the rapid onset and offset of inhibition; black bars show
times of application of Cu2+. See Table IV for parameters
that were fitted to these data.
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Table IV
Parameters fitted to the inhibition of the current of M2-H37G
mutant protein by 0.1 mM Cu2+ for voltages between
40 and +20 mV
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To determine the possible location of the low affinity
Cu2+-binding site in M2-H37A and
M2-H37G, we measured the voltage dependence and pH
dependence of their inhibition. The voltage dependence for both
proteins (see Fig. 8 and Table IV for M2-H37G) is similar and much less significant than for the wild-type protein (Fig. 5 and
Table III). Moreover, we found that the inhibition of neither mutant
protein was strongly dependent on pH (see Fig.
9 for M2-H37G), in contrast
to the greater pH dependence found for the wild-type protein (Fig. 4).
These results suggest that replacement of His-37 with a residue
incapable of coordinating Cu2+ leaves the ion channel with
a low affinity binding site that is located near the outside of the
electric field.

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Fig. 9.
Inhibition of the current of the
M2-H37G mutant protein by 0.1 mM
Cu2+ is relatively independent of pH. The inhibition
was measured for three values of pH: pH 6.2 ( ), pH 5.7 ( ), and pH
5.2 ( ).
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We also tested the possibility that the cysteine residues located at
positions 17 and 19 (29), the charged aspartate residue at position 24, or the polar serine residue at position 31 might provide a low affinity
binding site for Cu2+ that may be partly responsible for
the rapid recovery from inhibition by 1 mM Cu2+
(see Fig. 3, Table II, and "Discussion"). We applied
Cu2+ to oocytes expressing the cysteineless (29) mutant
protein and the mutant proteins M2-D24A,
M2-S31A, and M2-S31G. The only departure from
the behavior of wild-type protein for any of these proteins in response
to application of Cu2+ (0.1 and 1.0 mM) was
that the rate of onset of inhibition was slightly slower for the
M2-D24A mutant protein than for the wild-type protein.
Perhaps this difference was due to the reduced charge of the
M2-D24A protein at the mouth of the presumed pore region. These results suggest that neither Cys-17, Cys-19, Asp-24, nor Ser-31
participate directly in the inhibition of the channel by Cu2+.
Metal Ion Specificity--
To characterize further the ion
specificity of the low and high affinity binding sites in
M2, we evaluated a series of transition metal ions,
including Cd2+, Mg2+, Mn2+,
Ni2+, Pt2+, and Zn2+. As was
expected for the replacement of a relatively soft for a hard metal ion,
the replacement of Cu2+ with Mg2+ led to
essentially no inhibition, indicating that Mg2+ does not
interact with either site. Similarly, Mn2+, which has
ligand preferences similar to Mg2+, but also shows some
"soft" character, inhibited the channel by less than 10% at 1 mM concentration. We next examined Cu+ and
Zn2+, which have ligand preferences similar to
Cu2+ but have a preference for octahedral or tetrahedral
complexes and would be less likely to assume the distorted square
pyramidal complex hypothesized for Cu2+. These metal ions
showed partial inhibition of the channel (Table V), and recovery from inhibition by these
metal ions was nearly complete within 2-5 min. We therefore
tentatively assign their effects to interactions with the low affinity
site. In a similar manner, we examined Ni2+ and
Pt2+, which have a preference for forming square planar
complexes; again these ions gave rise to only partial, rapidly
reversible inhibition of the channel (Table V). All of these data
suggest that the high affinity metal ion-binding site is quite specific for Cu2+, whereas the remaining, low affinity site is less
specific and able to interact with a variety of metal ions. To confirm
this suggestion, we tested the ability of 1 mM
Cd2+, Ni2+, and Zn2+ to inhibit the
currents of the M2-H37A and M2-H37G mutant
proteins. Indeed, all three metal ions inhibited these mutants in a
manner similar to that observed for the wild-type protein (data not
shown).
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Table V
Inhibition of wild-type M2 ion channel activity by transition
metals
The inhibition of these metals was measured as described for
Cu2+ with the exception that Cu+ was dissolved as the
tetrakisacetonitrile salt in a medium equilibrated with N2 (see
"Experimental Procedures"). Time is the elapsed time between the
start of incubation with the metal ion and the time when the
measurement was made.
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DISCUSSION |
The experiments described here demonstrate that Cu2+
and other transition metals are capable of inhibiting the
M2 ion channel of influenza A virus. Inhibition and
recovery from inhibition can be explained by a model with two binding
sites, each capable of inhibiting the current upon binding a ligand.
One site, of low affinity and low specificity for Cu2+, is
located near the outside of the electric field of the membrane and only
partially impedes ionic current through the channel. The second site
has higher affinity for Cu2+, is located inside the applied
electric field, and more completely blocks current flow. The high
affinity site is probably formed by the association of imidazole side
chains of His-37 from the transmembrane helices of the M2 tetramer.
The strong voltage dependence of the level of inhibition of the current
of the wild-type channel by Cu2+ (Fig. 5 and Table III)
suggests that the imidazole of His-37 is tightly coupled to the high
affinity binding site for Cu2+. This is consistent with
models showing His-37 to be located well inside the presumed
transmembrane domain (18, 22), which begins with Pro-25. Also,
inhibition by low [Cu2+] is dependent on
pHout. The dependence of Cu2+ inhibition on
pHout occurs within a range of pH that modulates the
currents of the wild-type ion channel. This modulation of the ionic
current has been shown to depend on the presence of His-37. Since
His-37 is the only residue in the transmembrane domain that is
titratable in the range of pH values studied, it is likely that this
residue is also responsible for the pH dependence of inhibition rate. A
reasonable explanation for the slower onset of inhibition at low pH
(Fig. 4) is that competition of H+ and Cu2+ for
a binding site on the imidazole of His-37 favors H+ at low
pH. Finally, the presence of the reversible inhibitor BL-1743 prevents
inhibition by low [Cu2+] (Fig. 7). BL-1743 has been shown
to inhibit the M2 ion channel, and several mutants that are
not affected by the compound have been identified (15). Most of these
mutants map to residues of the transmembrane domain located between
Pro-25 and His-37, suggesting that the compound occupies the outer
region of the pore. It is probable that the compound prevented
inhibition by restricting access of Cu2+ to His-37.
These results point to the predictive power of the current model for
M2 and also add considerably to the mechanistic
understanding of this channel. We have recently proposed that His-37
lies within the conduction pathway of the channel, where it serves as a
relay to selectively shuttle protons down their concentration gradient into the virus. In this mechanism, the channel conducts more
efficiently at low pH primarily because of an increase in the
concentration of the protonated histidine. His-37 allows efficient
shuttling in vivo because it has a pK between pH
5 and 7 where the proton is not too strongly bound. The finding that
Cu2+ binds to a high affinity site associated with His-37
is consistent with this because the normal ligation would require the
histidine to be in its unprotonated, basic form.
Although our experiments indicate that the inner, high affinity binding
site is probably His-37, we were not able to identify a single low
affinity, exterior site. We mutated each residue external to His-37
whose side chain might be thought to bind divalent metal ions (Cys-17,
Cys-19, Asp-24, and Ser-31), and we found that inhibition by
Cu2+ was essentially the same as that found for the
wild-type M2 protein.
As many ligand- and voltage-gated ion channels are modified by
transition metals, it would be of interest to compare the effects of
transition metals on the M2 ion channel with the effects on other ion channels. Among the best-studied of these channels is the
Na+ channel; however, the inhibition of the Na+
channel by transition metals differs greatly from the inhibition of the
M2 ion channel by Cu2+. 1) Cu2+
inhibits both inward and outward currents of the M2 ion
channel, but transition metals inhibit the inward current of the
cardiac Na+ channel. 2) The Cu2+-binding site
in M2 is highly specific for Cu2+, whereas
Cd2+ and Mn2+ inhibit the cardiac
Na+ channel but do not inhibit the M2 ion
channel. 3) The high affinity binding site differs; the imidazole of
His-37 of the transmembrane domain of the M2 ion channel is
probably the high affinity binding site for Cu2+, but
Cys-401 of the pore region of the cardiac Na+ channel is
thought to be the high affinity binding site for Cd2+ and
Zn2+ (30, 31). Such differences in metal binding indicate
that these ion channel proteins have stable, but different, structures that present coordinating ligands of different affinities to permeating metal ions.
The results of our experiments have important implications for future
work. First, Cu2+ has an unpaired electron and thus could
serve as a probe for structural studies of the M2 molecule
using either electron paramagnetic resonance or ENDOR methods. Second,
the inhibition of the M2 ion channel by low
[Cu2+] shares several important properties with the
inhibition by amantadine; both are slowed by low pH, both inhibit
currents in both inward and outward directions, and both depend on the
presence of His-37. If Cu2+ and amantadine interact with
the imidazole of His-37 in a similar fashion, then information gained
using Cu2+ may be helpful in the design of inhibitors that
are more useful than amantadine.
 |
ACKNOWLEDGEMENTS |
We are very grateful to Drs. Thomas
O'Halloran and Christoph Fahrni for helpful discussions; to Dr. Mark
Krystal of Squibb, Wallingford, CT, for making available BL-1743; and
to Christina Bauer for constructing the M2 mutant S31G.
 |
FOOTNOTES |
*
This research was supported by Public Health Service
Research Grants AI-20201 (to R. A. L.) and AI-31882 (to L. H. P.)
from the 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.
Investigator of the Howard Hughes Medical Institute.
**
To whom correspondence should be addressed: Dept. of Neurobiology
and Physiology, Hogan Hall, 2153 North Campus Dr., Northwestern University, Evanston, IL 60208-3520. Tel.: 847-491-7915; Fax: 847-491-5211; E-mail: larry-pinto{at}nwu.edu.
 |
ABBREVIATIONS |
The abbreviation used is:
MES, 4-morpholineethanesulfonic acid.
 |
APPENDIX |
This Appendix derives kinetic models for two-state binding of
Cu2+ to the M2 ion channel protein. We envision
an outer site located in the pore of the channel close to the outside
of the membrane electric field and an inner site located near the
inside and well within the transmembrane electric field. Binding of
Cu2+ to the outer site is assumed to be capable of
inhibiting the current to a fraction of its initial value, whereas
binding to the inner site inhibits completely. States are defined by
their location and Cu2+ occupancy: Sa is the
state defined by unoccupied inner and outer sites, Sb by
occupancy of only the outer site, Sc by only the inner site,
and Sd by both sites occupied. Different, non-equivalent kinetic schemes can be devised to connect the states. We used two as
follows: the first (Scheme 1) prohibiting Sa to
Sc but allowing a direct, reversible Sa to
Sd transition; and the second (Scheme 2) allowing
Sa to Sc and prohibiting Sa to
Sd. Note that no distinction is made in this mechanism
between the two sides of the membrane so it is possible that the
Sd to Sa transition involves Cu2+
entering the cytoplasm of the oocyte.
The forward and reverse reaction rate constants for each transition are
those defined in the following equations of Schemes 1 and 2.
These equations, with the initial condition that
Sa = 1 and all other states are empty, were used (by
the program MLAB; Civilized Software, Inc., Bethesda, which has a
built-in differential equation solving routine (33)) to generate
current-time records for the three different Cu2+
concentrations (100, 500, and 1000 µM). Copper
concentrations were taken to be step functions with transitions
corresponding to those applied in each experiment, and the rate
constants were estimated by curve-fitting. The parameters fitted to the
data using each of these model schemes are shown in Table
IA.
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Table IA
The parameters of the two different model schemes shown in the Appendix
estimated by global curve-fitting of the three data sets, including the
washout phase
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Standard errors, as reported by the MLAB program, are for qualitative
comparison only because they depend on the unsupportable assumption
that the curve-fitting error function varies linearly with the fitting
parameters. The number of parameters in the models discourages
quantitative interpretation of these results.
To compare the site affinities for Cu2+, the reactions
Sa + Cu2+
Sb and Sa + Cu2+
Sc are the relevant equilibria. The
"operational" site dissociation constants
Kouter and Kinner
corresponding to the above reactions can be calculated from the rate
constant ratios. For Scheme 1, Kouter = k1b/k1f = 0.4 mM and KINNER = Kouter k2b/k2f = 1.9 µM. For Scheme 2, Kouter = k1b/k1f = 0.25 mM; Kinner
k2b/k2f = 1.6 µM.
 |
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