Expression and initial structural insights from solid-state NMR of the M2 proton channel from influenza A virus

The M2 protein from influenza A virus has been expressed, purified, and reconstituted into DMPC/DMPG liposomes. SDS-PAGE analysis of reconstituted M2 protein in DMPC/DMPG liposomes demonstrates a stable tetrameric preparation. Circular dichroism spectra of the intact M2 protein in detergent indicate 67% alpha-helix. The uniformly (15)N-labeled M2 protein and both (15)N-Val- and (15)N-Leu-labeled M2 protein have been expressed from defined M9 media. The (1)H-(15)N HSQC (heteronuclear single quantum correlation) solution NMR experiments have been performed on the amino acid specific labeled protein in 300 mM SDS-d(25) micelles, and the data indicate a homogeneous preparation. The reconstituted M2/DMPC/DMPG proteoliposomes were used for preparing uniformly aligned solid-state NMR samples for (15)N-(1)H dipolar/(15)N chemical shift correlation experiments. The spectra support a transmembrane helix in M2 protein having a tilt angle of approximate 25 degrees, quantitatively similar to results obtained on the isolated M2 transmembrane peptide reconstituted in DMPC bilayers (38 degrees ). In addition, the spectra suggest that the tetrameric protein forms a symmetric or at least pseudosymmetric bundle consistent with data obtained by other research groups based on electrophysiological measurements and substituted cysteine scanning mutagenesis experiments that characterize a tetrameric structure.     

Transmembrane helix orientation and dynamics: insights from ensemble dynamics with solid-state NMR observables

As the major component of membrane proteins, transmembrane helices embedded in anisotropic bilayer environments adopt preferential orientations that are characteristic or related to their functional states. Recent developments in solid-state nuclear magnetic resonance (SSNMR) spectroscopy have made it possible to measure NMR observables that can be used to determine such orientations in a native bilayer environment. A quasistatic single conformer model is frequently used to interpret the SSNMR observables, but important motional information can be missing or misinterpreted in the model. In this work, we have investigated the orientation of the single-pass transmembrane domain of viral protein ”u“ (VpuTM) from HIV-1 by determining an ensemble of structures using multiple conformer models based on the SSNMR ensemble dynamics technique. The resulting structure ensemble shows significantly larger orientational fluctuations while the ensemble-averaged orientation is compatible with the orientation based on the quasistatic model. This observation is further corroborated by comparison with the VpuTM orientation from comparative molecular dynamics simulations in explicit bilayer membranes. SSNMR ensemble dynamics not only reveals the importance of transmembrane helix dynamics in interpretation of SSNMR observables, but also provides a means to simultaneously extract both transmembrane helix orientation and dynamics information from the SSNMR measurements.     

Ion channel activity of influenza A virus M2 protein: characterization of the amantadine block.

The influenza A virus M2 integral membrane protein has ion channel activity which can be blocked by the antiviral drug amantadine. The M2 protein transmembrane domain is highly conserved in amino acid sequence for all the human, swine, equine, and avian strains of influenza A virus, and thus, known amino acid differences could lead to altered properties of the M2 ion channel. We have expressed in oocytes of Xenopus laevis the M2 protein of human influenza virus A/Udorn/72 and the avian virus A/chicken/Germany/34 (fowl plague virus, Rostock) and derivatives of the Rostock ion channel altered in the presumed pore region. The pH of activation of the M2 ion channels and amantadine block of the M2 ion channels were investigated. The channels were found to be activated by pH in a similar manner but differed in their apparent Kis for amantadine block.     

Diffusion-collision model study of misfolding in a four-helix bundle protein.

Proteins with complex folding kinetics will be susceptible to misfolding at some stage in the folding process. We simulate this problem by using the diffusion-collision model to study non-native kinetic intermediate misfolding in a four-helix bundle protein. We find a limit on the size of the pairwise hydrophobic area loss in non-native intermediates, such that burying above this limit creates long-lasting non-native kinetic intermediates that would disrupt folding and prevent formation of the native state. Our study of misfolding suggests a method for limiting the production of misfolded kinetic intermediates for helical proteins and could, perhaps, lead to more efficient production of proteins in bulk.     

A comparison of three- and four-helix bundle TASP molecules.

We have designed, synthesized and characterized three- and four-helix bundle template-assembled synthetic proteins CTASPs). The TASPs were synthesized using disulphide bonds between the peptides and either the cyclotribenzylene (CTB) template, or the cavitand (BOWL) template, to form the three- and four helix bundles, respectively. The TASPs were constructed using peptides that were linked via their N-termini (peptide CGGGEELLKKXEELLKKG, where X = L, I, Nle or V), or via their C-termini (peptide GEELLKKLEELLKKGGGC). Each TASP was assayed for its structure, stability, 'native-like' characteristics and whether it was a monomer in solution. All TASPs were found to be highly helical, and highly resistant to chemical denaturation using guanidine hydrochloride (GnHCl). Analysis of the GnHCl-induced unfolding curves of the different TASPs demonstrated stability differences based on the number of helices in the bundle, the end of the helix that was attached to the template, and the identity of the core amino acid. The TASPs all had molten-globule structure, which is (generally) consistent with a degenerate sequence in the core. The four-helix bundle TASPs appeared to be monomers in solution, whereas there is some evidence that the three-helix bundle TASPs are weakly self-associating.     

pH-driven helix rotations in the influenza M2 H+ channel: a potential gating mechanism

The pH activated M2 H⁺ channel from influenza A has been a subject of numerous studies due to following: (1) It serves as a target for the aminoadamantane drugs that block its channel activity. (2) M2’s small size makes it amenable to biophysical scrutiny. (3) A single histidine residue is thought to control the pH gating of the channel. Recent FTIR analysis proposed that the helices of the channel rotate about their directors during pH activation. Herein, we report on molecular dynamics simulations of the X-ray structure of the protein with three charged histidine residues, representing the open form of the protein and two rotated forms with neutral histidines, representing its closed form. We compare the channel stability, convergence, interaction with water and hydration of the histidine residues that have been implicated in channel gating. Taken together, we show that both forms of the protein are stable during the course of the MD simulation and that indeed a rotation of the helices leads to channel closure. Finally, we propose a mechanism for channel gating that involves protonation of the histidine residues that necessities their increased solvation.     

Helix tilt of the M2 transmembrane peptide from influenza A virus: an intrinsic property

Solid-state NMR has been used to study the influence of lipid bilayer hydrophobic thickness on the tilt of a peptide (M2-TMP) representing the transmembrane portion of the M2 protein from influenza A. Using anisotropic (15)N chemical shifts as orientational constraints, single-site isotopically labeled M2-TMPs were studied in hydrated dioleoylphosphatidylcholine (DOPC) and dimyristoylphosphatidylcholine (DMPC) lipid bilayers oriented between thin glass plates. These chemical shifts provide orientational information for the molecular frame with respect to the magnetic field in the laboratory frame. When modeled as a uniform ideal alpha-helix, M2-TMP has a tilt of 37(+/-3) degrees in DMPC and 33(+/-3) degrees in DOPC with respect to the bilayer normal in these lipid environments. The difference in helix tilt between the two environments appears to be small. This lack of a substantial change in tilt further suggests that significant interactions occur between the helices, as in an oligomeric state, to prevent a change in tilt in thicker lipid bilayers.     

Human interleukin 4. The solution structure of a four-helix bundle protein.

Heteronuclear 13C and 15N three-dimensional nuclear magnetic resonance (n.m.r.) techniques have been used to determine the solution structure of human interleukin 4, a four-helix bundle protein. A dynamical simulated annealing protocol was used to calculate an ensemble of structures from an n.m.r. data set of 1735 distance restraints, 101 phi angle restraints and 27 pairs of hydrogen bond restraints. The protein structure has a left-handed up-up-down-down topology for the four helices with the two long overhand loops in the structure being connected by a short section of irregular antiparallel beta-sheet. Analysis of the side-chains in the protein shows a clustering of hydrophobic residues, particularly leucines, in the core of the bundle with the side-chains of charged residues being located on the protein surface. The solution structure has been compared with a recent structure prediction for human interleukin 4 and with crystal structures of other helix bundle proteins.     

Defining the transmembrane helix of M2 protein from influenza A by molecular dynamics simulations in a lipid bilayer

Integral membrane proteins containing at least one transmembrane (TM) alpha-helix are believed to account for between 20% and 30% of most genomes. There are several algorithms that accurately predict the number and position of TM helices within a membrane protein sequence. However, these methods tend to disagree over the beginning and end residues of TM helices, posing problems for subsequent modeling and simulation studies. Molecular dynamics (MD) simulations in an explicit lipid and water environment are used to help define the TM helix of the M2 protein from influenza A virus. Based on a comparison of the results of five different secondary structure prediction algorithms, three different helix lengths (an 18mer, a 26mer, and a 34mer) were simulated. Each simulation system contained 127 POPC molecules plus approximately 3500-4700 waters, giving a total of approximately 18,000-21,000 atoms. Two simulations, each of 2 ns duration, were run for the 18mer and 26mer, and five separate simulations were run for the 34mer, using different starting models generated by restrained in vacuo MD simulations. The total simulation time amounted to 11 ns. Analysis of the time-dependent secondary structure of the TM segments was used to define the regions that adopted a stable alpha-helical conformation throughout the simulation. This analysis indicates a core TM region of approximately 20 residues (from residue 22 to residue 43) that remained in an alpha-helical conformation. Analysis of atomic density profiles suggested that the 18mer helix revealed a local perturbation of the lipid bilayer. Polar side chains on either side of this region form relatively long-lived H-bonds to lipid headgroups and water molecules.     

pH-dependent tetramerization and amantadine binding of the transmembrane helix of M2 from the influenza A virus

The M2 proton channel from the influenza A virus is a small protein with a single transmembrane helix that associates to form a tetramer in vivo. This protein forms proton-selective ion channels, which are the target of the drug amantadine. Here, we propose a mechanism for the pH-dependent association, and amantadine binding of M2, based on studies of a peptide representing the M2 transmembrane segment in dodecylphosphocholine micelles. Using analytical ultracentrifugation, we find that the sedimentation curves for the peptide depend on its concentration in the micellar phase. The data are well-described by a monomer-tetramer equilibrium, and the binding of amantadine shifts the monomer-tetramer equilibrium toward tetrameric species. Both tetramerization and the binding of amantadine lead to increases in the magnitude of the ellipticity at 223 nm in the circular dichroism spectrum of the peptide. The tetramerization and binding of amantadine are more favorable at elevated pH, with a pK(a) that is assigned to a His side chain, the only ionizable residue within the transmembrane helix. Our results, interpreted quantitatively in terms of a reversible monomer and tetramer protonation equilibrium model, suggest that amantadine competes with protons for binding to the deprotonated tetramer, thereby stabilizing the tetramer in a slightly altered conformation. This model accounts for the observed inhibition of proton flux by amantadine. Additionally, our measurements suggest that the M2 tetramer is substantially protonated at neutral pH and that both singly and doubly protonated states could be involved in M2's proton conduction at more acidic pHs.     

Use of a single glycine residue to determine the tilt and orientation of a transmembrane helix. A new structural label for infrared spectroscopy

Site-directed dichroism is an emerging technique for the determination of membrane protein structure. However, due to a number of factors, among which is the high natural abundance of (13)C, the use of this technique has been restricted to the study of small peptides. We have overcome these problems through the use of a double C-deuterated glycine as a label. The modification of a single residue (Gly) in the transmembrane segment of M2, a protein from the Influenza A virus that forms H(+)-selective ion channels, has allowed us to determine its helix tilt and rotational orientation. Double C-deuteration shifts the antisymmetric and symmetric stretching vibrations of the CD(2) group in glycine to a transparent region of the infrared spectrum where the dichroic ratio of these bands can be measured. The two dichroisms, along with the helix amide I dichroic ratio, have been used to determine the helix tilt and rotational orientation of M2. The results are entirely consistent with previous site-directed dichroism and solid-state NMR experiments, validating C-deuterated glycine (GlyCD(2)) as a structural probe that can now be used in the study of polytopic membrane proteins.     

Proton transport behavior through the influenza A M2 channel: insights from molecular simulation

The structural properties of the influenza A virus M2 transmembrane channel in dimyristoylphosphatidylcholine bilayer for each of the four protonation states of the proton-gating His-37 tetrad and their effects on proton transport for this low-pH activated, highly proton-selective channel are studied by classical molecular dynamics with the multistate empirical valence-bond (MS-EVB) methodology. The excess proton permeation free energy profile and maximum ion conductance calculated from the MS-EVB simulation data combined with the Poisson-Nernst-Planck theory indicates that the triply protonated His-37 state is the most likely open state via a significant side-chain conformational change of the His-37 tetrad. This proposed open state of M2 has a calculated proton permeation free energy barrier of 7 kcal/mol and a maximum conductance of 53 pS compared to the experimental value of 6 pS. By contrast, the maximum conductance for Na(+) is calculated to be four orders of magnitude lower, in reasonable agreement with the experimentally observed proton selectivity. The pH value to activate the channel opening is estimated to be 5.5 from dielectric continuum theory, which is also consistent with experimental results. This study further reveals that the Ala-29 residue region is the primary binding site for the antiflu drug amantadine (AMT), probably because that domain is relatively spacious and hydrophobic. The presence of AMT is calculated to reduce the proton conductance by 99.8% due to a significant dehydration penalty of the excess proton in the vicinity of the channel-bound AMT.     

Disulfide crosslinks to probe the structure and flexibility of a designed four-helix bundle protein.

The introduction of disulfide crosslinks is a generally useful method by which to identify regions of a protein that are close together in space. Here we describe the use of disulfide crosslinks to investigate the structure and flexibility of a family of designed 4-helix bundle proteins. The results of these analyses lend support to our working model of the proteins' structure and suggest that the proteins have limited main-chain flexibility.     

Redesign of a four-helix bundle protein by phage display coupled with proteolysis and structural characterization by NMR and X-ray crystallography

To test whether it is practical to use phage display coupled with proteolysis for protein design, we used this approach to convert a partially unfolded four-helix bundle protein, apocytochrome b(562), to a stably folded four-helix bundle protein. Four residues expected to form a hydrophobic core were mutated. One residue was changed to Trp to provide a fluorescence probe for studying the protein's physical properties and to partially fill the void left by the heme. The other three positions were randomly mutated. In addition, another residue in the region to be redesigned was substituted with Arg to provide a specific cutting site for protease Arg-c. This library of mutants was displayed on the surface of phage and challenged with protease Arg-c to select stably folded proteins. The consensus sequence that emerged from the selection included hydrophobic residues at only one of the three positions and non-hydrophobic residues at the other two. Nevertheless, the selected proteins were thermodynamically very stable. The structure of a selected protein was characterized using multi-dimensional NMR. All four helices were formed in the structure. Further, site-directed mutagenesis was used to change one of the two non-hydrophobic residues to a hydrophobic residue, which increased the stability of the protein, indicating that the selection result was not based solely on the protein's global stability and that local structural characteristics may also govern the selection. This conclusion is supported by the crystal structure of another mutant that has two hydrophobic residues substituted for the two non-hydrophobic residues. These results suggest that the hydrophobic interactions in the core are not sufficient to dictate the selection and that the location of the cutting site of the protease also influences the selection of structures.     

Potent neutralization of influenza A virus by a single-domain antibody blocking M2 ion channel protein

Influenza A virus poses serious health threat to humans. Neutralizing antibodies against the highly conserved M2 ion channel is thought to offer broad protection against influenza A viruses. Here, we screened synthetic Camel single-domain antibody (VHH) libraries against native M2 ion channel protein. One of the isolated VHHs, M2-7A, specifically bound to M2-expressed cell membrane as well as influenza A virion, inhibited replication of both amantadine-sensitive and resistant influenza A viruses in vitro, and protected mice from a lethal influenza virus challenge. Moreover, M2-7A showed blocking activity for proton influx through M2 ion channel. These pieces of evidence collectively demonstrate for the first time that a neutralizing antibody against M2 with broad specificity is achievable, and M2-7A may have potential for cross protection against a number of variants and subtypes of influenza A viruses.     

Influenza A virus M2 ion channel protein: a structure-function analysis.

A structure-function analysis of the influenza A virus M2 ion channel protein was performed. The M2 protein of human influenza virus A/Udorn/72 and mutants containing changes on one face of the putative alpha helix of the M2 transmembrane (TM) domain, several of which lead to amantadine resistance when found in virus, were expressed in oocytes of Xenopus laevis. The membrane currents of oocytes expressing mutant M2 ion channels were measured at both normal and low pH, and the amantadine-resistant mutant containing the change of alanine at residue 30 to threonine was found to have a significantly attenuated low pH activation response. The specific activity of the channel current of the amantadine-resistant mutants was investigated by measuring the membrane current of individual oocytes followed by quantification of the amount of M2 protein expressed in these single oocytes by immunoblotting analysis. The data indicate that changing residues on this face of the putative alpha helix of the M2 TM domain alters properties of the M2 ion channel. Some of the M2 proteins containing changes in the TM domain were found to be modified by addition of an N-linked carbohydrate chain at an asparagine residue that is membrane proximal and which is not modified in the wild-type M2 protein. These N-linked carbohydrate chains were further modified by addition of polylactosaminoglycan. A glycosylated M2 mutant protein (M2 + V, A30T) exhibited an ion channel activity with a voltage-activated, time-dependent kinetic component. Prevention of carbohydrate addition did not affect the altered channel activity. The ability of the M2 protein to tolerate deletions in the TM domain was examined by expressing three mutants (del29-31, del28-31, and del27-31) containing deletions of three, four, and five residues in the TM domain. No ion channel activity was detected from expression of M2 del29-31 and del27-31, whereas expression of M2 del28-31 resulted in an ion channel activity that was activated by hyperpolarization (and not low pH) and was resistant to amantadine block. Examination of the oligomeric form of M2 del28-31 indicated that the oligomer is different from wild-type M2, and the data were consistent with M2 del28-31 forming a pentamer.     

The influenza virus ion channel and maturation cofactor M2 is a cholesterol-binding protein

The influenza-virus M2 protein has proton channel activity required for virus uncoating and maturation of hemagglutinin (HA) through low-pH compartments. The proton channel is cytotoxic in heterologous expression systems and can be blocked with rimantadine. In an independent, rimantadine-resistant function, M2, interacting with the M1 protein, controls the shape of virus particles. These bud from cholesterol-rich membrane rafts where viral glycoproteins and matrix (M1)/RNP complexes assemble. We demonstrate that M2 preparations from influenza virus-infected cells and from a baculovirus expression system contain 0.5–0.9 molecules of cholesterol per monomer. Sequence analyses of the membrane-proximal M2 endodomain reveal interfacial hydrophobicity, a cholesterol-binding motif first identified in peripheral benzodiazepine receptor and human immunodeficiency virus gp41, and an overlapping phosphatidylinositol 4,5-bisphosphate-binding motif. M2 induced rimantadine-reversible cytotoxicity in intrinsically cholesterol-free E. coli, and purified E. coli-expressed M2 functionally reconstituted into cholesterol-free liposomes supported rimantadine-sensitive proton translocation. Therefore, cholesterol was nonessential for M2 ion-channel function and cytotoxicity and for the effect of rimantadine. Only about 5–8% of both M2 preparations, regardless of cholesterol content, associated with detergent-resistant membranes. Cholesterol affinity and palmitoylation, in combination with a short transmembrane segment suggest M2 is a peripheral raft protein. Preference for the raft/non-raft interface may determine colocalization with HA during apical transport, the low level of M2 incorporated into the viral envelope and its undisclosed role in virus budding for which a model is presented. M2 may promote clustering and merger of rafts and the pinching-off (fission) of virus particles.Abbreviations CRAC Cholesterol recognition/interaction amino acid consensus - DMPC l--dimyristoylphosphatidylcholine - DRM Detergent-resistant membrane - HA Hemagglutinin - HDL High-density lipoprotein - KPS Potassium phosphate buffer with K₂SO₄ - LDL Low-density lipoprotein - MDCK Madin-Darby canine kidney - NA Neuraminidase - NaPS Sodium phosphate buffer with Na₂SO₄ - Ni-NTA Nickel-nitrilotriacetic acid - OG N-octyl--d-glucopyranoside - PM Plasma membrane - PS Phosphatidylserine - RNP Ribonucleoprotein - Sf9 Spodoptera frugiperda - TDC Taurodeoxycholate - TGN trans-Golgi network - TM Transmembrane - T. ni Trichoplusia ni - TX-100 Triton X-100 - Hydrophobic amino acid     

Self-assembly of heme A and heme B in a designed four-helix bundle: implications for a cytochrome c oxidase maquette

Heme A, a prosthetic group of cytochrome c oxidase [EC 1.9.3.1], has been introduced into two de novo designed four helix bundle proteins, [H10A24](2) and [H10H24](2), known to bind 2-4 equiv of heme B, respectively [Robertson, D. E., Farid, R. S., Moser, C. C., Mulholland, S. E., Pidikiti, R., Lear, J. D., Wand, A., J., DeGrado, W. F., and Dutton, P. L. (1994) Nature 368, 425-432]. [H10A24](2), [Ac-CGGGELWKL x HEELLKK x FEELLKL x AEERLKK x L-CONH(2)](2)(2), binds two heme A molecules per four-helix unit via bis-histidine ligation at the 10,10' positions with measured K(d) values of <0.1 and 5 nM, values much lower than those measured for heme B (K(d) values of 50 and 800 nM). The heme A-protein complex, [heme A-H10A24](2), exhibits well-defined absorption spectra in both the ferric and ferrous states, and an electron paramagnetic resonance spectrum characteristic of a low spin heme in the ferric form. A single midpoint redox potential (E(m8)) was determined for [heme A-H10A24](2) at -45 mV (vs SHE), which is significantly higher than that of the protein bound heme B (-130 and -200 mV). The observation of a single midpoint redox potential for [heme A-H10A24](2) and a pair of midpoints for [heme B-H10A24](2) indicates that the di-alpha-helical monomers are oriented in an anti topology (disulfides on opposite sides of bundle) in the former (lacking heme-heme electrostatic interaction) and syn in the latter. A mixture of global topologies was indicated by the potentiometric titration of the related [heme A-H10H24](2) which possess two distinct reduction potentials of +41 (31%) and -65 mV (69%). Self-assembly of the mixed cofactor heme A-heme B-[H10A24](2) was accomplished by addition of a single equivalent of each heme A and heme B to [H10A24](2). The single midpoint redox potential of heme B, E(m8) = -200 mV, together with the split midpoint redox potential of heme A in heme A-heme B-[H10A24](2), E(m8) = +28 mV (33%) and -65 mV (67%), indicated the existence of both syn and anti topologies of the two di-alpha-helical monomers in this four helix bundle. Synthesis of the mixed cofactor [heme A-heme B-H10H24](2) was accomplished by addition of a 2 equiv of each heme A and heme B to [H10H24](2) and potentiometry indicated the pair of hemes B resided in the 10,10' sites and heme A occupied the 24,24' sites. The results indicate that heme peripheral structure controls the orientation of the di-alpha-helical monomers in the four-helix bundle which are interchangeable between syn and anti topologies. In the reduced form, [heme A-H10A24](2), reacts quantitatively to form [carbonmonoxy-heme A-H10A24](2) as evidenced by optical spectroscopy. The synthetic [heme A-H10A24](2) can be enzymatically reduced by NAD(P)H with natural reductases under anaerobic conditions, and reversibly oxidized by dioxygen to the ferric form.     

Hydrogel biopolymer created from the self-assembly of a designed protein containing a four-helix bundle forming motif

A protein hydrogel system based on the assembly of a four-helix bundle motif was proposed and synthesized by genetic engineering methods. This new polypeptide, named GBH1, consists of identical amphipathic helices of 22 residues in length oriented in opposite fashion to one another at each end of a polypeptide with a total length of 227 amino acids. The middle portion of the polypeptide (residues 79-147) is an unstructured random coil. The region between the amphipathic and unstructured segment is an α-helical stretch (23-78 and 148-204) not possessing a sequence compatible with a coiled-coil conformation, but rather possesses regions that have overwinding of the helix. The thermal unfolding of GBH1 shows more than one inflection point (T(m1) = 30.5 °C, T(m2) = 64.6 °C), indicative of a partially unfolded intermediate and, thus, multiple interactions in the folded state. A qualitative assessment of hydrogel formation with varying pH showed that acidic conditions did not support the gel state, indirectly indicating that the proposed four-helix bundle is the major cross-linking structure and not a leucine zipper motif. Scanning electron microscopy reveals a network of interacting protein molecules forming a spongelike matrix with numerous pores that would be occupied with water molecules.     

Permeation and activation of the M2 ion channel of influenza A virus

The M(2) ion channel protein of influenza A virus is essential for mediating protein-protein dissociation during the virus uncoating process that occurs when the virus is in the acidic environment of the lumen of the secondary endosome. The difficulty of determining the ion selectivity of this minimalistic ion channel is due in part to the fact that the channel activity is so great that it causes local acidification in the expressing cells and a consequent alteration of reversal voltage, V(rev). We have confirmed the high proton selectivity of the channel (1.5-2.0 x 10(6)) in both oocytes and mammalian cells by using four methods as follows: 1) comparison of V(rev) with proton equilibrium potential; 2) measurement of pH(in) and V(rev) while Na(+)(out) was replaced; 3) measurements with limiting external buffer concentration to limit proton currents specifically; and 4) comparison of measurements of M(2)-expressing cells with cells exposed to a protonophore. Increased currents at low pH(out) are due to true activation and not merely increased [H(+)](out) because increased pH(out) stops the outward current of acidified cells. Although the proton conductance is the biologically relevant conductance in an influenza virus-infected cell, experiments employing methods 1-3 show that the channel is also capable of conducting NH(4)(+), probably by a different mechanism from H(+).