Protonation of histidine and histidine-tryptophan interaction in the activation of the M2 ion channel from influenza a virus

The M2 protein of influenza A virus forms a homotetramer ion channel in the lipid membrane. The channel is specific for proton conductance and is activated by low pH with a transition midpoint at pH 5.7. We have studied the structure of the transmembrane domain of the M2 ion channel by using UV resonance Raman spectroscopy, with special attention to the side chains of histidine (His37) and tryptophan (Trp41) residues. The Raman spectra provide direct evidence that the imidazole ring of His37 is protonated upon channel activation at low pH. Concomitantly, the UV resonance Raman scattering from Trp41 shows an unusual intensity change, which is ascribed to a cation-pi interaction between the protonated (cationic) imidazole ring of His37 and the indole ring of Trp41. The protonation of His37 and the Raman intensity change of Trp41 do not occur in the presence of amantadine that blocks the M2 ion channel. These observations clearly show that the protonation of His37 and concomitant cation-pi interaction with Trp41 is a key step in the activation of the M2 ion channel. The His37-Trp41 interaction associated with the channel activation is explained by assuming a conformational transition of His37 induced by electrostatic repulsion among the protonated imidazole rings of four His37 residues in the tetramer channel. Trp41 may play a role in stabilizing the channel open state through cation-pi interaction with His37. A molecular model for the activation of M2 ion channel is proposed on the basis of the gating mechanism.     

Molecular origins of asymmetric proton conduction in the influenza M2 channel

The M2 proton channel of influenza A is embedded into the viral envelope and allows acidification of the virion when the external pH is lowered. In contrast, no outward proton conductance is observed when the internal pH is lowered, although outward current is observed at positive voltage. Residues Trp41 and Asp44 are known to play a role in preventing pH-driven outward conductance, but the mechanism for this is unclear. We investigate this issue using classical molecular dynamics simulations with periodic proton hops. When all key His37 residues are neutral, inward proton movement is much more facile than outward movement if the His are allowed to shuttle the proton. The preference for inward movement increases further as the charge on the His37 increases. Analysis of the trajectories reveals three factors accounting for this asymmetry. First, in the outward direction, Asp44 traps the hydronium by strong electrostatic interactions. Secondly, Asp44 and Trp41 orient the hydronium with the protons pointing inward, hampering outward Grotthus hopping. As a result, the effective barrier is lower in the inward direction. Trp41 adds to the barrier by weakly H-bonding to potential H⁺ acceptors. Finally, for charged His, the H₃O⁺ in the inner vestibule tends to get trapped at lipid-lined fenestrations of the cone-shaped channel. Simulations qualitatively reproduce the experimentally observed higher outward conductance of mutants. The ability of positive voltage, unlike proton gradient, to induce an outward current appears to arise from its ability to bias H₃O⁺ and the waters around it toward more H-outward orientations.     

Computational studies of proton transport through the M2 channel

The M2 ion channel is an essential component of the influenza A virus. This low-pH gated channel has a high selectivity for protons. Evidence from various experimental data has indicated that the essential structure responsible for the channel is a parallel homo-tetrameric alpha-helix bundle having a left-handed twist with each helix tilted with respect to the membrane normal. A backbone structure has been determined by solid state nuclear magnetic resonance (NMR). Though detailed structures for the side chains are not available yet, evidence has indicated that His37 and Trp41 in the alpha-helix are implicated in the local molecular structure responsible for the selectivity and channel gate. More definitive conformations for the two residues were recently suggested based on the known backbone structure and recently obtained NMR data. While two competitive proton-conductance mechanisms have been proposed, the actual proton-conductance mechanism remains an unsolved problem. Computer simulations of an excess proton in the channel and computational studies of the His37/Trp41 conformations have provided insights into these structural and mechanism issues.     

Roles of the histidine and tryptophan side chains in the M2 proton channel from influenza A virus

The M2 protein form influenza A virus forms a tetrameric ion channel, which enables proton passage across biological membranes when the N-terminal side is acidified. Among the amino acid residues in the transmembrane domain of the M2 protein, His37 and Trp41 are essential for the pH-regulated proton conductance. Current knowledge about the structures and interactions of His37 and Trp41 suggests a model for the M2 ion channel, in which the channel is closed by a network of His37 hydrogen bonds at neutral pH and is opened by a His37-Trp41 cation-pi interaction at acidic pH.     

A computational study of the closed and open states of the influenza a M2 proton channel

In this study, four possible conformations of the His-37 and Trp-41 residues for the closed state of the influenza M2 ion channel were identified by a conformation scan based on a solid-state NMR restraint. In the four conformations, the His-37 residue can be of either the t-160 or t60 rotamer, whereas Trp-41 can be of either the t-105 or t90 rotamer. These conformations were further analyzed by density functional theory calculations and molecular dynamics simulations, and the data indicate that the His-37 residue most likely adopts the t60 rotamer and should be monoprotonated at the delta-nitrogen site, whereas Trp-41 adopts the t90 rotamer. This result is consistent with published experimental data and points to a simple gating mechanism: in the closed state, the His-37 and Trp-41 residues adopt the (t60, t90) conformation, which nearly occludes the pore, preventing nonproton ions from passing through due to the steric and desolvation effects. Moreover, the His-37 tetrad interrupts the otherwise continuous hydrogen-bonding network of the pore water by forcing the water molecules above and below it to adopt opposite orientations, thus adding to the blockage of proton shuttling. The channel can be easily opened by rotating the His-37 chi2 angle from 60 to 0 degrees . This open structure allows pore water to penetrate the constrictive region and to form a continuous water wire for protons to shuttle through, while being still narrow enough to exclude other ions.     

Chemical rescue of histidine selectivity filter mutants of the M2 ion channel of influenza A virus

The influenza virus M2 proton-selective ion channel activity facilitates virus uncoating, a process that occurs in the acidic environment of the endosome. The M2 channel causes acidification of the interior of the virus particle, which results in viral protein-protein dissociation. The M2 protein is a homotetramer that contains in its aqueous pore a histidine residue (His-37) that acts as a selectivity filter and a tryptophan residue (Trp-41) that acts as a channel gate. Substitution of His-37 modifies M2 ion channel properties drastically. However, the results of such experiments are difficult to interpret because substitution of His-37 could cause gross structural changes to the channel pore. We described here experiments in which partial or, in some cases, full rescue of specific M2 ion channel properties of His-37 substitution mutants was achieved by addition of imidazole to the bathing medium. Chemical rescue was demonstrated for three histidine substitution mutant ion channels (M2-H37G, M2-H37S, and M2-H37T) and for two double mutants in which the Trp-41 channel gate was also mutated (H37G/W41Y and H37G/W41A). Currents of the M2-H37G mutant ion channel were inhibited by Cu(II), which has been shown to coordinate with His-37 in the wild-type channel. Chemical rescue was very specific for imidazole. Buffer molecules that were neutral when protonated (4-morpholineethanesulfonic acid and 3-morpholino-2-hydroxypropanesulfonic acid) did not rescue ion channel activity of the M2-H37G mutant ion channel, but 1-methylimidazole did provide partial rescue of function. These results were consistent with a model for proton transport through the pore of the wild-type channel in which the imidazole side chain of His-37 acted as an intermediate proton acceptor/donor group.     

The gate of the influenza virus M2 proton channel is formed by a single tryptophan residue

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 Trp(41) blocking the pore of the M2 channel when pH(out) is high and with this side chain leaving the pore when pH(out) is low. Thus, the Trp(41) side chain acts as the gate that opens and closes the pore.     

Designing Inhibitors of M2 Proton Channel against H1N1 Swine Influenza Virus

Background

M2 proton channel of H1N1 influenza A virus is the target protein of anti-flu drugs amantadine and rimantadine. However, the two once powerful adamantane-based drugs lost their 90% bioactivity because of mutations of virus in recent twenty years. The NMR structure of the M2 channel protein determined by Schnell and Chou (Nature, 2008, 451, 591–595) may help people to solve the drug-resistant problem and develop more powerful new drugs against H1N1 influenza virus.

Methodology

Docking calculation is performed to build the complex structure between receptor M2 proton channel and ligands, including existing drugs amantadine and rimantadine, and two newly designed inhibitors. The computer-aided drug design methods are used to calculate the binding free energies, with the computational biology techniques to analyze the interactions between M2 proton channel and adamantine-based inhibitors.

Conclusions

1) The NMR structure of M2 proton channel provides a reliable structural basis for rational drug design against influenza virus. 2) The channel gating mechanism and the inhibiting mechanism of M2 proton channel, revealed by the NMR structure of M2 proton channel, provides the new ideas for channel inhibitor design. 3) The newly designed adamantane-based inhibitors based on the modeled structure of H1N1-M2 proton channel have two pharmacophore groups, which act like a “barrel hoop”, holding two adjacent helices of the H1N1-M2 tetramer through the two pharmacophore groups outside the channel. 4) The inhibitors with such binding mechanism may overcome the drug resistance problem of influenza A virus to the adamantane-based drugs.     

Spontaneous mobility of GABAA receptor M2 extracellular half relative to noncompetitive antagonist action

The gamma-aminobutyric acid type A receptor beta(3) homopentamer is spontaneously open and highly sensitive to many noncompetitive antagonists(NCAs) and Zn(2+). Our earlier study of the M2 cytoplasmic half (-1' to 10') established a model in which NCAs bind at pore-lining residues Ala(2)', Thr(6)', and Leu(9)'. To further define transmembrane 2 (M2) structure relative to NCA action, we extended the Cys scanning to the extra cellular half of the beta(3) homopentamer (11' to 20'). Spontaneous disulfides formed with T13'C, L18'C, and E20'C from M2/M2 cross-linking and with I14'C (weak), H17'C, and R19'Con bridging M2/M3 intersubunits, based on single (M2 Cys only) and dual (M2 Cys plus M3 C289S) mutations. Induced disulfides also formed with T16'C, but there were few or none with M11'C, T12'C, and N15'C. These findings show conformational flexibility/mobility in the M2 extracellular half 17' to 20' region interpreted as a deformed beta-like conformation in the open channel. The NCA radioligands used were [(3)H]1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]octane ([(3)H]EBOB) and [(3)H]3,3-bis-trifluoromethylbicyclo[2.2.1]heptane-2,2-dicarbonitrile with essentially the same results. NCA binding was disrupted by individual Cys substitutions at 13',14',16',17', and 19'. The inactivity of T13'C/T13'S may have been due to disturbance of the channel gate; I14'S and T16'S showed much better binding activity than their Cys counterparts, and the low activities of H17'C and R19'C were reversed by dithiothreitol. Zn(2+) potency for inhibition of [(3)H]EBOB binding was lowered 346-fold by the mutation H17'A. We propose that NCAs enter their binding site both directly, through the channel pore, and indirectly, through the water cavity of adjacent subunits.     

Comparisons of the influenza virus A M2 channel binding affinities, anti-influenza virus potencies and NMDA antagonistic activities of 2-alkyl-2-aminoadamantanes and analogues

The new 2-alkyl-2-aminoadamantanes and analogues 4-10 were designed and synthesized by simplification of the structure of the potent anti-influenza virus A spiranic aminoadamantane heterocycles 2 and 3. The aim of the present work was to examine the effects of bulky and extended lipophilic moieties attached to amantadine 1 on binding to the M2 channel and the resulting antiviral potency. The binding affinities of the compounds to the M2 protein of influenza virus A/chicken/Germany/27 (Weybridge strain; H7N7) were measured for the first time using an assay based on quenching of Trp-41 fluorescence by His-37 protonation, and their antiviral potencies were evaluated against the replication of influenza virus A H2N2 and H3N2 subtypes and influenza virus B in MDCK cells. Of the various 2-alkyl-2-aminoadamantanes, and analogues, spiro[piperidine-2,2'-adamantane] 3 had the strongest M2 binding and antiviral potency, which were similar those of amantadine 1. The relative binding affinities suggested that the rigid carbon framework provided by the pyrrolidine or piperidine rings results in a more favorable orientation inside the M2 channel pore as compared to large, freely rotating alkyl groups. The aminoadamantane derivatives exhibited similar NMDA antagonistic activity to amantadine 1. A striking finding was the antiviral activity of the adamantanols 4, and 6, which lack any NMDA antagonist activity.     

Insights from investigating the interactions of adamantane-based drugs with the M2 proton channel from the H1N1 swine virus

The M2 proton channel is one of indispensable components for the influenza A virus that plays a vital role in its life cycle and hence is an important target for drug design against the virus. In view of this, the three-dimensional structure of the H1N1-M2 channel was developed based on the primary sequence taken from a patient recently infected by the H1N1 (swine flu) virus. With an explicit water-membrane environment, molecular docking studies were performed for amantadine and rimantadine, the two commercial drugs generally used to treat influenza A infection. It was found that their binding affinity to the H1N1-M2 channel is significantly lower than that to the H5N1-M2 channel, fully consistent with the recent report that the H1N1 swine virus was resistant to the two drugs. The findings and the relevant analysis reported here might provide useful structural insights for developing effective drugs against the new swine flu virus.     

Self-assembly of a simple membrane protein: coarse-grained molecular dynamics simulations of the influenza M2 channel

The transmembrane (TM) domain of the M2 channel protein from influenza A is a homotetrameric bundle of α-helices and provides a model system for computational approaches to self-assembly of membrane proteins. Coarse-grained molecular dynamics (CG-MD) simulations have been used to explore partitioning into a membrane of M2 TM helices during bilayer self-assembly from lipids. CG-MD is also used to explore tetramerization of preinserted M2 TM helices. The M2 helix monomer adopts a membrane spanning orientation in a lipid (DPPC) bilayer. Multiple extended CG-MD simulations (5 × 5 μs) were used to study the tetramerization of inserted M2 helices. The resultant tetramers were evaluated in terms of the most populated conformations and the dynamics of their interconversion. This analysis reveals that the M2 tetramer has 2× rotationally symmetrical packing of the helices. The helices form a left-handed bundle, with a helix tilt angle of ∼16°. The M2 helix bundle generated by CG-MD was converted to an atomistic model. Simulations of this model reveal that the bundle's stability depends on the assumed protonation state of the H37 side chains. These simulations alongside comparison with recent x-ray (3BKD) and NMR (2RLF) structures of the M2 bundle suggest that the model yielded by CG-MD may correspond to a closed state of the channel.     

The apolar channel in Cerebratulus lacteus hemoglobin is the route for O2 entry and exit

The major pathway for O2 binding to mammalian myoglobins (Mb) and hemoglobins (Hb) involves transient upward movement of the distal histidine (His-64(E7)), allowing ligand capture in the distal pocket. The mini-globin from Cerebratulus lacteus (CerHb) appears to have an alternative pathway between the E and H helices that is made accessible by loss of the N-terminal A helix. To test this pathway, we examined the effects of changing the size of the E7 gate and closing the end of the apolar channel in CerHb by site-directed mutagenesis. Increasing the size of Gln-44(E7) from Ala to Trp causes variation of association (k'O2) and dissociation (kO2) rate coefficients, but the changes are not systematic. More significantly, the fractions (Fgem approximately 0.05-0.19) and rates (kgem approximately 50-100 micros(-1)) of geminate CO recombination in the Gln-44(E7) mutants are all similar. In contrast, blocking the entrance to the apolar channel by increasing the size of Ala-55(E18) to Phe and Trp causes the following: 1) both k'O2 and kO2 to decrease roughly 4-fold; 2) Fgem for CO to increase from approximately 0.05 to 0.45; and 3) kgem to decrease from approximately 80 to approximately 9 micros(-1), as ligands become trapped in the channel. Crystal structures and low temperature Fourier-transform infrared spectra of Phe-55 and Trp-55 CerHb confirm that the aromatic side chains block the channel entrance, with little effect on the distal pocket. These results provide unambiguous experimental proof that diatomic ligands can enter and exit a globin through an interior channel in preference to the more direct E7 pathway.     

An in-depth analysis of the biological functional studies based on the NMR M2 channel structure of influenza A virus

The long-sought three-dimensional structure of the M2 proton channel of influenza A virus was successfully determined recently by the high-resolution NMR [J.R. Schnell, J.J. Chou, Structure and mechanism of the M2 proton channel of influenza A virus, Nature 451 (2008) 591-595]. Such a milestone work has provided a solid structural basis for studying drug-resistance problems. However, the action mechanism revealed from the NMR structure is completely different from the traditional view and hence prone to be misinterpreted as “conflicting” with some previous biological functional studies. To clarify this kind of confusion, an in-depth analysis was performed for these functional studies, particularly for the mutations D44N, D44A and N44D on position 44, and the mutations on positions 27-38. The analyzed results have provided not only compelling evidences to further validate the NMR structure but also very useful clues for dealing with the drug-resistance problems and developing new effective drugs against H5N1 avian influenza virus, an impending threat to human beings.     

Relative contribution of OAT1 and OAT3 transport activities in isolated perfused rabbit renal proximal tubules

The expression of both OAT1 and OAT3 along the isolated rabbit renal proximal tubule (RPT) was determined using RT-PCR. They were found to be very strong in S2 segment and weak in S1 and S3 segments. We further examined the relative transport activity of these transporters in isolated perfused rabbit RPT using [³H]para-aminohippurate ([³H]PAH), and estrone sulfate ([³H]ES) as specific substrates for rbOAT1 and rbOAT3, respectively. The transport activity of OAT1 was in the order S2 > S1 = S3 segments and that of OAT3 was in the order S1 = S2>>S3 segments. The addition of α-ketoglutarate (100 μM) in the bathing medium increased both OAT1 and OAT3 transport activities in all segments of proximal tubule. The kinetics of [³H]succinic acid transport, used to measure the activity of sodium dicarboxylate transporter 3 (NaDC3), were examined. The J_max for succinic acid was in the order S2 > S3 and unmeasurable in the S1 segment. Our data indicate that both OAT1 and OAT3 play quantitatively significant roles in the renal transport of organic anions along the proximal tubule but predominately in S2 segment. The relative contribution of both transporters depends on their relative expression levels and may possibly be affected by the activity of NaDC3 in RPT.     

Mutations in the M4 Domain of the Torpedo californica Nicotinic Acetylcholine Receptor Alter Channel Opening and Closing

We studied the functional effects of single amino acid substitutions in the postulated M4 transmembrane domains of Torpedo californica nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes at the single-channel level. At low ACh concentrations and cold temperatures, the replacement of wild-type α418Cys residues with the large, hydrophobic amino acids tryptophan or phenylalanine increased mean open times 26-fold and 3-fold, respectively. The mutation of a homologous cysteine in the β subunit (β447Trp) had similar but smaller effects on mean open time. Coexpression of α418Trp and β447Trp had the largest effect on channel open time, increasing mean open time 58-fold. No changes in conductance or ion selectivity were detected for any of the single subunit amino acid substitutions tested. However, the coexpression of the α418Trp and β447Trp mutated subunits also produced channels with at least two additional conductance levels. Block by acetylcholine was apparent in the current records from α418Trp mutants. Burst analysis of the α418Trp mutations showed an increase in the channel open probability, due to a decrease in the apparent channel closing rate and a probable increase in the effective opening rate. Our results show that modifications in the primary structure of the α- and β subunit M4 domain, which are postulated to be at the lipid-protein interface, can significantly alter channel gating, and that mutations in multiple subunits act additively to increase channel open time. Received: 27 September 1996/Revised: 28 January 1997     

Ion selectivity and activation of the M2 ion channel of influenza virus.

The influenza A virus-associated M2 ion channel is generally believed to function during uncoating of virions in infected cells. On endocytosis of a virion into the lumen of endosomes, the M2 ion channel is thought to cause acidification of the virion interior. In addition, the influenza virus M2 ion channel is thought to function in the exocytic pathway by equilibrating the pH gradient between the acidic lumen of the trans-Golgi network and the neutral cytoplasm. A necessary test of the proposed roles of the influenza virus M2 ion channel in the virus life cycle is to show directly that the M2 ion channel conducts protons. We have measured the ionic selectivity and activation of three subtypes (Udorn, Weybridge, and Rostock) of the M2 ion channel in oocytes of Xenopus laevis by measurement of 1) the intracellular pH (pHin) of voltage-clamped oocytes, 2) the current-voltage relationship in solutions of various pH and ionic composition, and 3) the flux of 86Rb. We took advantage of the low pHin achieved during incubation in low pH medium to study the effects of low pHin on M2 activation. Oocytes expressing each of the three subtypes of the M2 protein a) underwent a slow acidification when incubated in medium of low pH (acidification was blocked by the M2 ion channel inhibitor, amantadine); b) had current-voltage relationships that shifted to more positive values and had greater conductance when the pHout was lowered (this relationship was modified when Na- was replaced by NH4+ or Li+); c) had an amantadine-sensitive influx of Rb+. The effects on the current-voltage relationship of reduced pHin were opposed to the increased conductance found with reduced pHout. We interpret these results to indicate that the M2 ion channel is capable of conducting H+ and that other ions may also be conducted. Moreover, the channel conductance is reduced by decreased pHin. These findings are consistent with the proposed roles of the M2 protein in the life cycle of influenza A virus.     

Two possible conducting states of the influenza A virus M2 ion channel

Molecular dynamics simulations have been performed on protonated four-helix bundles based on the 25-residue Duff-Ashley transmembrane sequence of the M2 channel of the influenza A virus. Well-equilibrated tetrameric channels, with one, two and four of the H37 residues protonated, were investigated. The protonated peptide bundles were immersed in the octane portion of a phase-separated water/octane system, which provided a membrane-mimetic environment. The simulations suggest that there could be two conducting states of the M2 channel corresponding to tetramers containing one or two protonated histidines. The more open structure of the doubly protonated state suggests it would have the higher conductance.     

Mechanism for proton conduction of the M(2) ion channel of influenza A virus

The M(2) integral membrane protein of influenza A virus forms a proton-selective ion channel. We investigated the mechanism for proton transport of the M(2) protein in Xenopus oocytes using a two-electrode voltage clamp and in CV-1 cells using the whole cell patch clamp technique. Membrane currents were recorded while manipulating the external solution to alter either the total or free proton concentration or the solvent itself. Membrane conductance decreased by approximately 50% when D(2)O replaced H(2)O as the solvent. From this, we conclude that hydrogen ions do not pass through M(2) as hydronium ions, but instead must interact with titratable groups that line the pore of the channel. M(2) currents measured in solutions of low buffer concentration (<15 mM in oocytes and <0.15 mM in CV-1 cells) were smaller than those studied in solutions of high buffer concentration. Furthermore, the reversal voltage measured in low buffer was shifted to a more negative voltage than in high buffer. Also, at a given pH, M(2) current amplitude in 15 mM buffer decreased when pH-pK(a) was increased by changing the buffer pK(a). Collectively, these results demonstrate that M(2) currents can be limited by external buffer capacity. The data presented in this study were also used to estimate the maximum single channel current of the M(2) ion channel, which was calculated to be on the order of 1-10 fA.     

Structure and dynamics of the influenza A M2 Channel: a comparison of three structures

The M2 protein is an essential component of the Influenza virus’ infectivity cycle. It is a homo-tetrameric bundle forming a pH-gated H⁺ channel. The structure of M2 was solved by three different groups, using different techniques, protein sequences and pH environment. For example, solid-state NMR spectroscopy was used on a protein in lipid bilayers, while X-ray crystallography and solution NMR spectroscopy were applied on a protein in detergent micelles. The resulting structures from the above efforts are rather distinct. Herein, we examine the different structures under uniform conditions such as a lipid bilayer and specified protonation state. We employ extensive molecular dynamics simulations, in several protonation states, representing both closed and open forms of the channel. Exploring the properties of each of these structures has shown that the X-ray structure is more stable than the other structures according to various criteria, although its water conductance and water-wire formation do not correlate to the protonation state of the channel.