Influenza virus proton channels

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Abstract

The M2 ion channel proteins of influenza A and B viruses are essential to viral replication. The two ion channels share a common motif, HXXXW, that is responsible for proton selectivity and activation. The ion channel for the influenza A virus, but not influenza B virus, is inhibited by the antiviral drug amantadine and amantadine-resistant escape mutants form in treated influenza A patients. The studies reviewed suggest that an antiviral compound directed against the conserved motif would be more useful than amantadine in inhibiting viral replication.

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Introduction

The proton channels of influenza A virus and influenza B virus (A/M2 and BM2, respectively) are of interest because (1) they are essential for viral replication and (2) they are among the smallest bona fide ion channel proteins, possessing the properties of ion selectivity and activation. They succeed in functioning as proton channels with only a meager similarity in primary amino acid sequence in a single turn of the transmembrane helix. Moreover, one of these proteins, the A/M2 protein from influenza A virus, is the target for action of the antiviral drug amantadine. Thus, these proteins are also important because they are important therapeutic targets.¹

The proton channels of both viruses must function in order for viral replication to occur.^2,3 Both viruses enter the infected cell by endocytosis (Fig. 1), and the interior of the membrane-bound virion (virus particle) must become acidified while it is contained in the endosome as a prerequisite for uncoating (release of genetic material to the cytoplasm).^4–6 The proton channels serve this acidification function. This review will summarize evidence that the apparatus for proton selectivity and for turning-on (activating) these channels resides in a single turn of the transmembrane helix.

Fig. 1 Life cycle of influenza virus showing the steps in which the M2 ion channel functions. The life cycle and role of the M2 protein are similar for both influenza A virus and influenza B virus. The M2 proton channels function while the virion is contained in the endosome (step 3) to permit subsequent uncoating. For certain subtypes of influenza A virus the M2 protein also shunts the pH gradient of the trans-Golgi apparatus to prevent premature conformational change in hemagglutinin (step 6).

Biochemical properties of the A/M2 and BM2 proteins

Both proteins are homotetrameric, type III integral membrane proteins containing a small N-terminal ectodomain, a single transmembrane domain, and C-terminal cytoplasmic tail. The A/M2 ion channel protein of influenza A virus is found in the virion.^7,8 The transmembrane domain acts as a signal sequence to target the nascent polypeptide domain to the membrane of the endoplasmic reticulum as it emerges from the ribosome, and this hydrophobic domain also serves as the stop-transfer sequence to anchor the protein in the membrane.⁹ Lastly, the transmembrane domain becomes the pore of the channel. The predicted membrane-spanning domains of A/M2 and BM2 are 20 amino acids long and the N-terminal domain of the BM2 protein (7 residues) is shorter than that of the A/M2 protein (23 residues). Long cytoplasmic C-terminal domains characterize both the A/M2 protein (53 residues) and the BM2 protein (82 residues). The only homology between the amino acid sequences of these two proteins is found in the HXXXW motif of the inner membrane-spanning residues; this motif proves to be critical to the ion channel activity (see below). Both A/M2 and BM2 behave as homotetramers in cross-linking and sedimentation experiments,^6,10–12 and the active oligomeric state of the A/M2 protein was demonstrated to be a tetramer.¹³ Post-translational modifications occur to the A/M2 protein but do not seem to be important for ion channel function.¹⁴ Thus, the flux of protons across the membrane must occur within the pore formed by the four identical subunits of the transmembrane domain of the protein, and protons must interact with the amino acids that form the lining of the pore.

Proton selectivity and activation are subserved by residues lying in a single helical turn in the transmembrane domain

Both ion channels are very selective for protons,^15–19 and their selectivity depends on a histidine residue in the transmembrane domain. Ion selectivity measurements have been made using in vitro expression systems^15,19–22 and by reconstitution of ion channel activity from recombinant protein in bilayers^23,24 or liposomes.¹⁸ The high proton selectivity of the A/M2 channel is lost when transmembrane domain His37 is replaced with glycine, alanine, glutamic acid, serine or threonine,^25,26 making the mutant channel capable of transporting Na⁺ and K⁺ as well. The ion selectivity of histidine substitution mutant proteins is partially restored by adding imidazole buffer to the solution bathing the expressing cell.²⁶ Thus, the imidazole side chain of histidine plays an essential role in the specificity for proton transport.

The mechanism for transport of protons through the aqueous pore of the channel has not been established with certainty, but two observations are informative. First, the specific activity (single channel conductance) of the wild-type (wt) A/M2 ion channel is very low (it transports roughly 10⁵ protons per tetramer per second at pH 5.7, the pH found in endosomes).^18,22 Second, the kinetic isotope effect measured when deuterium replaces hydrogen shows that this replacement results in a decrease in conductance by an amount greater than the ratio of diffusion coefficients of the two isotopes. These observations suggest that bulk transport of hydronium ions is not responsible for proton transport.²² Two other mechanisms have been suggested for proton transport. First, imidazole might serve as a “relay” molecule, binding protons presented from one end of the channel and releasing them to the other end by dissociation; this mechanism might be assisted by tautomerization of imidazole.²⁷ Second, short-lived proton ‘wires’ might open to allow shuttling of protons from one water molecule to another in the pore, without the water molecules themselves moving.²² Energy minimization simulations support the former model²⁸ and molecular dynamic simulations support the latter model.²⁹ Thus, the exact mechanism for transport of protons with high selectivity is not known.

Mechanism for opening of the A/M2 ion channel

The A/M2 channel does not conduct protons under all conditions; in order to do so the pH of the medium bathing the N-terminal ectodomain, pHout, must be lowered below ca. pH 7. This ability to open and close is dependent on the action of a single transmembrane domain residue, Trp41. Two observations suggest that the channel is closed when pHout exceeds pH 7.5 and is opened when pHout is lower than pH 6.5. First, oocytes that express the channel become rapidly acidified when they are bathed in solutions of low pH but upon restoration of pHout to its normal value their internal pH recovers only very slowly. This restoration of pH occurs much more slowly than the re-alkalinization of cells treated with the protonophore FCCP,³⁰ suggesting efflux of protons from M2 expressing cells is impaired by elevated pHout. Second, cells expressing A/M2 protein that are injected with acid (e.g., 1 N HCl) while pHout is above pH 7.5 do not experience an efflux of protons, but when pHout is low, efflux of protons can be brought about by applying a positive voltage to the inside of the cell.^15,17,20,22 Thus, high pHout closes the channel and low pHout opens (activates) the channel.

Several lines of evidence point to Trp41 as the key residue in opening and closing the channel pore.³⁰ (1) For the wt ion channel, outward currents are not observed when pHout is high, regardless of the means taken to establish an outward electrochemical gradient for protons. Unlike the case for the wt ion channel, it is possible to observe outward proton currents under these conditions for mutant ion channel proteins in which Trp41 is replaced with amino acids having a small side-chain. (2) It is possible to improve the closing of a mutant ion channel in which Trp41 is replaced with Cys by placing a functional group resembling the Trp side chain on the Cys sulfur atom. (3) Cu(II) injected intracellularly is able to coordinate with His37 in a mutant ion channel in which Trp is replaced by Ala, but is not able to coordinate with His37 when injected into cells expressing the wt channel. An explanation for these observations is that the bulky indole side chain of Trp41 interferes with the passage of protons when pHout is high.³⁰ This interpretation is supported by oxidative disulfide cross-linking analysis showing that a structural rearrangement occurs in this region of the protein when pHout is altered.³¹ Furthermore, resonance Raman spectroscopy has shown that pH-dependent interactions occur between His37 and Trp41, perhaps between protonated imidazole and the π electrons of indole.³² Thus, opening and closing of the channel depends on pHout and probably involves structural alterations that encompass Trp41. It is noteworthy that the key functional elements of the channel, His37 and Trp41, are contained in a single turn of the transmembrane helix of this very compact channel.

Inhibition of the A/M2 ion channel

Amantadine inhibits the A/M2 ion channel,^15,20,33 but not the BM2 channel,¹⁹ which is consistent with amantadine inhibiting influenza A virus but not influenza B virus replication. Five observations, taken together, suggest a mechanism for inhibition by amantadine. (1) Two of the mutations that result in resistance to amantadine occur on residues that have been found by cysteine scanning mutagenesis to line the aqueous pore. Both of these mutations result in the replacement of the native residue with ones that are less hydrophobic (A30T and G34E).³⁴ (2) Inhibition of the channel occurs more readily when pH of the bathing medium is high.³³ (3) Amantadine only inhibits when it is applied to the medium bathing the N-terminal ectodomain, and not when applied to the C-terminal cytoplasmic tail; injection of as much as 1 mM amantadine into cells expressing the A/M2 channel does not inhibit the channel, whereas application of 10 µM to the solution bathing the ectodomain inhibits fully.³³ (4) Neutron diffraction studies of amantadine applied to the M2 transmembrane peptide show the compound to lie in the outer region of the membrane.³⁵ (5) Amantadine inhibits with 1 : 1 stoichiometry.³³ These observations suggest that amantadine acts from the outside of the aqueous pore, that the hydrophobic adamantane group interacts with hydrophobic pore-lining residues and that perhaps the ammonium nitrogen of amantadine shares a hydrogen bond with an unprotonated imidazole histidine.²² If such hydrogen bonding occurred it would interrupt the interactions formed by the ring of His and Trp residues from adjacent subunits.³⁶ Thus, amantadine probably resides within the aqueous pore of the channel when it inhibits but probably does not inhibit by simply blocking the pore. It has been suggested that the reason that the BM2 ion channel is not inhibited by amantadine is probably because the BM2 channel pore region is lined with polar, and not hydrophobic, amino acids.¹⁹

Summary of structure–function relationship of the A/M2 ion channel

The available biochemical and solid state NMR data indicate that the transmembrane domain of the A/M2 protein is comprised of a four helix bundle with a tilt of about 25 degrees.^37,38 Functional studies showed that residues Val27, Ala30, Gly34, His37 and Trp41 line the aqueous pore.^31,39,40 Amantadine inhibition requires interaction with these residues. Further functional studies have indicated (1) that His37 forms a barrier to large molecules and that Trp41 functions as a gate that closes with high pHout^27,30,39 and (2) that the portion of the cytoplasmic domain nearest the transmembrane is important for normal ion channel function.⁴¹ Solid state NMR results are consistent with this domain lying in close proximity to the inner membrane leaflet.³⁷ The most remarkable aspect of this channel is the conclusion that much of its functionality is provided for by the His37 and Trp41 residues found in one turn of the transmembrane helical bundle.

Acknowledgements

We thank Drs Paul Loach and R. Gundlach for reading the manuscript. RAL is an Investigator of the Howard Hughes Medical Institute. Research in the authors’ laboratories is supported by research grants R01AI-31882 (LHP) and R37AI-20201 (RAL) from the National Institutes of Allergy and Infectious Diseases.

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Footnote

† This paper was published as part of the special issue on Proton Transfer in Biological Systems.

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