The M2 Proton Channels of Influenza A and B Viruses

Influenza A virus and influenza B virus are two different enveloped, negative stranded RNA viruses that cause epidemic infections. The virion (virus particle) of each virus contains a small integral membrane protein (A/M2 and BM2, respectively) that functions as a proton channel and is essential to viral replication. These proton channels are of interest because they are among the smallest bona fide ion channel proteins (with the properties of ion selectivity and activation), and they succeed in accomplishing the same function with only a meager similarity in primary amino acid sequence. This similarity is contained in a single turn of the transmembrane helix that appears to impart the channels with their key proton transport properties. 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 (1Lamb R.A. Krug R.M. Knipe D.M. Howley P.M. Fields Virology. 4th Ed. Lippincott Williams 76: 1391-1399Crossref PubMed Scopus (207) Google Scholar, 3Jackson D. Cadman A. Zurcher T. Barclay W.S. J. Virol. 2002; 76: 11744-11747Crossref PubMed Scopus (66) Google Scholar). Both viruses enter the infected cell by endocytosis, and the interior of the virion must become acidified while it is contained in the endosome as a prerequisite for uncoating (release of genetic material to the cytoplasm) (4Zhirnov O.P. Virology. 1990; 176: 274-279Crossref PubMed Scopus (91) Google Scholar, 5Zhirnov O.P. Virology. 1992; 186: 324-330Crossref PubMed Scopus (109) Google Scholar, 6Paterson R.G. Takeda M. Ohigashi Y. Pinto L.H. Lamb R.A. Virology. 2003; 306: 7-17Crossref PubMed Scopus (63) Google Scholar). The proton channels serve this acidification function. This review will discuss the mechanisms for proton selectivity, for turning-on (activating) and for inhibiting these proton channels. Both A/M2 and BM2 proteins are homotetrameric, type III integral membrane proteins containing a small N-terminal ectodomain, a single transmembrane domain, and C-terminal cytoplasmic tail. The transmembrane domain acts as both a signal sequence and a membrane anchor during protein synthesis. 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 (6Paterson R.G. Takeda M. Ohigashi Y. Pinto L.H. Lamb R.A. Virology. 2003; 306: 7-17Crossref PubMed Scopus (63) Google Scholar, 7Holsinger L.J. Lamb R.A. Virology. 1991; 183: 32-43Crossref PubMed Scopus (291) Google Scholar, 8Panayotov P.P. Schlesinger R.W. Virology. 1992; 186: 352-355Crossref PubMed Scopus (37) Google Scholar, 9Sugrue R.J. Hay A.J. Virology. 1991; 180: 617-624Crossref PubMed Scopus (393) Google Scholar), and the active oligomeric state of the A/M2 protein was demonstrated to be a tetramer (10Sakaguchi T. Leser G.P. Lamb R.A. J. Cell Biol. 1996; 133: 733-747Crossref PubMed Scopus (170) Google Scholar). Post-translational modifications occur to the A/M2 protein but do not seem to be important for ion channel function (11Holsinger L.J. Shaughnessy M.A. Micko A. Pinto L.H. Lamb R.A. J. Virol. 1995; 69: 1219-1225Crossref PubMed Google Scholar). 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. Both ion channels are very selective for protons (12Chizhmakov I.V. Geraghty F.M. Ogden D.C. Hayhurst A. Antoniou M. Hay A.J. J. Physiol. (Lond.). 1996; 494: 329-336Crossref Scopus (260) Google Scholar, 13Mould J.A. Drury J.E. Frings S.M. Kaupp U.B. Pekosz A. Lamb R.A. Pinto L.H. J. Biol. Chem. 2000; 275: 31038-31050Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 14Shimbo K. Brassard D.L. Lamb R.A. Pinto L.H. Biophys. J. 1996; 70: 1336-1346Abstract Full Text PDF Scopus (123) Google Scholar, 15Lin T.I. Schroeder C. J. Virol. 2001; 75: 3647-3656Crossref PubMed Scopus (105) Google Scholar, 16Mould J.A. Paterson R.G. Takeda M. Ohigashi Y. Venkataraman P. Lamb R.A. Pinto L.H. Dev. Cell. 2003; 5: 175-184Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), and their selectivity depends on a histidine residue in the transmembrane domain. Ion selectivity measurements have been made using in vitro expression systems (12Chizhmakov I.V. Geraghty F.M. Ogden D.C. Hayhurst A. Antoniou M. Hay A.J. J. Physiol. (Lond.). 1996; 494: 329-336Crossref Scopus (260) Google Scholar, 16Mould J.A. Paterson R.G. Takeda M. Ohigashi Y. Venkataraman P. Lamb R.A. Pinto L.H. Dev. Cell. 2003; 5: 175-184Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 17Pinto L.H. Holsinger L.J. Lamb R.A. Cell. 1992; 69: 517-528Abstract Full Text PDF PubMed Scopus (995) Google Scholar, 18Wang C. Lamb R.A. Pinto L.H. Virology. 1994; 205: 133-140Crossref PubMed Scopus (92) Google Scholar, 19Mould J.A. Li H.-C. Dudlak C.S. Lear J.D. Pekosz A. Lamb R.A. Pinto L.H. J. Biol. Chem. 2000; 275: 8592-8599Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar) and by reconstitution of ion channel activity from recombinant protein in bilayers (20Vijayvergiya V. Wilson R. Chorak A. Gao P.F. Cross T.A. Busath D.D. Biophys. J. 2004; 87: 1697-1704Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 21Tosteson M.T. Pinto L.H. Holsinger L.J. Lamb R.A. J. Membr. Biol. 1994; 142: 117-126Crossref PubMed Scopus (75) Google Scholar) or liposomes (15Lin T.I. Schroeder C. J. Virol. 2001; 75: 3647-3656Crossref PubMed Scopus (105) Google Scholar). The high proton selectivity of the channel is lost when transmembrane domain His37 is replaced with glycine, alanine, glutamic acid, serine, or threonine (22Wang C. Lamb R.A. Pinto L.H. Biophys. J. 1995; 69: 1363-1371Abstract Full Text PDF PubMed Scopus (223) Google Scholar, 23Venkataraman P. Lamb R.A. Pinto L.H. J. Biol. Chem. 2005; 280: 21463-21472Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), 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 (23Venkataraman P. Lamb R.A. Pinto L.H. J. Biol. Chem. 2005; 280: 21463-21472Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). 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) 3The abbreviation used is: wt, wild-type. 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) (15Lin T.I. Schroeder C. J. Virol. 2001; 75: 3647-3656Crossref PubMed Scopus (105) Google Scholar, 19Mould J.A. Li H.-C. Dudlak C.S. Lear J.D. Pekosz A. Lamb R.A. Pinto L.H. J. Biol. Chem. 2000; 275: 8592-8599Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). 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 (19Mould J.A. Li H.-C. Dudlak C.S. Lear J.D. Pekosz A. Lamb R.A. Pinto L.H. J. Biol. Chem. 2000; 275: 8592-8599Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Two other mechanisms have been suggested for proton transport. First, imidazole might serve as a “relay” molecule (Fig. 2), 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 (24Pinto L.H. Dieckmann G.R. Gandhi C.S. Shaughnessy M.A. Papworth C.G. Braman J. Lear J.D. Lamb R.A. DeGrado W.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11301-11306Crossref PubMed Scopus (319) Google Scholar). 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 (19Mould J.A. Li H.-C. Dudlak C.S. Lear J.D. Pekosz A. Lamb R.A. Pinto L.H. J. Biol. Chem. 2000; 275: 8592-8599Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Energy minimization simulations support the former model (25Lear J.D. FEBS Lett. 2003; 552: 17-22Crossref PubMed Scopus (44) Google Scholar) and molecular dynamic simulations support the latter model (26Smondyrev A.M. Voth G.A. Biophys. J. 2002; 83: 1987-1996Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Thus, the exact mechanism for transport of protons with high selectivity is not known. The A/M2 channel does not conduct protons under all conditions; to do so the pH of the medium bathing the N-terminal ectodomain, pHout, must be lowered below ∼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 carbonyl cyanide p-trifluoromethoxyphenylhydrazone (27Tang Y. Zaitseva F. Lamb R.A. Pinto L.H. J. Biol. Chem. 2002; 277: 39880-39886Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar), 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 (12Chizhmakov I.V. Geraghty F.M. Ogden D.C. Hayhurst A. Antoniou M. Hay A.J. J. Physiol. (Lond.). 1996; 494: 329-336Crossref Scopus (260) Google Scholar, 14Shimbo K. Brassard D.L. Lamb R.A. Pinto L.H. Biophys. J. 1996; 70: 1336-1346Abstract Full Text PDF Scopus (123) Google Scholar, 17Pinto L.H. Holsinger L.J. Lamb R.A. Cell. 1992; 69: 517-528Abstract Full Text PDF PubMed Scopus (995) Google Scholar, 19Mould J.A. Li H.-C. Dudlak C.S. Lear J.D. Pekosz A. Lamb R.A. Pinto L.H. J. Biol. Chem. 2000; 275: 8592-8599Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). 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 (27Tang Y. Zaitseva F. Lamb R.A. Pinto L.H. J. Biol. Chem. 2002; 277: 39880-39886Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar) (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 (27Tang Y. Zaitseva F. Lamb R.A. Pinto L.H. J. Biol. Chem. 2002; 277: 39880-39886Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). 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 (28Bauer C.M. Pinto L.H. Cross T.A. Lamb R.A. Virology. 1999; 254: 196-209Crossref PubMed Scopus (61) Google Scholar). Furthermore, resonance Raman spectroscopy has shown that pH-dependent interactions occur between His37 and Trp41, perhaps between protonated imidazole and the pi electrons of indole (29Okada A. Miura T. Takeuchi H. Biochemistry. 2001; 40: 6053-6060Crossref PubMed Scopus (205) Google Scholar). 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. Because protons must pass through the membrane via the pore of an ion channel, it is very important to identify the residues that line the pore and to obtain a rough idea of the secondary structure of the transmembrane domain. The former has been done for the A/M2 ion channel by combining cysteine and that a to cysteine residues K. Lamb R.A. Pinto L.H. J. Virol. 2000; PubMed Scopus Google Scholar). the conductance of a mutant channel with a cysteine residue in a is by the it is that the of the cysteine the aqueous pore at that The of cysteine mutant proteins and when the was to the medium bathing the N-terminal The conductance of the cysteine mutant protein was when the was injected into the but not when to the bathing the mutant protein was not by cytoplasmic of the These results are with His37 a to molecules and are also with the role for Trp41 as a capable of closing the channel. was also used with oxidative disulfide cross-linking (28Bauer C.M. Pinto L.H. Cross T.A. Lamb R.A. Virology. 1999; 254: 196-209Crossref PubMed Scopus (61) Google Scholar) to that residues and formed was the that when was at low pHout cross-linking was much lower for residues and than when at pHout suggesting that a pH-dependent of occurs in this region of the The secondary structure of the transmembrane domain of the A/M2 channel has been with state spectroscopy of the transmembrane Cross T.A. J. Biol. 2000; PubMed Scopus Google Scholar, J. Cross T.A. Biophys. J. 2000; Full Text Full Text PDF PubMed Scopus (87) Google Scholar, J. S. F. Cross T.A. Sci. 2001; PubMed Scopus Google Scholar, K. S. Cross T.A. Biochemistry. 2002; PubMed Scopus Google Scholar) and the A/M2 ion channel protein C. K. Lamb R.A. Pinto L.H. Cross T.A. Biochemistry. 2002; PubMed Scopus Google Scholar, C. Gao P.F. Pinto L.H. Lamb R.A. Cross T.A. Sci. 2003; PubMed Scopus Google Scholar). These that the of the the transmembrane domain has a of and that transmembrane domain Trp of one with of an K. S. Cross T.A. Biochemistry. 2002; PubMed Scopus Google Scholar). Furthermore, measurements that residues in the transmembrane helix more rapidly than residues in the cytoplasmic domain, with the of an aqueous pore. Thus, the aqueous pore of the A/M2 ion channel is formed by and Trp41, and these residues are found on a transmembrane having a of The cytoplasmic is the domain of both the A/M2 and BM2 proteins, and this domain has been found to be essential for the function of the A/M2 channel. of the A/M2 channel at residue or shorter in mutant ion channels with activity that not be K. Pinto L.H. Lamb R.A. J. Virol. 1999; PubMed Google Scholar). state experiments have the structural about the cytoplasmic domain for residues C. Gao P.F. Pinto L.H. Lamb R.A. Cross T.A. Sci. 2003; PubMed Scopus Google Scholar). These that an helix is found in this with the inner membrane This helix was found in the same region of the cytoplasmic that been found to be essential for ion channel activity K. Pinto L.H. Lamb R.A. J. Virol. 1999; PubMed Google Scholar). Thus, ion channel activity has been demonstrated with only transmembrane Virology. 1992; PubMed Scopus Google Scholar), the cytoplasmic plays an essential role in the function of the wt ion channel The and state that the transmembrane domain of the A/M2 protein is of a with a of about These experiments are also with the functional that that that residues and Trp41 line the aqueous pore. Furthermore, these results that His37 a to molecules and that Trp41 functions as a that closes with high pHout. that the of the cytoplasmic domain the membrane is important for normal ion channel and state that the structure of this domain it into close to the inner membrane The of this channel is the that much of its is for by the His37 and Trp41 residues found in one turn of the transmembrane This is important because antiviral that the ion channel also replication of the influenza A virus, and the mechanism of be in The antiviral drug and its the replication of the influenza A virus but not influenza B virus M. PubMed Scopus Google Scholar). the A/M2 ion channel (12Chizhmakov I.V. Geraghty F.M. Ogden D.C. Hayhurst A. Antoniou M. Hay A.J. J. Physiol. (Lond.). 1996; 494: 329-336Crossref Scopus (260) Google Scholar, 17Pinto L.H. Holsinger L.J. Lamb R.A. Cell. 1992; 69: 517-528Abstract Full Text PDF PubMed Scopus (995) Google Scholar, C. Takeuchi K. Pinto L.H. Lamb R.A. J. Virol. PubMed Google Scholar) but not the BM2 channel J.A. Paterson R.G. Takeda M. Ohigashi Y. Venkataraman P. Lamb R.A. Pinto L.H. Dev. Cell. 2003; 5: 175-184Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), which is with inhibiting influenza A virus but not influenza B virus replication. Several lines of evidence that of viral replication by results from the of A/M2 proton channel The evidence from mutant viruses in which the of the was and found to in the A/M2 transmembrane domain A.J. A.J. J. PubMed Scopus Google Scholar). these proteins in oocytes L.H. Holsinger L.J. Lamb R.A. Cell. 1992; 69: 517-528Abstract Full Text PDF PubMed Scopus (995) Google Scholar, L.J. D. Pinto L.H. Lamb R.A. J. Virol. 1994; PubMed Google Scholar) or cells (12Chizhmakov I.V. Geraghty F.M. Ogden D.C. Hayhurst A. Antoniou M. Hay A.J. J. Physiol. (Lond.). 1996; 494: 329-336Crossref Scopus (260) Google Scholar, 18Wang C. Lamb R.A. Pinto L.H. Virology. 1994; 205: 133-140Crossref PubMed Scopus (92) Google Scholar) their currents found to be to amantadine. The second line of evidence from of the virus uncoating acidification of the virus to with the membrane K. A. Cell. 1991; Full Text PDF PubMed Scopus Google Scholar, M. A. J. Virol. 1996; 70: PubMed Google Scholar, A. Cell. 1992; 69: Full Text PDF PubMed Scopus Google Scholar), and the protein is capable of the acidification when the virion is contained in the The viral integral membrane proteins R.A. Cell. 40: Full Text PDF PubMed Scopus Google Scholar), and only A/M2 is capable of proton transport. Thus, of the essential proton transport function of the A/M2 ion channel results in of replication of the taken suggest a mechanism for by amantadine. 1) Two of the that in to occur on residues that have been found by cysteine to line the aqueous pore. Both of these are to residues that are and than the residue A.J. A.J. J. PubMed Scopus Google Scholar). of the channel occurs more when pH of the bathing medium is high C. Takeuchi K. Pinto L.H. Lamb R.A. J. Virol. PubMed Google Scholar). only when it is to the medium bathing the N-terminal and not when to the C-terminal cytoplasmic of as much as 1 into cells expressing the A/M2 channel does not the channel, of to the solution bathing the C. Takeuchi K. Pinto L.H. Lamb R.A. J. Virol. PubMed Google Scholar). of to the transmembrane the to in the region of the membrane A.M. Virology. 1994; PubMed Scopus (91) Google Scholar). with C. Takeuchi K. Pinto L.H. Lamb R.A. J. Virol. PubMed Google Scholar). These observations suggest that acts from the of the aqueous pore, that the group with and that perhaps the of a hydrogen with an imidazole histidine (19Mould J.A. Li H.-C. Dudlak C.S. Lear J.D. Pekosz A. Lamb R.A. Pinto L.H. J. Biol. Chem. 2000; 275: 8592-8599Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). hydrogen it the interactions formed by the of and Trp residues from subunits K. S. Cross T.A. Biochemistry. 2002; PubMed Scopus Google Scholar). Thus, probably within the aqueous pore of the channel when it but probably does not by the pore. It is to the amino acid sequences of the region of the transmembrane domain of these two ion channels because the A/M2 channel by in the region of the pore (see The residues of A/M2 have been in K. Lamb R.A. Pinto L.H. J. Virol. 2000; PubMed Scopus Google Scholar, C.S. Shuck K. Lear J.D. Dieckmann G.R. DeGrado W.F. Lamb R.A. Pinto L.H. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). It is to the that and of the BM2 protein are residues because of these residues alterations in BM2 function that are to found by of His37 and Trp41 in the A/M2 ion channel. these residues as and that the BM2 transmembrane domain a (Fig. of the sequences of the two channels shows that residues occur in the BM2 channel in that are predicted to be The of residues be to the of the of the It is of interest to that replacement of a residue with a residue occurs in an of the A/M2 ion channel. The is and in active L.J. D. Pinto L.H. Lamb R.A. J. Virol. 1994; PubMed Google Scholar). Thus, the that the BM2 ion channel is not by is probably because the BM2 channel pore region is with and not amino This explanation shows that the of to the BM2 protein is in of the primary amino acid sequence of its transmembrane domain. One of the properties of the A/M2 ion channel is its high selectivity for protons, and two from this The is selectivity is by a mechanism or by a mechanism in which the imidazole of transmembrane His37 protons, binding and releasing them but as a for other this it will be of to the channel is not to K+ or it is to and J.A. Drury J.E. Frings S.M. Kaupp U.B. Pekosz A. Lamb R.A. Pinto L.H. J. Biol. Chem. 2000; 275: 31038-31050Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). The second by the very high proton selectivity of the channel is can it transport protons across the virion membrane to acidification to allow uncoating to protons into the virion they will with them a positive the only for ions to across the virion membrane is the ion channel their will impart a positive voltage on the inside of the It only a ions the virion to its voltage positive to proton and this small of ions to the virion the of protons occurs at the very of virion with the and ion channels of a positive A has to do with the cytoplasmic of the A/M2 and BM2 The cytoplasmic of both of these proteins is than the other and its does not seem to be for ion channel the cytoplasmic another of the an important to be the of a that the function of the HXXXW motif of both proteins without with and R. for the