Symmetry, Selectivity, and the 2003 Nobel Prize

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 & Wilkins, Philadelphia2001: 1487-1531Google Scholar). The proton channels of both viruses must function for viral replication to occur (Fig. 1) (2Takeda M. Pekosz A. Shuck K. Pinto L.H. Lamb R.A. J. Virol. 2002; 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 scanning mutagenesis and water-soluble sulfhydryl-specific reagents that attach a large hydrophobic adduct to cysteine residues (30Shuck K. Lamb R.A. Pinto L.H. J. Virol. 2000; 74: 7755-7761Crossref PubMed Scopus (41) Google Scholar). If the conductance of a mutant channel with a cysteine residue in a particular location is diminished by the reagent, then it is concluded that the sulfhydryl moiety of the cysteine faces the aqueous pore at that location. The conductances of cysteine mutant proteins A30C and G34C were diminished when the reagent was applied to the medium bathing the N-terminal ectodomain. The conductance of the cysteine mutant protein W41C was decreased when the reagent was injected into the cytoplasm but not when applied to the bathing medium. Moreover, the G34C mutant protein was not affected by cytoplasmic injection of the reagent. These results are consistent with His37 forming a barrier to large molecules and are also consistent with the proposed role for Trp41 as a gate capable of closing the channel. Cysteine scanning mutagenesis was also used together 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 show that residues 27, 30, 34, 37, and 41 formed dimers most rapidly. Most interesting, however, was the finding that when oxidation was performed at low pHout (pH 5.2) cross-linking was much lower for residues 40, 42, and 43 than when performed at neutral pHout (pH 7.4), suggesting that a pH-dependent alteration of conformation occurs in this region of the molecule. The secondary structure of the transmembrane domain of the A/M2 channel has been studied with solid state NMR spectroscopy of the transmembrane peptide (31Kovacs F.A. Denny J.K. Song Z. Quine J.R. Cross T.A. J. Mol. Biol. 2000; 295: 117-125Crossref PubMed Scopus (131) Google Scholar, 32Song Z. Kovacs F.A. Wang J. Denny J.K. Shekar S.C. Quine J.R. Cross T.A. Biophys. J. 2000; 79: 767-775Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 33Wang J. Kim S. Kovacs F. Cross T.A. Protein Sci. 2001; 10: 2241-2250Crossref PubMed Scopus (225) Google Scholar, 34Nishimura K. Kim S. Zhang L. Cross T.A. Biochemistry. 2002; 41: 13170-13177Crossref PubMed Scopus (198) Google Scholar) and the full-length A/M2 ion channel protein (35Tian C. Tobler K. Lamb R.A. Pinto L.H. Cross T.A. Biochemistry. 2002; 41: 11294-11300Crossref PubMed Scopus (55) Google Scholar, 36Tian C. Gao P.F. Pinto L.H. Lamb R.A. Cross T.A. Protein Sci. 2003; 12: 2597-2605Crossref PubMed Scopus (124) Google Scholar). These studies concluded that the orientation of the helical bundle forming the transmembrane domain has a tilt angle of ∼25 degrees and that transmembrane domain Trp of one subunit interacts with His of an adjacent subunit (34Nishimura K. Kim S. Zhang L. Cross T.A. Biochemistry. 2002; 41: 13170-13177Crossref PubMed Scopus (198) Google Scholar). Furthermore, hydrogen-deuterium exchange measurements showed that residues in the transmembrane helix exchange more rapidly than residues located in the cytoplasmic domain, consistent with the presence of an aqueous pore. Thus, the aqueous pore of the A/M2 ion channel is formed by Ala30, Gly34, His37, and Trp41, and these residues are found on a transmembrane helical bundle having a tilt angle of ∼25 degrees. The cytoplasmic tail is the largest 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. Truncation of the A/M2 channel at residue 61 or shorter resulted in mutant ion channels with activity that could not be sustained (37Tobler K. Kelly M.L. Pinto L.H. Lamb R.A. J. Virol. 1999; 73: 9695-9701Crossref PubMed Google Scholar). Solid state NMR experiments have provided the first direct structural information about the cytoplasmic domain for residues 45–62 (36Tian C. Gao P.F. Pinto L.H. Lamb R.A. Cross T.A. Protein Sci. 2003; 12: 2597-2605Crossref PubMed Scopus (124) Google Scholar). These studies indicated that an amphipathic helix is found in this region, associated with the inner membrane leaflet. This amphipathic helix was found in the same region of the cytoplasmic tail that had been found to be essential for ion channel activity (37Tobler K. Kelly M.L. Pinto L.H. Lamb R.A. J. Virol. 1999; 73: 9695-9701Crossref PubMed Google Scholar). Thus, even though ion channel activity has been demonstrated with only transmembrane peptides (38Duff K.C. Ashley R.H. Virology. 1992; 190: 485-489Crossref PubMed Scopus (219) Google Scholar), the cytoplasmic tail certainly plays an essential role in the function of the wt ion channel protein. 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. These experiments are also consistent with the functional studies that showed that that residues Val27, Ala30, Gly34, His37, and Trp41 line the aqueous pore. Furthermore, these results indicate that His37 forms a barrier to large molecules and that Trp41 functions as a gate that closes with high pHout. Functional studies indicate that the portion of the cytoplasmic domain nearest the membrane is important for normal ion channel function, and solid state NMR studies indicate that the structure of this domain brings it into close proximity to the inner membrane leaflet. The most remarkable aspect of this channel is the observation that much of its functionality is provided for by the His37 and Trp41 residues found in one turn of the transmembrane helical bundle. This topic is important because antiviral drugs that inhibit the M2 ion channel also inhibit replication of the influenza A virus, and understanding the mechanism of inhibition would be helpful in developing better inhibitors. The antiviral drug amantadine and its derivative rimantadine inhibit the replication of the influenza A virus but not influenza B virus (39Davies W.L. Grunert R.R. Haff R.F. McGahen J.W. Neumayer E.M. Paulshock M. Watts J.C. Wood T.R. Herman E.C. Hoffman C.E. Science. 1964; 144: 862-863Crossref PubMed Scopus (646) Google Scholar). Amantadine inhibits 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, 40Wang C. Takeuchi K. Pinto L.H. Lamb R.A. J. Virol. 1993; 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 consistent with amantadine inhibiting influenza A virus but not influenza B virus replication. Several lines of evidence indicate that inhibition of viral replication by amantadine results from the inhibition of A/M2 proton channel The first 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 were 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 were 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 M2 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, inhibition of the essential proton transport function of the A/M2 ion channel results in inhibition of replication of the taken suggest a mechanism for inhibition by amantadine. 1) Two of the that in to amantadine occur on residues that have been found by cysteine scanning mutagenesis to line the aqueous pore. Both of these are to residues that are hydrophobic 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. 1993; PubMed Google Scholar). Amantadine only inhibits when it is applied to the medium bathing the N-terminal and not when applied to the C-terminal cytoplasmic injection of as much as 1 amantadine into cells expressing the A/M2 channel does not inhibit the channel, of to the solution bathing the inhibits C. Takeuchi K. Pinto L.H. Lamb R.A. J. Virol. 1993; PubMed Google Scholar). studies of amantadine applied to the M2 transmembrane peptide show the to in the region of the membrane K.C. A.M. Virology. 1994; PubMed Scopus (91) Google Scholar). Amantadine inhibits with C. Takeuchi K. Pinto L.H. Lamb R.A. J. Virol. 1993; PubMed Google Scholar). These observations suggest that amantadine acts from the of the aqueous pore, that the hydrophobic group interacts with hydrophobic and that perhaps the of amantadine 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). If hydrogen it would the interactions formed by the of His and Trp residues from adjacent subunits (34Nishimura K. Kim S. Zhang L. Cross T.A. Biochemistry. 2002; 41: 13170-13177Crossref PubMed Scopus (198) Google Scholar). Thus, amantadine probably within the aqueous pore of the channel when it inhibits but probably does not inhibit by the pore. It is to the amino acid sequences of the region of the transmembrane domain of these two ion channels because amantadine inhibits the A/M2 channel by in the region of the pore (see The residues of A/M2 have been in studies (30Shuck K. Lamb R.A. Pinto L.H. J. Virol. 2000; 74: 7755-7761Crossref PubMed Scopus (41) 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 forms a helical bundle (Fig. of the sequences of the two channels shows that residues occur in the BM2 channel in that are predicted to be The presence of residues would be to the of the hydrophobic moiety of the It is of interest to that replacement of a hydrophobic 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 amantadine is probably because the BM2 channel pore region is with and not amino This explanation shows that the of amantadine to inhibit the BM2 protein is in of the primary amino acid sequence of its transmembrane domain. One of the remarkable properties of the A/M2 ion channel is its high selectivity for protons, and two from this The first 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 barrier 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 M2 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 tail of the A/M2 and BM2 The cytoplasmic tail of both of these proteins is than the other and its full-length does not seem to be for ion channel the cytoplasmic tail another of the an important to would be the of a that inhibits the function of the HXXXW motif of both proteins without with and R. for the

The channel kinases TRPM6 and TRPM7 have recently been discovered to play important roles in Mg2+ and Ca2+ homeostasis, which is critical to both human health and cell viability. However, the molecular basis underlying these channels' unique Mg2+ and Ca2+ permeability and pH sensitivity remains unknown. Here we have created a series of amino acid substitutions in the putative pore of TRPM7 to evaluate the origin of the permeability of the channel and its regulation by pH. Two mutants of TRPM7, E1047Q and E1052Q, produced dramatic changes in channel properties. The I-V relations of E1052Q and E1047Q were significantly different from WT TRPM7, with the inward currents of 8- and 12-fold larger than TRPM7, respectively. The binding affinity of Ca2+ and Mg2+ was decreased by 50- to 140-fold in E1052Q and E1047Q, respectively. Ca2+ and Mg2+ currents in E1052Q were 70% smaller than those of TRPM7. Strikingly, E1047Q largely abolished Ca2+ and Mg2+ permeation, rendering TRPM7 a monovalent selective channel. In addition, the ability of protons to potentiate inward currents was lost in E1047Q, indicating that E1047 is critical to Ca2+ and Mg2+ permeability of TRPM7, and its pH sensitivity. Mutation of the corresponding residues in the pore of TRPM6, E1024Q and E1029Q, produced nearly identical changes to the channel properties of TRPM6. Our results indicate that these two glutamates are key determinants of both channels' divalent selectivity and pH sensitivity. These findings reveal the molecular mechanisms underpinning physiological/pathological functions of TRPM6 and TRPM7, and will extend our understanding of the pore structures of TRPM channels.

There is intense interest in comprehensive proteomic approaches for analyzing integral membrane proteins and lipoproteins. Key features of mass spectrometric analysis center on enriching biological material for proteins of interest, efficiently digesting them, extracting the resulting peptides, and using fractionation methods to comprehensively sample proteins or peptides by tandem mass spectrometry. However, lipid-associated proteins are generally rich in hydrophobic domains and are often low in abundance. These features, together with the associated lipid, make their mass spectrometric analysis technically challenging. In this article, we review analytical strategies for successful proteomic analysis of lipid-associated proteins.

Voltage-gated potassium channels are transmembrane proteins made up of four subunits, each comprising six transmembrane (S1-S6) segments. S1-S4 form the voltage-sensing domain and S5-S6 the pore domain with its central pore. The sensor domain detects membrane depolarization and transmits the signal to the activation gates situated in the pore domain, thereby leading to channel opening. An understanding of the mechanism by which the sensor communicates the signal to the pore requires knowledge of the structure of the interface between the voltage-sensing and pore domains. Toward this end, we have introduced single cysteine mutations into the extracellular end of S4 (positions 356 and 357) in conjunction with a cysteine in S5 (position 418) of the Shaker channel and expressed the mutants in Xenopus oocytes. We then examined the propensity of each pair of engineered cysteines to form a metal bridge or a disulfide bridge, respectively, by examining the effect of Cd2+ ions and copper phenanthroline on the K+ conductance of a whole oocyte. Both reagents reduced currents through the S357C,E418C double mutant channel, presumably by restricting the movements necessary for coupling the voltage-sensing function to pore opening. This inhibitory effect was seen in the closed state of the channel and with heteromers composed of S357C and E418C single mutant subunits; no effect was seen with homomers of any of the single mutant channels. These data indicate that the extracellular end of S4 lies in close proximity to the extracellular end of the S5 of the neighboring subunit in closed channels.

ATP-gated P2X(2) channels undergo activation-dependent permeability increases as they proceed from the selective I(1) state to the I(2) state that is readily permeable to organic cations. There are two main models about how permeability changes may occur. The first proposes that permeability change-competent P2X channels are clustered or redistribute to form such regions in response to ATP. The second proposes that permeability changes occur because of an intrinsic conformational change in P2X channels. In the present study we experimentally tested these views with total internal reflection fluorescence microscopy, electrophysiology, and mutational perturbation analysis. We found no evidence for clusters of P2X(2) channels within the plasma membrane or for cluster formation in response to ATP, suggesting that channel clustering is not an obligatory requirement for permeability changes. We next sought to identify determinants of putative intrinsic conformational changes in P2X(2) channels by mapping the transmembrane domain regions involved in the transition from the relatively selective I(1) state to the dilated I(2) state. Initial channel opening to the I(1) state was only weakly affected by Ala substitutions, whereas dramatic effects were observed for the higher permeability I(2) state. Ten residues appeared to perturb only the I(1)-I(2) transition (Phe(31), Arg(34), Gln(37), Lys(53), Ile(328), Ile(332), Ser(340), Gly(342), Trp(350), Leu(352)). The data favor the hypothesis that permeability changes occur because of permissive motions at the interface between first and second transmembrane domains of neighboring subunits in pre-existing P2X(2) channels.

Protein S-palmitoylation, the reversible thioester linkage of a 16-carbon palmitate lipid to an intracellular cysteine residue, is rapidly emerging as a fundamental, dynamic, and widespread post-translational mechanism to control the properties and function of ligand- and voltage-gated ion channels. Palmitoylation controls multiple stages in the ion channel life cycle, from maturation to trafficking and regulation. An emerging concept is that palmitoylation is an important determinant of channel regulation by other signaling pathways. The elucidation of enzymes controlling palmitoylation and developments in proteomics tools now promise to revolutionize our understanding of this fundamental post-translational mechanism in regulating ion channel physiology.

NAADP is a potent second messenger that mobilizes Ca(2+) from acidic organelles such as endosomes and lysosomes. The molecular basis for Ca(2+) release by NAADP, however, is uncertain. TRP mucolipins (TRPMLs) and two-pore channels (TPCs) are Ca(2+)-permeable ion channels present within the endolysosomal system. Both have been proposed as targets for NAADP. In the present study, we probed possible physical and functional association of these ion channels. Exogenously expressed TRPML1 showed near complete colocalization with TPC2 and partial colocalization with TPC1. TRPML3 overlap with TPC2 was more modest. TRPML1 and to some extent TRPML3 co-immunoprecipitated with TPC2 but less so with TPC1. Current recording, however, showed that TPC1 and TPC2 did not affect the activity of wild-type TRPML1 or constitutively active TRPML1(V432P). N-terminally truncated TPC2 (TPC2delN), which is targeted to the plasma membrane, also failed to affect TRPML1 and TRPML1(V432P) channel function or TRPML1(V432P)-mediated Ca(2+) influx. Whereas overexpression of TPCs enhanced NAADP-mediated Ca(2+) signals, overexpression of TRPML1 did not, and the dominant negative TRPML1(D471K) was without affect on endogenous NAADP-mediated Ca(2+) signals. Furthermore, the single channel properties of NAADP-activated TPC2delN were not affected by TRPML1. Finally, NAADP-evoked Ca(2+) oscillations in pancreatic acinar cells were identical in wild-type and TRPML1(-/-) cells. We conclude that although TRPML1 and TPCs are present in the same complex, they function as two independent organellar ion channels and that TPCs, not TRPMLs, are the targets for NAADP.

Although many of the details of GlyR dynamics still remain elusive, the extensive studies conducted on GlyR and other members of the nicotinicoid superfamily in recent years provide us with an emerging picture of the structure and function of these receptors. As novel studies continue to examine these receptors, we are confident that these ion channels will reveal their molecular mechanisms. These details are significant in that they will allow us to develop novel therapeutics and pharmacological tools to modulate channel activity in the central nervous system.

Cotranslational Membrane Protein Biogenesis at the Endoplasmic Reticulum

A growing body of evidence shows that membrane phosphatidylinositol 4,5-bisphosphates (PtdIns(4,5)P(2), PIP(2)) play an important role in cell signaling. The presence of PIP(2) is fundamentally important for maintaining the functions of a large number of ion channels and transporters, and for other cell processes such as vesicle trafficking, mobility, and endo- and exocytosis. PIP(2) levels in the membrane are dynamically modulated, which is an important signaling mechanism for modulation of PIP(2)-dependent cellular processes. In this study, we describe a novel mechanism of membrane PIP(2) modulation. Membrane depolarization induces an elevation in membrane PIP(2), and subsequently increases functions of PIP(2)-sensitive KCNQ potassium channels expressed in Xenopus oocytes. Further evidence suggests that the depolarization-induced elevation of membrane PIP(2) occurs through increased activity of PI4 kinase. With increased recognition of the importance of PIP(2) in cell function, the effect of membrane depolarization in PIP(2) metabolism is destined to have important physiological implications.

Epithelial Na+ channels facilitate the transport of Na+ across high resistance epithelia. Proteolytic cleavage has an important role in regulating the activity of these channels by increasing their open probability. Specific proteases have been shown to activate epithelial Na+ channels by cleaving channel subunits at defined sites within their extracellular domains. This minireview addresses the mechanisms by which proteases activate this channel and the question of why proteolysis has evolved as a mechanism of channel activation.

Voltage-gated ion channels are responsible for the generation of action potentials in our nervous system. Conformational rearrangements in their voltage sensor domains in response to changes of the membrane potential control pore opening and thus ion conduction. Crystal structures of the open channel in combination with a wealth of biophysical data and molecular dynamics simulations led to a consensus on the voltage sensor movement. However, the coupling between voltage sensor movement and pore opening, the electromechanical coupling, occurs at the cytosolic face of the channel, from where no structural information is available yet. In particular, the question how far the cytosolic pore gate has to close to prevent ion conduction remains controversial. In cells, spectroscopic methods are hindered because labeling of internal sites remains difficult, whereas liposomes or detergent solutions containing purified ion channels lack voltage control. Here, to overcome these problems, we controlled the state of the channel by varying the lipid environment. This way, we directly measured the position of the S4-S5 linker in both the open and the closed state of a prokaryotic Kv channel (KvAP) in a lipid environment using Lanthanide-based resonance energy transfer. We were able to reconstruct the movement of the covalent link between the voltage sensor and the pore domain and used this information as restraints for molecular dynamics simulations of the closed state structure. We found that a small decrease of the pore radius of about 3-4 Å is sufficient to prevent ion permeation through the pore.

Glycine receptors are Cys loop ligand-gated ion channels that mediate fast inhibitory synaptic transmission in the mammalian central nervous system. The functionally distinct splice variants alpha3L and alpha3K of the human glycine receptor differ by a 15-amino acid insert within the long intracellular TM3-4 loop, a region of high intersubunit diversity. In a mutational study, effects of the insert on ion channel function and secondary structure of the TM3-4 loop were investigated. Whole cell current responses and protein surface expression data indicated that the major effect of mutations within the insert was on channel gating. Changes in channel gating correlated with the distribution of charged residues about the splice region. Analysis of complex molecular weight indicated that recombinant TM3-4 loops of alpha3L and alpha3K associated into oligomers of different stoichiometry. Secondary structure analysis suggested that the insert stabilized the overall fold of the large cytoplasmic domain of alpha3L subunits. The absence of the insert resulted in a channel that was still functional, but the TM3-4 cytoplasmic domain appeared not stably folded. Thus, our data identified the spliced insert within the large TM 3-4 loop of alpha3 Gly receptors as a novel regulatory motif that serves a 2-fold role: (i) the presence of the insert stabilizes the overall spatial structure of the domain, and (ii) the insert presents a control unit that regulates gating of the receptor ion channel.

Extracellular Determinants of Anion Discrimination of the Cl−/H+ Antiporter Protein CLC-5

The WD-repeat protein receptor for activated C-kinase (RACK1) was identified by its interaction with the cyclic AMP-specific phosphodiesterase (PDE4) isoform PDE4D5 in a yeast two-hybrid screen. The interaction was confirmed by co-immunoprecipitation of native RACK1 and PDE4D5 from COS7, HEK293, 3T3-F442A, and SK-N-SH cell lines. The interaction was unaffected by stimulation of the cells with the phorbol ester phorbol 2-myristate 3-acetate. PDE4D5 did not interact with two other WD-repeat proteins, beta'-coatomer protein and Gsbeta, in two-hybrid tests. RACK1 did not interact with other PDE4D isoforms or with known PDE4A, PDE4B, and PDE4C isoforms. PDE4D5 and RACK1 interacted with high affinity (Ka approximately 7 nM) [corrected] when they were expressed and purified from Escherichia coli, demonstrating that the interaction does not require intermediate proteins. The binding of the E. coli-expressed proteins did not alter the kinetics of cAMP hydrolysis by PDE4D5 but caused a 3-4-fold change in its sensitivity to inhibition by the PDE4 selective inhibitor rolipram. The subcellular distributions of RACK1 and PDE4D5 were extremely similar, with the major amount of both proteins (70%) in the high speed supernatant (S2) fraction. Analysis of constructs with specific deletions or single amino acid mutations in PDE4D5 demonstrated that a small cluster of amino acids in the unique amino-terminal region of PDE4D5 was necessary for its interaction with RACK1. We suggest that RACK1 may act as a scaffold protein to recruit PDE4D5 and other proteins into a signaling complex.