FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2014763114> ?p ?o ?g. }

• W2014763114 endingPage "21472" @default.
• W2014763114 startingPage "21463" @default.
• W2014763114 abstract "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 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 M2 protein of influenza A virus permits protons to enter virus particles during virion uncoating in endosomes, and the M2 channel also causes the equilibration of pH between the acidic lumen of the trans-Golgi network and the cytoplasm (1Hay A.J. Semin. Virol. 1992; 3: 21-30Google Scholar, 2Lamb R.A. Holsinger L.J. Pinto L.H. Wimmer E. Receptor-mediated Virus Entry into Cells. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994: 303-321Google Scholar, 3Lamb R.A. Pinto L.H. Kawaoka Y. Contemporary Topics in Influenza Virology. Horizon Scientific Press, Wymondham, Norfolk, UK2005Google Scholar). The activity of the M2 ion channel is inhibited by the antiviral drug amantadine (4Pinto L.H. Holsinger L.J. Lamb R.A. Cell. 1992; 69: 517-528Abstract Full Text PDF PubMed Scopus (1000) Google Scholar, 5Wang C. Takeuchi K. Pinto L.H. Lamb R.A. J. Virol. 1993; 67: 5585-5594Crossref PubMed Google Scholar, 6Chizhmakov 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). The mature M2 protein consists of a 23-residue N-terminal extracellular domain, a single internal hydrophobic domain of 19 residues that acts as a transmembrane (TM) 1The abbreviations used are: TM, transmembrane; MES, 4-morpholineethanesulfonic acid; MOPSO, 3-morpholino-2-hydroxypropanesulfonic acid; NMDG, N-methyl-d-glucamine; wt, wild type. domain and forms the pore of the channel, and a 54-residue cytoplasmic tail (7Lamb R.A. Zebedee S.L. Richardson C.D. Cell. 1985; 40: 627-633Abstract Full Text PDF PubMed Scopus (441) Google Scholar). Chemical cross-linking studies (8Sugrue R.J. Hay A.J. Virology. 1991; 180: 617-624Crossref PubMed Scopus (394) Google Scholar, 9Holsinger L.J. Lamb R.A. Virology. 1991; 183: 32-43Crossref PubMed Scopus (291) Google Scholar, 10Panayotov P.P. Schlesinger R.W. Virology. 1992; 186: 352-355Crossref PubMed Scopus (37) Google Scholar) and statistical analysis of the ion channel activity of mixed oligomers (11Sakaguchi T. Tu Q. Pinto L.H. Lamb R.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5000-5005Crossref PubMed Scopus (146) Google Scholar) showed the active state of the M2 ion channel protein to exist minimally as a homotetramer. Despite the small size of the active M2 oligomer, several lines of evidence indicate that ion channel activity is intrinsic to the M2 protein. First, ion channel activity has been observed in three different expression systems, Xenopus oocytes (4Pinto L.H. Holsinger L.J. Lamb R.A. Cell. 1992; 69: 517-528Abstract Full Text PDF PubMed Scopus (1000) Google Scholar, 6Chizhmakov 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), mammalian cells (6Chizhmakov 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, 12Wang C. Lamb R.A. Pinto L.H. Virology. 1994; 205: 133-140Crossref PubMed Scopus (92) Google Scholar), and yeast (13Tu Q. Pinto L.H. Luo G. Shaughnessy M.A. Mullaney D. Kurtz S. Krystal M. Lamb R.A. J. Virol. 1996; 70: 4246-4252Crossref PubMed Google Scholar, 14Kurtz S. Luo G. Hahnenberger K.M. Brooks C. Gecha O. Ingalls K. Numata K. Krystal M. Antimicrob. Agents Chemother. 1995; 39: 2204-2209Crossref PubMed Scopus (72) Google Scholar). Second, M2 channel activity has also been recorded in artificial lipid bilayers from a reconstituted peptide corresponding to the TM domain of the M2 protein (15Duff K.C. Ashley R.H. Virology. 1992; 190: 485-489Crossref PubMed Scopus (220) Google Scholar) and from purified M2 protein (16Tosteson M.T. Pinto L.H. Holsinger L.J. Lamb R.A. J. Membr. Biol. 1994; 142: 117-126Crossref PubMed Scopus (75) Google Scholar, 17Lin T.I. Schroeder C. J. Virol. 2001; 75: 3647-3656Crossref PubMed Scopus (106) Google Scholar). Thus, due to its structural simplicity, the M2 ion channel is a potentially useful model for studying ion channels in general. The ion selectivity of the M2 ion channel has been studied with voltage clamp (18Mould 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 (135) Google Scholar, 19Shimbo K. Brassard D.L. Lamb R.A. Pinto L.H. Biophys. J. 1996; 70: 1336-1346Abstract Full Text PDF Scopus (123) Google Scholar) and ion flux studies (17Lin T.I. Schroeder C. J. Virol. 2001; 75: 3647-3656Crossref PubMed Scopus (106) Google Scholar, 19Shimbo K. Brassard D.L. Lamb R.A. Pinto L.H. Biophys. J. 1996; 70: 1336-1346Abstract Full Text PDF Scopus (123) Google Scholar) indicating that the channel is nearly perfectly selective for protons. The channel is inactive for extracellular pH values higher than pH 7.5 but becomes active when extracellular pH is lowered. This greater activity is due to two factors, greater abundance of protons and increased channel opening due to an interaction between protonated His-37 and the indole side chain of Trp-41, the putative “gate” of the ion channel (20Okada A. Miura T. Takeuchi H. Biochemistry. 2001; 40: 6053-6060Crossref PubMed Scopus (206) Google Scholar, 21Tang Y. Zaitseva F. Lamb R.A. Pinto L.H. J. Biol. Chem. 2002; 277: 39880-39886Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Experiments in which His-37 of the TM domain was replaced by site-directed mutagenesis with Gly, Ala, Glu, Lys, and Arg (22Wang C. Lamb R.A. Pinto L.H. Biophys. J. 1995; 69: 1363-1371Abstract Full Text PDF PubMed Scopus (224) Google Scholar) indicate that His-37 is essential for the proton selectivity of the channel and its activation by low pH. However, it is possible that substitution of even a single residue in the closely packed pore of this protein might bring about large structural changes in the protein, making a comparison of the mutant and wild-type (wt) channels difficult. For some enzymes that transport protons as part of their catalytic cycle, substitution of histidine in the active site with alanine or glycine resulted in loss of enzymatic activity, e.g. carbonic anhydrase II (23Tu C.K. Silverman D.N. Forsman C. Jonsson B.H. Lindskog S. Biochemistry. 1989; 28: 7913-7918Crossref PubMed Scopus (351) Google Scholar, 24Tu C. Rowlett R.S. Tripp B.C. Ferry J.G. Silverman D.N. Biochemistry. 2002; 41: 15429-15435Crossref PubMed Scopus (43) Google Scholar, 25Duda D. Govindasamy L. Agbandje-McKenna M. Tu C. Silverman D.N. McKenna R. Acta Crystallogr. Sect. D. Biol. Crystallogr. 2003; 59: 93-104Crossref PubMed Scopus (50) Google Scholar), copper amine oxidase (26Matsunami H. Okajima T. Hirota S. Yamaguchi H. Hori H. Kuroda S. Tanizawa K. Biochemistry. 2004; 43: 2178-2187Crossref PubMed Scopus (18) Google Scholar), aldolase (27Hopkins C.E. O'Connor P.B. Allen K.N. Costello C.E. Tolan D.R. Protein Sci. 2002; 11: 1591-1599Crossref PubMed Scopus (33) Google Scholar), (S)-mandelate dehydrogenase (28Lehoux I.E. Mitra B. Biochemistry. 1999; 38: 9948-9955Crossref PubMed Scopus (41) Google Scholar), bacterial luciferase (29Huang S. Tu S.C. Biochemistry. 1997; 36: 14609-14615Crossref PubMed Scopus (53) Google Scholar), the reaction center of photosynthetic bacteria Rhodobacter spheroides (30Adelroth P. Paddock M.L. Tehrani A. Beatty J.T. Feher G. Okamura M.Y. Biochemistry. 2001; 40: 14538-14546Crossref PubMed Scopus (66) Google Scholar), and protein kinases (31Admiraal S.J. Schneider B. Meyer P. Janin J. Veron M. Deville-Bonne D. Herschlag D. Biochemistry. 1999; 38: 4701-4711Crossref PubMed Scopus (61) Google Scholar). However, one way to overcome the argument that the amino acid substitution caused gross structural changes in the protein was achieved by partial or complete chemical rescue of the mutant protein on introduction of imidazole or an imidazole analog into the buffer solution. This chemical rescue demonstrated that the loss of catalytic function resulted from the lack of the histidine residue and not from large scale structural changes. Chemical rescue experiments also provided insight into the function of the histidine residue in the wt enzyme, providing direct evidence that the imidazole side chain of histidine acts as an intermediate proton acceptor/donor, in which protons were accepted by the histidine molecule from one chemical moiety and subsequently donated to a second chemical moiety. Chemical rescue of mutant ion channels has been reported in only a few instances. However, an important property of the M2 ion channel suggested that histidine substitution mutants might be susceptible to chemical rescue by imidazole. This property is the ability of the channel to be inhibited by amantadine, a molecule of approximately the same size as imidazole. We reasoned that it was likely that the diameter of the channel pore might be large enough to accommodate the imidazole molecule, and we applied imidazole buffer to three histidine substitution mutants. It was found that partial rescue was possible for each mutant M2 ion channel and that the imidazole-enhanced currents were able to be inhibited by Cu(II). These results confirm the role of the imidazole side chain of histidine in proton transport across the M2 channel pore and support the hypothesis that the imidazole side chain of histidine acts as an intermediate proton acceptor/donor to relay protons through the aqueous pore of the M2 ion channel while obstructing the flow of larger cations. Mutant and mRNA Synthesis, Culture, and Microinjection of Oocytes—Mutations were introduced by PCR in a high expression vector that has a portion of the Xenopus 5′ globin untranslated region (32Swanson R. Marshall J. Smith J.S. Williams J.B. Boyle M.B. Folander K. Luneau C.J. Antanavage J. Oliva C. Buhrow S.A. Bennett C. Stein R.B. Kaczmarek L.M. Neuron. 1990; 4: 929-939Abstract Full Text PDF PubMed Scopus (265) Google Scholar). Oocytes were removed from female Xenopus laevis (Nasco, Fort Atkinson, WI), defolliculated, microinjected with 50 nl of mRNA, and incubated in ND96 (pH 8.5) at 19 °C before use (18Mould 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 (135) Google Scholar). The amounts of mRNA injected were as follows: wt (∼100 ng), M2-H37G (∼44 ng), M2-H37S (∼22.5 ng), M2-H37T (∼39 ng), M2-H37G/W41A (∼180 ng), and M2-H37G/W41Y (∼308 ng). Oocytes expressing the histidine substitution mutant proteins were incubated in ND96 for at least 24 h before recording, whereas recordings from oocytes expressing the wt protein were made ∼65 h after injection. Recording Solutions—Occytes were bathed in either normal Barth's solution or modified Barth's solution during recording. Normal Barth's solution contained (in mm) 88 NaCl, 1 KCl, 2.4 NaHCO3, 0.3 NaNO3, 0.71 CaCl2, 0.82 MgSO4, 15 HEPES (for pH 8.5) or 15 MES (for pH 5.5), osmolality ∼202 mosm/kg. For ion substitution experiments in Tables I and II, Na+ ions were substituted with equimolar concentrations of N-methyl-d-glucamine (NMDG) or K+. Cl- ions were substituted with equimolar concentrations of methanesulfonic acid. Titration of 50 mm imidazole buffer used in chemical rescue and ion substitution experiments required the addition of significant amounts of HCl, resulting in a concomitant increase in osmolality. In this case, control solutions were made isoosmotic by addition of mannitol. In experiments with 50 mm buffer (either MES or imidazole), the solutions contained (in mm)88 Na+, 1 KCl, 2.4 NaHCO3, 50 buffer (MES or imidazole or HEPES), 0.3 NaNO3, 0.71 CaCl2, and 0.82 MgSO4. The osmolality of solution with imidazole buffer was ∼260 mosm/kg. The osmolality of the control solutions buffered with MES and HEPES was adjusted with mannitol to ∼260 mosm/kg. The composition of solutions used in Na+ ion substitution experiments (Table III and Table IV) performed in the presence of 50 mm buffer (MES or imidazole) was (in mm) 8.33 Na+, 79.66 NMDG, 1 KCl, 2.4 NaHCO3, 50 buffer (MES or imidazole or HEPES), 0.3 NaNO3, 0.71 CaCl2, and 0.82 MgSO4. In experiments to identify compounds capable of chemical rescue, the composition of the solutions was (in mm) 88 NaCl, 1 KCl, 2.4 NaHCO3, 0.3 NaNO3, 0.71 CaCl2, 0.82 MgSO4, 15 HEPES (for pH 8.5) or 15 MES/MOPSO/imidazole/lutidine/1,2,4-triazole/1-methylimidazole (for pH 5.5), osmolality ∼202 mosm/kg. The order of presentation of test solutions, the time of recovery in normal solution, and the time during which oocytes were exposed to low pH solutions were important for obtaining recordings in which reversible changes in current flow and acidification occurred and are described in Supplemental Information 1.Table IReversal voltage of M2-H37G mutant ion channel measured at pH 7.5 with 15 mm HEPES buffer Vrev of the amantadine-sensitive component of current was measured. Note that, unlike wt, this mutant ion channel has significant Na+ conductance at pH 7.5.GenotypeVrev (88 mm Na+)Vrev (88 mm NMDG)mVmVH37G-7.6 ± 0.2-42 ± 1.7ap < 0.0001(n = 3)(n = 3)a p < 0.0001 Open table in a new tab Table IIReversal voltages measured at pH 5.5 with 15 mm MES buffer Ion selectivity of the wt and the M2-H37G mutant ion channels. Vrev of the amantadine-sensitive component of current was measured. Vrev measured in oocytes expressing the wt ion channel is always more negative than predicted from extracellular pH and resting intracellular pH because oocytes acidify rapidly when bathed in low pH medium (18Mould 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 (135) Google Scholar). Note that Vrev for the mutant ion channel is significantly decreased by lowering [Na+], but the difference is less at pH 5.5 than at pH 7.5 (see Table I). Reversal voltage for the wt channel can only be measured at low pH when the channel is open and is unaffected by [Na+]. Reversal voltage for the M2-W41A channel is not affected by [Na+]; the Vrev shown was for oocytes with an average current of approximately the same amplitude at pH 5.5 as for oocytes expressing the wt channel; Vrev for this very active channel quickly changes due to oocyte acidification.GenotypeVrev (88 mm Na+, 1 mm K+)Vrev (88 mm NMDG, 1 mm K+)Vrev (89 mm K+)mVmVmVWT56 ± 2.055 ± 2.151 ± 1.8ap < 0.05(n = 9)(n = 9)(n = 5)H37G8.6 ± 1.0-14 ± 1.2bp < 0.0001. The p values refer to NMDG11 ± 1.9cp < 0.001. The p values refer to NMDG(n = 11)(n = 11)(n = 5)W41A44 ± 5.744 ± 6.040 ± 6.8(n = 4)(n = 4)(n = 4)a p < 0.05b p < 0.0001. The p values refer to NMDGc p < 0.001. The p values refer to NMDG Open table in a new tab Table IIIReversal voltage of M2-H37G mutant ion channel measured at pH 7.5 with 50 mm imidazole buffer Effect of imidazole buffer on the ion selectivity of the M2-H37G mutant ion channel. Vrev was measured using the amantadine-sensitive component of current. Note that Vrev for the mutant channel was less affected by lowering [Na+] than when HEPES buffer was used (Table I) and that oocyte acidification (induced by lowering pH of the bathing medium from pH 8.5 (HEPES) to imidazole-buffered solution at pH 7.5 for 30 sec) was not affected by reduced [Na+] (see Supplemental Information 2).Genotype88 mm Na+88 mm NMDGVrev (mV)ΔpHi (pH units)Vrev (mV)ΔpHi (pH units)H37G15 ± 0.60.26 ± 0.0210 ± 0.4ap < 0.00010.24 ± 0.07(n = 8)(n = 3)(n = 8)(n = 3)a p < 0.0001 Open table in a new tab Table IVReversal voltages measured at pH 5.5 with 50 mm buffer Effect of imidazole buffer on the ion selectivity of the wt M2 ion channel and the M2-H37G mutant ion channel. Vrev was measured using the amantadine-sensitive component of current. Note that Vrev for the mutant ion channel changes much less when Na+ is replaced in imidazole buffer than in MES buffer.GenotypeVrev (MES buffer, 88 mm Na+)Vrev (MES buffer, 79.66 mm NMDG)Vrev (imidazole buffer, 88 mm Na+)Vrev (imidazole buffer, 79.66 mm NMDG)mVmVmVmVWT55 ± 5.960 ± 5.360 ± 2.069 ± 1.3(n = 3)(n = 3)(n = 3)(n = 3)H37G5.3 ± 0.2-11 ± 0.8ap < 0.0541 ± 1.845 ± 1.4(n = 3)(n = 3)(n = 3)(n = 3)a p < 0.05 Open table in a new tab Measurement of Membrane Current and Intracellular pH of Oocytes—Whole-cell currents were measured using a two-electrode voltage clamp amplifier (Dagan TEV 200A) at ∼25 °C using electrodes filled with 3 m KCl. Oocyte holding potential was -20 mV unless stated otherwise. Recording of intracellular pH was done with silanized microelectrodes filled with protonophore as described previously (19Shimbo K. Brassard D.L. Lamb R.A. Pinto L.H. Biophys. J. 1996; 70: 1336-1346Abstract Full Text PDF Scopus (123) Google Scholar). Immunofluorescence of Living Oocytes—The relative expression levels of the mutant M2 proteins at the surface membrane of the oocytes were measured with respect to that of the wild-type protein. This was done by incubating the oocytes after recording with a solution containing a monoclonal antibody directed against the N-terminal ectodomain of the influenza A M2 protein (monoclonal antibody 14C2) (33Zebedee S.L. Lamb R.A. J. Virol. 1988; 62: 2762-2772Crossref PubMed Google Scholar). Individual oocytes were washed with ND96 lacking pyruvate and gentamycin (4 °C, three times), incubated in ND96 containing 2% bovine serum albumin (4 °C, 1 h), incubated in primary antibody (1:500 in 2% bovine serum albumin, 4 °C, 1 h), washed with ND96 (4 °C, three times for 10 min), incubated in secondary antibody (goat anti-mouse IgG1 (γ1) labeled with Alexa Fluor® 546 (catalog number A21123, Molecular Probes (Medford, OR), 10 or 20 μg/ml in 2% bovine serum albumin in ND96, 4 °C), and washed with ND96 (4 °C, five times for 10 min). Fluorescence was quantified using a PTI image master microfluorometer (London, Ontario, Canada) with a 20× 0.5 NA objective. Approximately 20% of the lower oocyte surface was imaged upon the photocathode of the CCD camera. The emission spectrum of the fluorescence was confirmed to peak at 546 nm, consistent with acceptably low autofluorescence of the oocyte. For quantification of the fluorescence of each oocyte, the excitation wavelength was 556 nm, and emission was measured for wavelengths longer than 574 nm. Calculation of Free [Cu2+]—The HYSS program (www.chem.leeds.ac.uk/People/Peter_Gans/HySS.htm) was used to calculate the amount of total Cu(II) salt needed to achieve the desired free concentration. This program uses dissociation constants from the NIST data base (version 46). When oocytes expressing the wt M2 protein are bathed in solutions of low pH, but not solutions of high pH, an inward proton current flows across their membrane, and the cytoplasm becomes acidified (Fig. 1A). Previous work had shown that oocytes expressing histidine substitution mutant proteins have membrane currents, but these currents flowed at both high pH and low pH (22Wang C. Lamb R.A. Pinto L.H. Biophys. J. 1995; 69: 1363-1371Abstract Full Text PDF PubMed Scopus (224) Google Scholar). We tested whether proton flow was included in the currents of oocytes expressing the histidine substitution mutant proteins by recording intracellular pH of the oocytes and found that acidification occurred when the oocytes were bathed in solutions of low pH (Fig. 1B), showing that protons can pass through the pore of the mutant ion channel in the absence of histidine (see Supplemental Information 2). However, results with mutant enzymes that normally contain histidine as part of a proton transport pathway indicated that proton transport rates are increased when imidazole buffer is used (23Tu C.K. Silverman D.N. Forsman C. Jonsson B.H. Lindskog S. Biochemistry. 1989; 28: 7913-7918Crossref PubMed Scopus (351) Google Scholar, 24Tu C. Rowlett R.S. Tripp B.C. Ferry J.G. Silverman D.N. Biochemistry. 2002; 41: 15429-15435Crossref PubMed Scopus (43) Google Scholar, 25Duda D. Govindasamy L. Agbandje-McKenna M. Tu C. Silverman D.N. McKenna R. Acta Crystallogr. Sect. D. Biol. Crystallogr. 2003; 59: 93-104Crossref PubMed Scopus (50) Google Scholar). We thus applied imidazole buffer to oocytes expressing histidine substitution mutant proteins, and we found that current amplitude and acidification were increased significantly (p < 0.05, n = 4) (Fig. 1B). Increases were not observed for oocytes expressing the wt protein (Fig. 1A). These observations suggested that the presence of imidazole buffer might cause the properties of the mutant ion channels to resemble more closely those of the wild-type ion channel (i.e.“rescue” the channel properties). We thus analyzed the key properties of the mutant ion channels with and without imidazole buffer. Our studies focused on the M2-H37G mutant ion channel because the currents of oocytes expressing this genotype were more reproducible from experiment-to-experiment than those of oocytes expressing the other mutant ion channels. The ion selectivity for the M2-H37G, M2-H37S, and M2-H37T mutant ion channels was studied by measuring their current-voltage relationships in media of various pH, buffer, and ionic composition (Fig. 2 and Tables I and II). These mutant ion channels displayed much less proton selectivity than the wt ion channel when studied at high pH (pH 7.5 or pH 8.5) and low pH (pH 5.5) in MES buffer. Measurements at High pH—Oocytes expressing these mutant ion channels had standing currents when bathed at pH 7.5 or pH 8.5 (Fig. 2A). The standing current for the M2-H37G mutant ion channel became outward when Na+ in the bathing medium was replaced with NMDG (holding voltage -20 mV). This suggested that the mutant ion channel conducted Na+, unlike the wt channel that does not conduct Na+ at all. To test if Na+ carried the inward current of the M2-H37G mutant ion channel at pH 7.5, reversal voltage (Vrev, Table I) was measured and found to be less negative in media containing Na+ (-7.6 ± 0.2 mV, n = 3 mean ± S.E.) than in media containing NMDG (-42 ± 1.7 mV, n = 3), consistent with the conclusion that Na+ is conducted through the mutant channel. Measurements at Low pH—Oocytes expressing the wt M2 ion channel and the M2-H37G mutant ion channel had steady inward currents at pH 5.5 (-20 mV holding voltage, Fig. 2A). The inward current amplitude of oocytes expressing the M2-H37G mutant ion channel decreased when Na+ in the medium was replaced by NMDG, suggesting that the mutant ion channel has Na+ conductance at low pH. However, the amplitude of the decrease achieved by this replacement was not as large when studied at pH 5.5 (0.55 ± 0.05 μA, n = 6) as it was at pH 7.5 (0.99 + 0.2 μA, n = 3; p < 0.05). Consistent with the interpretation that the channel has Na+ conductance at pH 5.5, Na+ replacement made the Vrev considerably more negative for oocytes expressing the M2-H37G mutant ion channel (Table II). In contrast, neither inward current amplitude nor the Vrev (Table II) of oocytes expressing the highly proton-selective wt channel was affected by replacement of Na+ in the bathing medium with NMDG (6Chizhmakov 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, 18Mould 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 (135) Google Scholar, 19Shimbo K. Brassard D.L. Lamb R.A. Pinto L.H. Biophys. J. 1996; 70: 1336-1346Abstract Full Text PDF Scopus (123) Google Scholar). To test for the possibility that the mutant ion channels also conduct K+ at pH 5.5, [K+] in the bathing medium was increased to 88 mm by replacing Na+. The value of Vrev found under this condition was considerably more positive than for bathing medium containing NMDG and approximately equal to that for medium containing Na+. Moreover, the amplitude of the inward current was larger in the medium with 88 mm K+ than in medium with 88 mm Na+. The results of these alterations of Vrev with the composition of the bathing medium and the larger amplitude of inward current in high K+ medium indicate that the mutant channel also has significant conductance for K+ (Table II). To test for possible conduction of Cl- by the M2-H37G mutant ion channel, [Cl-]in the bathing medium was replaced with methane sulfonate, but the Vrev did not change significantly (data not shown). Thus, the M2-H37G mutant protein forms a poorly selective cation channel. To test the dependence of ion selectivity of the M2-H37G mutant ion channel on the concentration of imidazole buffer in the bathing medium, we measured the current-voltage relationship (Fig. 2B) and Vrev for oocytes that were bathed in solutions with each of several concentrations of imidazole buffer between 1.5 and 50 mm at pH 5.5 (Fig. 3). For these measurements we studied the amantadine-sensitive component of current. Neither the amplitude of the inward current (Fig. 2B) nor the Vrev of oocytes expressing the wt channel varied significantly with imidazole concentration (Fig. 3A), nor was Vrev affected by replacement of Na+ in the bathing medium with NMDG (6Chizhmakov 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, 18Mould 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 (135) Google Scholar). However, the Vrev of oocytes expressing the M2-H37G mutant ion channel became more positive with increased imidazole concentration (Fig. 2B and Fig. 3B), consistent with increased proton conductance resulting from higher imidazole concentration. The current-voltage relationship for the M2-H37G mutant ion channel was increased by replacing MES buffer with imidazole buffer (Fig. 2B); this increase in slope occurred for both inward and outward current. In contrast, the current-voltage relationship for the wt ion channel was changed very little by imidazole buffer (Fig. 2B). To test ion selectivity further, we measured the effect on Vrev of replacement of Na+ in the bathing medium with NMDG (50 mm buffer, pH 5.5; Table IV). This was done while the cells were bathed in" @default.
• W2014763114 created "2016-06-24" @default.
• W2014763114 creator A5025281918 @default.
• W2014763114 creator A5029287017 @default.
• W2014763114 creator A5038382113 @default.
• W2014763114 date "2005-06-01" @default.
• W2014763114 modified "2023-10-11" @default.
• W2014763114 title "Chemical Rescue of Histidine Selectivity Filter Mutants of the M2 Ion Channel of Influenza A Virus" @default.
• W2014763114 cites W1538591251 @default.
• W2014763114 cites W1585610099 @default.
• W2014763114 cites W1969628201 @default.
• W2014763114 cites W1969651682 @default.
• W2014763114 cites W1972065621 @default.
• W2014763114 cites W1974452612 @default.
• W2014763114 cites W1988295340 @default.
• W2014763114 cites W1993934877 @default.
• W2014763114 cites W2001099462 @default.
• W2014763114 cites W2004067557 @default.
• W2014763114 cites W2006428261 @default.
• W2014763114 cites W2008617981 @default.
• W2014763114 cites W2016920258 @default.
• W2014763114 cites W2018878611 @default.
• W2014763114 cites W2019069561 @default.
• W2014763114 cites W2028372889 @default.
• W2014763114 cites W2034453538 @default.
• W2014763114 cites W2045053284 @default.
• W2014763114 cites W2047260093 @default.
• W2014763114 cites W2047486233 @default.
• W2014763114 cites W2058292477 @default.
• W2014763114 cites W2058412945 @default.
• W2014763114 cites W2060899129 @default.
• W2014763114 cites W2071135420 @default.
• W2014763114 cites W2073140843 @default.
• W2014763114 cites W2076219046 @default.
• W2014763114 cites W2088786994 @default.
• W2014763114 cites W2093034908 @default.
• W2014763114 cites W2093447936 @default.
• W2014763114 cites W2109910734 @default.
• W2014763114 cites W2128375777 @default.
• W2014763114 cites W2136223709 @default.
• W2014763114 cites W2143276100 @default.
• W2014763114 cites W2150500510 @default.
• W2014763114 cites W2151592106 @default.
• W2014763114 cites W2162316896 @default.
• W2014763114 cites W2165133395 @default.
• W2014763114 cites W2167063739 @default.
• W2014763114 cites W4252988873 @default.
• W2014763114 doi "https://doi.org/10.1074/jbc.m412406200" @default.
• W2014763114 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15784624" @default.
• W2014763114 hasPublicationYear "2005" @default.
• W2014763114 type Work @default.
• W2014763114 sameAs 2014763114 @default.
• W2014763114 citedByCount "90" @default.
• W2014763114 countsByYear W20147631142012 @default.
• W2014763114 countsByYear W20147631142013 @default.
• W2014763114 countsByYear W20147631142014 @default.
• W2014763114 countsByYear W20147631142015 @default.
• W2014763114 countsByYear W20147631142016 @default.
• W2014763114 countsByYear W20147631142017 @default.
• W2014763114 countsByYear W20147631142018 @default.
• W2014763114 countsByYear W20147631142019 @default.
• W2014763114 countsByYear W20147631142020 @default.
• W2014763114 countsByYear W20147631142021 @default.
• W2014763114 countsByYear W20147631142022 @default.
• W2014763114 countsByYear W20147631142023 @default.
• W2014763114 crossrefType "journal-article" @default.
• W2014763114 hasAuthorship W2014763114A5025281918 @default.
• W2014763114 hasAuthorship W2014763114A5029287017 @default.
• W2014763114 hasAuthorship W2014763114A5038382113 @default.
• W2014763114 hasBestOaLocation W20147631141 @default.
• W2014763114 hasConcept C104317684 @default.
• W2014763114 hasConcept C118792377 @default.
• W2014763114 hasConcept C143065580 @default.
• W2014763114 hasConcept C159047783 @default.
• W2014763114 hasConcept C161790260 @default.
• W2014763114 hasConcept C185592680 @default.
• W2014763114 hasConcept C2522874641 @default.
• W2014763114 hasConcept C2777546802 @default.
• W2014763114 hasConcept C2778460671 @default.
• W2014763114 hasConcept C515207424 @default.
• W2014763114 hasConcept C55493867 @default.
• W2014763114 hasConcept C86803240 @default.
• W2014763114 hasConceptScore W2014763114C104317684 @default.
• W2014763114 hasConceptScore W2014763114C118792377 @default.
• W2014763114 hasConceptScore W2014763114C143065580 @default.
• W2014763114 hasConceptScore W2014763114C159047783 @default.
• W2014763114 hasConceptScore W2014763114C161790260 @default.
• W2014763114 hasConceptScore W2014763114C185592680 @default.
• W2014763114 hasConceptScore W2014763114C2522874641 @default.
• W2014763114 hasConceptScore W2014763114C2777546802 @default.
• W2014763114 hasConceptScore W2014763114C2778460671 @default.
• W2014763114 hasConceptScore W2014763114C515207424 @default.
• W2014763114 hasConceptScore W2014763114C55493867 @default.
• W2014763114 hasConceptScore W2014763114C86803240 @default.
• W2014763114 hasIssue "22" @default.
• W2014763114 hasLocation W20147631141 @default.
• W2014763114 hasOpenAccess W2014763114 @default.
• W2014763114 hasPrimaryLocation W20147631141 @default.

• next