During an average year, 10-20 percent of the US population becomes infected with influenza virus, but the only antiviral treatment available for those infected with influenza A virus is rimantadine, the target for which is the M2 ion channel protein. More effective inhibitors of the channel are needed because rimantadine-resistant mutants are generated rapidly. In the previous grant period we found that inhibition of the channel occurs when the pH of the cytoplasmic domain is lowered and transition metal complexes are formed with the M2 molecule. We propose to determine the mechanism for these inhibitions in order to identify new targets for drug action on the M2 molecule. From functional measurements we have found that the active state of the M2 ion channel is a homotetramer and we have identified residues that line the channel pore. We propose to determine the structure of the molecule with direct measurements because this structural information will allow for the design of more useful inhibitors. The individual aims are: 1. To understand the mechanism for inhibition of M2 ion channel as a result of decreased pH of the C-terminal domain. 2. To determine the M2 ion channel pore structure with the substituted cysteine accessibility method. 3. To use transition metal complexes to probe the pore structure of the M2 ion channel. 4. To apply the approach of site-directed spin labeling of the M2 ion channel transmembrane domain to determine its resting structure and structural changes that occur on activation by low pH. 5. To understand the role of transmembrane histidine 37 in proton transport by the M2 ion channel. 6. In a separate set of experiments we will test the hypothesis that the NB protein of influenza B virus functions as an ion channel.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Experimental Virology Study Section (EVR)
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Lambert, Linda C
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Northwestern University at Chicago
Schools of Arts and Sciences
United States
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Stouffer, Amanda L; Ma, Chunlong; Cristian, Lidia et al. (2008) The interplay of functional tuning, drug resistance, and thermodynamic stability in the evolution of the M2 proton channel from the influenza A virus. Structure 16:1067-76
Pinto, Lawrence H; Lamb, Robert A (2007) Controlling influenza virus replication by inhibiting its proton channel. Mol Biosyst 3:18-23
Pinto, Lawrence H; Lamb, Robert A (2006) Influenza virus proton channels. Photochem Photobiol Sci 5:629-32
Pinto, Lawrence H; Lamb, Robert A (2006) The M2 proton channels of influenza A and B viruses. J Biol Chem 281:8997-9000
Venkataraman, Padmavati; Lamb, Robert A; Pinto, Lawrence H (2005) Chemical rescue of histidine selectivity filter mutants of the M2 ion channel of influenza A virus. J Biol Chem 280:21463-72
Kelly, Marie L; Cook, James A; Brown-Augsburger, Patricia et al. (2003) Demonstrating the intrinsic ion channel activity of virally encoded proteins. FEBS Lett 552:61-7
Mould, Jorgen A; Paterson, Reay G; Takeda, Makoto et al. (2003) Influenza B virus BM2 protein has ion channel activity that conducts protons across membranes. Dev Cell 5:175-84
Takeda, Makoto; Pekosz, Andrew; Shuck, Kevin et al. (2002) Influenza a virus M2 ion channel activity is essential for efficient replication in tissue culture. J Virol 76:1391-9
Tang, Yajun; Zaitseva, Florina; Lamb, Robert A et al. (2002) The gate of the influenza virus M2 proton channel is formed by a single tryptophan residue. J Biol Chem 277:39880-6
Mould, J A; Li, H C; Dudlak, C S et al. (2000) Mechanism for proton conduction of the M(2) ion channel of influenza A virus. J Biol Chem 275:8592-9

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