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J. Biol. Chem., Vol. 283, Issue 23, 15921-15931, June 6, 2008

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Identification of the Pore-lining Residues of the BM2 Ion Channel Protein of Influenza B Virus^*

Chunlong Ma, Cinque S. Soto, Yuki Ohigashi, Albert Taylor¹, Vasilios Bournas, Brett Glawe, Maria K. Udo^¶, William F. DeGrado^||, Robert A. Lamb^**2, and Lawrence H. Pinto³

From the Department of Neurobiology and Physiology, ^**Department of Biochemistry, Molecular Biology, and Cell Biology, and Howard Hughes Medical Institute, Northwestern University, Evanston, Illinois 60208-3500, the Department of Biochemistry and Biophysics, School of Medicine, and ^||Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6059, and ^¶Physics Department, Loyola University, Chicago, Illinois 60626

The influenza B virus BM2 proton-selective ion channel is essential for virus uncoating, a process that occurs in the acidic environment of the endosome. The BM2 channel causes acidification of the interior of the virus particle, which results in dissociation of the viral membrane protein from the ribonucleo-protein core. The BM2 protein is similar to the A/M2 protein ion channel of influenza A virus (A/M2) in that it contains an HXXXW motif. Unlike the A/M2 protein, the BM2 protein is not inhibited by the antiviral drug amantadine. We used mutagenesis to ascertain the pore-lining residues of the BM2 ion channel. The specific activity (relative to wild type), reversal voltage, and susceptibility to modification by (2-aminoethyl)-methane thiosulfonate and N-ethylmaleimide of cysteine mutant proteins were measured in oocytes. It was found that mutation of transmembrane domain residues Ser⁹, Ser¹², Phe¹³, Ser¹⁶, His¹⁹, and Trp²³ to cysteine were most disruptive for ion channel function. These cysteine mutants were also most susceptible to (2-aminoethyl)-methane thiosulfonate and N-ethylmaleimide modification. Furthermore, considerable amounts of dimer were formed in the absence of oxidative reagents when cysteine was introduced at positions Ser⁹, Ser¹², Ser¹⁶, or Trp²³. Based on these experimental data, a BM2 transmembrane domain model is proposed. The presence of polar residues in the pore is a probable explanation for the amantadine insensitivity of the BM2 protein and suggests that related but more polar compounds might serve as useful inhibitors of the protein.

Received for publication, December 18, 2007 , and in revised form, April 9, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants R01 AI-57363 (to L. H. P.), R01 AI-20201 (to R. A. L.), and R01 GM-56423 (to W. F. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables 1 and 2 and Figs. 1–7.

¹ Present address: Dept. of Neurobiology, Pharmacology, and Physiology, University of Chicago, Chicago, IL 60637.

² An Investigator of the Howard Hughes Medical Institute.

³ To whom correspondence should be addressed: Dept. of Neurobiology and Physiology, Hogan Hall, 2205 Tech Dr., Northwestern University, Evanston, IL 60208-3500. Tel.: 847-491-7915; Fax: 847-491-5211; E-mail: larry-pinto{at}northwestern.edu.

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