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J. Biol. Chem., Vol. 280, Issue 22, 21463-21472, June 3, 2005

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Chemical Rescue of Histidine Selectivity Filter Mutants of the M2 Ion Channel of Influenza A Virus^*

Padmavati Venkataraman, Robert A. Lamb¶||, and Lawrence H. Pinto**

From the Department of Neurobiology and Physiology and the Department of Biochemistry, Molecular Biology, and Cell Biology, ^¶Howard Hughes Medical Institute, Northwestern University, Evanston, Illinois 60208-3500

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.

Received for publication, November 2, 2004 , and in revised form, March 7, 2005.

^* This work was supported by NIAID Research Grants R01AI-31882 (to L. H. P.) and R37AI-20201 (to R. A. L.) from the National Institutes of Health. 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 Information 1–4.

^|| Investigator of the Howard Hughes Medical Institute.

^** 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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