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Journal of Virology, September 2000, p. 7755-7761, Vol. 74, No. 17
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Analysis of the Pore Structure of the Influenza A Virus M2 Ion Channel by the Substituted-Cysteine Accessibility Method

Kevin Shuck,1 Robert A. Lamb,1,2,* and Lawrence H. Pinto3

Department of Biochemistry, Molecular Biology and Cell Biology,1 Howard Hughes Medical Institute,2 and Department of Neurobiology and Physiology,3 Northwestern University, Evanston, Illinois 60208-3500

Received 4 April 2000/Accepted 30 May 2000

The M2 ion channel of influenza A virus is a small integral membrane protein whose active form is a homotetramer with each polypeptide chain containing 96-amino-acid residues. To identify residues of the transmembrane (TM) domain that line the presumed central ion-conducting pore, a set of mutants was generated in which each residue of the TM domain (residues 25 to 44) was replaced by cysteine. The accessibility of the cysteine mutants to modification by the sulfhydryl-specific reagents methane thiosulfonate ethylammonium (MTSEA) and MTS tetraethylammonium (MTSET) was tested. Extracellular application of MTSEA evoked decreases in the conductances measured from two mutants, M2-A30C and M2-G34C. The changes observed were not reversible on washout, indicative of a covalent modification. Inhibition by MTSEA, or by the larger reagent MTSET, was not detected for residues closer to the extracellular end of the channel than Ala-30, indicating the pore may be wider near the extracellular opening. To investigate the accessibility of the cysteine mutants to reagents applied intracellularly, oocytes were microinjected directly with reagents during recordings. The conductance of the M2-W41C mutant was decreased by intracellular injection of a concentrated MTSET solution. However, intracellular application of MTSET caused no change in the conductance of the M2-G34C mutant, a result in contrast to that obtained when the reagent was applied extracellularly. These data suggest that a constriction in the pore exists between residues 34 and 41 which prevents passage of the MTS reagent. These findings are consistent with the proposed role for His-37 as the selectivity filter. Taken together, these data confirm our earlier model that Ala-30, Gly-34, His-37, and Trp-41 line the channel pore (L. H. Pinto, G. R. Dieckmann, C. S. Gandhi, C. G. Papworth, J. Braman, M. A. Shaughnessy, J. D. Lear, R. A. Lamb, and W. F. DeGrado, Proc. Natl. Acad. Sci. USA 94:11301-11306, 1997).

* Corresponding author. Mailing address: Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, 2153 North Campus Dr., Evanston, IL 60208-3500. Phone: (847) 491-5433. Fax: (847) 491-2467. E-mail: ralamb{at}northwestern.edu.

Journal of Virology, September 2000, p. 7755-7761, Vol. 74, No. 17
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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