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J Biol Chem, Vol. 274, Issue 9, 5474-5482, February 26, 1999

Cu(II) Inhibition of the Proton Translocation Machinery of the Influenza A Virus M₂ Protein

Chris S. Gandhi, Kevin Shuck^§, James D. Lear^¶, Gregg R. Dieckmann^¶, William F. DeGrado^¶, Robert A. Lamb^§, and Lawrence H. Pinto

From the  Department of Neurobiology and Physiology, Northwestern University, Evanston, Illinois 60208-3520, the ^§ Howard Hughes Medical Institute and Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208-3500, and the ^¶ Department of Biochemistry and Biophysics, The Johnson Foundation, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6059

The homotetrameric M₂ integral membrane protein of influenza virus forms a proton-selective ion channel. An essential histidine residue (His-37) in the M₂ transmembrane domain is believed to play an important role in the conduction mechanism of this channel. Also, this residue is believed to form hydrogen-bonded interactions with the ammonium group of the anti-viral compound, amantadine. A molecular model of this channel suggests that the imidazole side chains of His-37 from symmetry-related monomers of the homotetrameric pore converge to form a coordination site for transition metals. Thus, membrane currents of oocytes of Xenopus laevis expressing the M₂ protein were recorded when the solution bathing the oocytes contained various transition metals. Membrane currents were strongly and reversibly inhibited by Cu²⁺ with biphasic reaction kinetics. The biphasic inhibition curves may be explained by a two-site model involving a fast-binding peripheral site with low specificity for divalent metal ions, as well as a high affinity site (K_diss ~2 µM) that lies deep within the pore and shows rather slow-binding kinetics (k_on = 18.6 ± 0.9 M¹ s¹). The pH dependence of the interaction with the high affinity Cu²⁺-binding site parallels the pH dependence of inhibition by amantadine, which has previously been ascribed to protonation of His-37. The voltage dependence of the inhibition at the high affinity site indicates that the binding site lies within the transmembrane region of the pore. Furthermore, the inhibition by Cu²⁺ could be prevented by prior application of the reversible blocker of M₂ channel activity, BL-1743, providing further support for the location of the site within the pore region of M₂. Finally, substitutions of His-37 by alanine or glycine eliminated the high affinity site and resulted in membrane currents that were only partially inhibited at millimolar concentrations of Cu²⁺. Binding of Cu²⁺ to the high affinity site resulted in an approximately equal inhibition of both inward and outward currents. The wild-type protein showed very high specificity for Cu²⁺ and was only partially inhibited by 1 mM Ni²⁺, Pt²⁺, and Zn²⁺. These data are discussed in terms of the functional role of His-37 in the mechanism of proton translocation through the channel.

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