Permeation and Activation of the M2 Ion Channel of Influenza A Virus

Abstract

The M 2 ion channel protein of influenza A virus is essential for mediating protein-protein dissociation during the virus uncoating process that occurs when the virus is in the acidic environment of the lumen of the secondary endosome. The difficulty of determining the ion selectivity of this minimalistic ion channel is due in part to the fact that the channel activity is so great that it causes local acidification in the expressing cells and a consequent alteration of reversal voltage, V rev . We have confirmed the high proton selectivity of the channel (1.5-2.0 ؋ 10 6 ) in both oocytes and mammalian cells by using four methods as follows: 1) comparison of V rev with proton equilibrium potential; 2) measurement of pH in and V rev while Na The M 2 protein of influenza A virus is thought to function as an ion channel that permits protons to enter virus particles during virion uncoating in endosomes. In addition, in influenza virus-infected cells, the M 2 protein causes the equilibration of pH between the acidic lumen of the trans-Golgi network and the cytoplasm (reviewed in Refs. 1 and 2). The activity of the M 2 ion channel is inhibited by the antiviral drug amantadine (3-5). The mature M 2 protein consists of a 23-residue N-terminal extracellular domain, a single internal hydrophobic domain of 19 residues that acts as a transmembrane domain and forms the pore of the channel, and a 54-residue cytoplasmic tail (6). Chemical cross-linking studies showed the M 2 protein to exist minimally as a homotetramer (7-9). Statistical analysis of the ion channel activity of mixed oligomers also indicated that a homotetramer is the minimal active oligomeric form of the protein (10).Despite the small size of the active M 2 oligomer, several lines of evidence indicate that ion channel activity is intrinsic to the M 2 protein. First, ion channel activity has been observed in three different expression systems, Xenopus oocytes (3, 11, 12), mammalian cells (5, 13), and yeast (14). Second, M 2 channel activity has also been recorded in artificial lipid bilayers from a reconstituted peptide corresponding to the transmembrane domain of the M 2 protein (15) and from purified M 2 protein (16). Thus, due to its structural simplicity, the M 2 ion channel is a potentially useful model for the study of ion channels in general.Although a great deal of evidence indicates H ϩ is the biologically relevant ion for the role of M 2 protein in the life cycle of the influenza virus (1, 3, 17-22), other ions have been shown to be capable of flowing through the channel (12). In addition, the ion selectivity measured for the M 2 channel has been found to differ depending on whether the activity was measured in Xenopus oocytes or mammalian cells. When M 2 protein was expressed in oocytes, V rev was found to differ from the proton equilibrium potential, E Hϩ as [H ϩ ] out was varied (12). On the other hand, when M 2 protein was expressed in MEL cells, V rev was found to agree with E Hϩ (5). In a recent study (23), we found I Hϩ of the M 2 ion channel to...

The hepatitis C virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives

“…Conductance levels of the 6,000-Da protein encoded by ␣-viruses cover a range from Ϸ50 pS to Ϸ800 pS (30). Other viral ion channels such as the tetrameric proton channel M2 from influenza A virus (31,32) exhibit conductance levels in the femtosiemen range (33,34). Channel-forming peptides show conductance levels as high as the ones we observed for HCV p7 when five or more transmembrane helices assemble to form a pore (26,27).…”

Structure and mechanism of proton transport through the transmembrane tetrameric M2 protein bundle of the influenza A virus

“…In the absence of strong buffer catalysis (which is not observed for M2) (47), the rate of deprotonation (k off ) of a conjugate acid is defined by its equilibrium dissociation constant (K diss ¼ 10 −pKa ) and the second order rate constant for protonation (k on ) according to the equation K diss ¼ k off ∕k on . Given M2's conducting pK a of approximately 6 and values of k on between 10 7 to 10 9 M −1 sec −1 for diffusion through a pore the size of M2, the expected value of k off is 10 1 to 10 3 sec −1 in good agreement with the observed maximal rate (47).…”

Viroporins: structure, function and potential as antiviral targets

“…Slow conductance (*200 H + s 21 ) and a lack of alkali metal ion conductivity pointed to the presence of a selectivity filter, which was highly likely to involve protonation of ionizable residues based upon the induction of activity by reduced external pH (Lin & Schroeder, 2001;Mould et al, 2000). The highly conserved His37 residue within the TMD was shown by mutagenesis to govern M2 selectivity, although His37 mutants retained amantadine sensitivity (Chizhmakov et al, 1996;Wang et al, 1995).…”