ACTIVATION OF THE M(2) ION-CHANNEL OF INFLUENZA-VIRUS - A ROLE FOR THE TRANSMEMBRANE DOMAIN HISTIDINE RESIDUE

To test the hypothesis that transmembrane domain histidine residue 37 of the M(2) ion channel of influenza A virus mediates the low pH-induced activation of the channel, the residue was changed to glycine, glutamate, arginine, or lysine. The wild-type and altered M(2) proteins were expressed in oocytes of Xenopus laevis and membrane currents were recorded. The mass of protein expressed in individual oocytes was measured using quantitative immunoblotting and correlated with membrane currents. Oocytes expressing the M(2)-H(37)G protein had a voltage-independent conductance with current-voltage relationship similar to that of the wild-type M(2) channel. The conductance of the M(2)-H(37)G protein was reversibly inhibited by the M(2) ion channel blocker amantadine and was only very slightly modulated by changes in pH(out) over the range pH 5.4 to pH 8.2. Oocytes expressing the M(2)-H(37)E protein also had a voltage-independent conductance with a current-voltage relationship similar to that of the wild-type M(2) channel. The conductance of the M(2)-H(37)E protein was reversibly inhibited by amantadine and was also only very slightly modulated by changes in pH(out) over the range pH 5.4 to pH 8.2. These slight alterations in conductance of the mutant ion channels on changes in pH(out) are in striking contrast to the 50-fold change in conductance seen for the wild-type M(2) channel over the range pH 4.5 to pH 8.2. The specific activity of the M(2)-H(37)G protein was 1.36 +/- 0.37 mu A/ng and the specific activity of the M(2)-H(37)E protein was 30 +/- 3 mu A/ng at pH 6.2. These values of specific activity greatly exceed that of the wild-type protein at the same pH (0.16 +/- 0.01 mu A/ng). Oocytes expressing the M(2)-H37K and M(2)-H(37)R mutant proteins could not be studied because the oocytes did not survive more than a few hours in culture. Oocytes expressing the M(2)-H(37)E mutant protein also had a voltage-activated Cl- conductance that was observed only for oocytes that expressed a mass of protein exceeding a large threshold value. These results are consistent with protonation of histidine residue 37 as an essential step in the activation of the wild-type M(2) ion channel.