Ion selectivity and activation of the M₂ ion channel of influenza virus

The influenza A virus-associated M₂ ion channel is generally believed to function during uncoating of virions in infected cells. On endocytosis of a virion into the lumen of endosomes, the M₂ ion channel is thought to cause acidification of the virion interior. In addition, the influenza virus M₂ ion channel is thought to function in the exocytic pathway by equilibrating the pH gradient between the acidic lumen of the trans-Golgi network and the neutral cytoplasm. A necessary test of the proposed roles of the influenza virus M₂ ion channel in the virus life cycle is to show directly that the M₂ ion channel conducts protons. We have measured the ionic selectivity and activation of three subtypes (Udorn, Weybridge, and Rostock) of the M₂ ion channel in oocytes of Xenopus laevis by measurement of 1) the intracellular pH (pH(in)) of voltage-clamped oocytes, 2) the current-voltage relationship in solutions of various pH and ionic composition, and 3) the flux of ⁸⁶Rb. We took advantage of the low pH(in) achieved during incubation in low pH medium to study the effects of low pH(in) on M₂ activation. Oocytes expressing each of the three subtypes of the M₂ protein a) underwent a slow acidification when incubated in medium of low pH (acidification was blocked by the M₂ ion channel inhibitor, amantadine); b) had current-voltage relationships that shifted to more positive values and had greater conductance when the pH(out) was lowered (this relationship was modified when Na⁺ was replaced by NH₄⁺ or Li⁺); c) had an amantadine-sensitive influx of Rb⁺. The effects on the current-voltage relationship of reduced pH(in) were opposed to the increased conductance found with reduced pH(out). We interpret these results to indicate that the M₂ ion channel is capable of conducting H⁺ and that other ions may also be conducted. Moreover, the channel conductance is reduced by decreased pH(in). These findings are consistent with the proposed roles of the M₂ protein in the life cycle of influenza A virus.