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.
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