Definitive assignment of proton selectivity and attoampere unitary current to the M2 ion channel protein of influenza A virus

The viral ion channel protein M2 supports the transit of influenza virus and its glycoproteins through acidic compartments of the cell, M2 conducts endosomal protons into the virion to initiate uncoating and, by equilibrating the pH at trans-Golgi membranes, preserves the native conformation of acid-sensitive viral hemagglutinin, The exceptionally low conductance of the M2 channel thwarted resolution of single channels by electrophysiological techniques. Assays of liposome-reconstituted M2 yielded the average unitary channel current of the M2 tetramer-1.2 aA (1.2 x 10(-18) A) at neutral pH and 2.7 to 4.1 aA at pH 5.7 - which activates the channel. Extrapolation to physiological temperature predicts 4.8 and 40 aA, respectively, and a unitary conductance of 0.03 versus 0.4 fS, This minute activity, below previous estimates, appears sufficient for virus reproduction, but low enough to avert abortive cytotoxicity, The unitary permeability of M2 was within the range reported for other proton channels, To address the ion selectivity of M2, we exploited the coupling of ionic influx and efflux in sealed liposomes, Metal ion fluxes were monitored by proton counterflow, employing a pH probe 1,000 times more sensitive than available Na(+) or Kf probes. Even low-pH-activated M2 did not conduct Na(+) and K(+), The proton selectivity of M2 was estimated to be at least 3 x 10(6) (over sodium or potassium ions), in agreement with electrophysiological studies. The stringent proton selectivity of M2 suggests that the cytopathology of influenza virus does not involve direct perturbation of cellular sodium or potassium gradients.