Mechanism for proton conduction of the M-2 ion channel of influenza A virus

The M-2 integral membrane protein of influenza A virus forms a proton-selec tive ion channel. We investigated the mechanism for proton transport of the M-2 protein in Xenopus oocytes using a two-electrode voltage clamp and in CV-1 cells using the whole cell patch clamp technique. Membrane currents me re recorded while manipulating the external solution to alter either the to tal or free proton concentration or the solvent itself. Membrane conductanc e decreased by similar to 50% when D2O replaced H2O as the solvent. From th is, me conclude that hydrogen ions do not pass through M-2 as hydronium ion s, but instead must interact with titratable groups that line the pore of t he channel. M-2 currents measured in solutions of low buffer concentration (<15 mM in oocytes and <0.15 mM in CV-1 cells) were smaller than those stud ied in solutions of high buffer concentration. Furthermore, the reversal vo ltage measured in low buffer was shifted to a more negative voltage than in high buffer. Also, at a given pH, M-2 current amplitude in 15 mar buffer d ecreased when pH-pK(a) was increased by changing the buffer pK(a). Collecti vely, these results demonstrate that M-2 currents can be limited by externa l buffer capacity. The data presented in this study were also used to estim ate the maximum single channel current of the M-2 ion channel, which was ca lculated to be on the order of 1-10 fA.