Influenza M2 Inhibits CFTR Activity through its Ion Channel Function

We previously reported that co‐expression of the Influenza M2 hydrogen (H + ) channel, and CFTR in Xenopus oocytes downregulates CFTR activity and expression. We now hypothesize that the H + ion channel activity of M2 decreases vesicular pH which in turn alters the maturation or degradation of CFTR. To test this hypothesis we constructed M2 mutant cRNAs containing bulky amino acid side chain substitutions blocking H + transport. When injected in Xenopus oocytes, these cRNAs expressed similar levels of protein as the wild type (wt) (determined by western blotting) and trafficked to the plasma membrane (confirmed by immunoluminescence). When these mutant cRNAs were co‐expressed with CFTR, little to no decrease in cAMP‐activated, glyH‐101 sensitive, Cl‐ currents were observed. The decrease in total membrane protein was also significantly attenuated. Next we made an M2 cRNA endoplasmic reticulum (ER) targeted construct which does not reach the plasma membrane (Sakaguchi et al. 1996). No decrease in CFTR activity was observed when coexpressed with this construct. Further, incubation of CFTR cRNA injected Xenopus oocytes with ammonium chloride, which increases the pH vesicular organelles, significantly decreased CFTR activity in a dose dependent manner. Overall, these studies suggest that activation of M2 by acidic organelles, and the subsequent alkalization of vesicular pH, is essential to CFTR downregulation. Funding provided by NIHR01‐ HL031197