The influenza virus M₂ ion channel protein: Probing the structure of the transmembrane domain in intact cells by using engineered disulfide cross- linking

The influenza A virus M₂ integral membrane protein is an ion channel that permits protons to enter virus particles during uncoating of virions in endosomes, and it also modulates the pH of the trans-Golgi network in virus- infected cells. M₂ protein is a homo-oligomer of 97 residues with a single transmembrane (TM) domain whose residues encompass the pore region of the channel and the biologically active form of the channel is a homotetramer. To understand the structural arrangement of the TM domains, each residue of the TM domain was changed in turn to cysteine, and oxidative disulfide cross- linking used to identify residues in close proximity. Oxidative treatment of M₂ protein in membranes using iodine resulted in maximum cross-linking at TM domain residues 27, 34, and 41. Oxidation of M₂ protein in membranes using the catalyst Cu(II)(1,10-phenanthroline)₃ resulted in cross-linking of many TM domain residues when the reaction was allowed to proceed at 37°C, suggesting that rotational movements of the TM domains in the membrane can occur. However, analysis of the kinetics of disulfide-linked dimer formation showed that TM domain residues 27, 30, 34, 37, and 41 formed most rapidly. Furthermore, when oxidation was performed at 4°C, maximum cross-linking occurred at TM domain residues 27, 30, 34, 37, and 41. These positions correspond to the a and d positions of a heptad repeat. Thus these biochemical data are consistent with the TM domain region of the M₂ tetramer forming a four-helix bundle. Analysis of the disulfide bonds that formed when oxidation of M₂ protein in membranes was performed at pH 5.2 showed greatly reduced cross-linking at TM domain residues 40, 42, and 43 than that found at pH 7.4. This pH-dependent change in cross-linking of residues toward the cytoplasmic side of the TM domain parallels with the activation of the M₂ ion channel at low pH.