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 degreesC, 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 degreesC, 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. Copyright 1999 Academic Press.