title: Positional editing of transmembrane domains during ion channel assembly.
abstract: The integration of transmembrane (TM)-spanning regions of many channels and ion transporters is potentially compromised by the presence of polar and charged residues required for biological function. Although the two TMs of the ATP-gated ion channel subunit P2X2 each contain charged/polar amino acids, we found that each TM is efficiently membrane inserted when it is analysed in isolation, and uncovered no evidence for cooperativity between these two TMs during P2X2 integration. However, using minimal N-glycosylation distance mapping, we find that the positioning of TM2 in newly synthesized P2X2 monomers is distinct from that seen in subunits of the high-resolution structures of assembled homologous trimers. We conclude that P2X2 monomers are initially synthesised at the endoplasmic reticulum in a distinct conformation, where the extent of the TM-spanning regions is primarily defined by the thermodynamic cost of their membrane integration at the Sec61 translocon. In this model, TM2 of P2X2 subsequently undergoes a process of positional editing within the membrane that correlates with trimerisation of the monomer, a process requiring specific polar/charged residues in both TM1 and TM2. We postulate that the assembly process offsets any energetic cost of relocating TM2, and find evidence that positional editing of TM2 in the acid-sensing ion channel (ASIC1a) is even more pronounced than that observed for P2X2. Taken together, these data further underline the potential complexities involved in accurately predicting TM domains. We propose that the orchestrated repositioning of TM segments during subunit oligomerisation plays an important role in generating the functional architecture of active ion channels, and suggest that the regulation of this underappreciated biosynthetic step may provide an elegant mechanism for maintaining ER homeostasis.
authors.affiliation: Department of Biochemistry and Biophysics, Center for Biomembrane Research, Stockholm University, SE-10691 Stockholm, Sweden.
authors.forename: Karin
authors.initials: K
authors.lastname: Öjemalm
authors.forename: Helen R
authors.initials: HR
authors.lastname: Watson
authors.forename: Peristera
authors.initials: P
authors.lastname: Roboti
authors.forename: Benedict C S
authors.initials: BC
authors.lastname: Cross
authors.forename: Jim
authors.initials: J
authors.lastname: Warwicker
authors.forename: Gunnar
authors.initials: G
authors.lastname: von Heijne
authors.forename: Stephen
authors.initials: S
authors.lastname: High
mesh_terms: D062565:Acid Sensing Ion Channels / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007473:Ion Channels / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D051381:Rats; D058476:Receptors, Purinergic P2X2 / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D013816:Thermodynamics
chemical_list: C568266:ASIC1 protein, human; D062565:Acid Sensing Ion Channels; D007473:Ion Channels; D021122:Protein Subunits; D058476:Receptors, Purinergic P2X2; D000255:Adenosine Triphosphate