title: Electrostatic interactions between transmembrane segments mediate folding of Shaker K+ channel subunits.
abstract: In voltage-dependent Shaker K+ channels, charged residues E293 in transmembrane segment S2 and R365, R368, and R371 in S4 contribute significantly to the gating charge movement that accompanies activation. Using an intragenic suppression strategy, we have now probed for structural interaction between transmembrane segments S2, S3, and S4 in Shaker channels. Charge reversal mutations of E283 in S2 and K374 in S4 disrupt maturation of the protein. Maturation was specifically and efficiently rescued by second-site charge reversal mutations, indicating that electrostatic interactions exist between E283 in S2 and R368 and R371 in S4, and between K374 in S4 and E293 in S2 and D316 in S3. Rescued subunits were incorporated into functional channels, demonstrating that a native structure was restored. Our data indicate that K374 interacts with E293 and D316 within the same subunit. These electrostatic interactions mediate the proper folding of the protein and are likely to persist in the native structure. Our results raise the possibility that the S4 segment is tilted relative to S2 and S3 in the voltage-sensing domain of Shaker channels. Such an arrangement might provide solvent access to voltage-sensing residues, which we find to be highly tolerant of mutations.
authors.affiliation: Department of Physiology, School of Medicine, University of California, Los Angeles 90095-1751, USA.
authors.forename: S K
authors.initials: SK
authors.lastname: Tiwari-Woodruff
authors.forename: C T
authors.initials: CT
authors.lastname: Schulteis
authors.forename: A F
authors.initials: AF
authors.lastname: Mock
authors.forename: D M
authors.initials: DM
authors.lastname: Papazian
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004594:Electrophysiology; D015870:Gene Expression / Q000235:genetics; D015640:Ion Channel Gating; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D016133:Polymerase Chain Reaction; D015221:Potassium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D051637:Shaker Superfamily of Potassium Channels; D014981:Xenopus
chemical_list: D015221:Potassium Channels; D051637:Shaker Superfamily of Potassium Channels