Hanging gondola structure of the T1 domain in a voltage-gated K(+) channel.
The T1 domain is a approximately 100-residue sequence in the cytoplasmic N-terminal region of K(v)-type K(+) channels. The structure of the isolated domain is known, but it is uncertain whether the structure of this domain is maintained in the fully assembled, membrane-associated, homotetrameric channel protein. We use the structure of the isolated domain as a guide for designing disulfide bonds to cross-link Shaker K(+) channels through the T1 domain. Six pairs of residues with side chains closely apposed across the T1 subunit interface were selected for replacement by cysteine. Of these, three pairs formed cross-links upon air oxidation of cysteine-substituted Shaker channels expressed in Xenopus oocyte membranes. Two of these cross-linked channels were examined electrophysiologically and were found to have gating properties only slightly altered from wild-type. The results show that the structure of the isolated T1 domain exists in the mature ion channel. They also demand that this domain is attached to the membrane-embedded part of the protein as a cytoplasmic "hanging gondola", and that ions gain access to the pore through four "windows" formed by the linker connecting T1 to the channel's first transmembrane helix.
Kobertz
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WR
Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, Massachusetts 02454, USA.
Williams
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C
Miller
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D000818:Animals; D001048:Aplysia / Q000378:metabolism; D003432:Cross-Linking Reagents; D004220:Disulfides / Q000737:chemistry; D005260:Female; D066298:In Vitro Techniques; D051662:Kv1.1 Potassium Channel; D008958:Models, Molecular; D009865:Oocytes / Q000378:metabolism; D015221:Potassium Channels / Q000737:chemistry*; D024642:Potassium Channels, Voltage-Gated; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D014981:Xenopus
D003432:Cross-Linking Reagents; D004220:Disulfides; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D011994:Recombinant Proteins; D051662:Kv1.1 Potassium Channel