Conserved cysteine residues in the shaker K+ channel are not linked by a disulfide bond.
Many voltage-activated K+ channels contain two conserved cysteine residues in putative transmembrane segments S2 and S6. It has been proposed that these cysteines form an intrasubunit disulfide bond [Guy, H.R., & Conti, F. (1990) Trends Neurosci. 13, 201-206]. This proposal was tested using site-directed mutagenesis followed by electrophysiological and biochemical analysis of the Shaker B K+ channel. Each Shaker B subunit contains seven cysteine residues, including the conserved residues C286 and C462 and a less conserved cysteine, C245. Each cysteine in the Shaker B protein can be mutated individually without eliminating functional activity, indicating that the protein does not contain a disulfide bond that is essential for protein folding or the assembly of active channels. To determine whether there is a nonessential disulfide bond, Shaker B protein was subjected to limited proteolysis. Fragments were analyzed by electrophoresis under reducing and nonreducing conditions followed by immunoblotting. The results indicate that the two conserved residues C286 and C462 do not form a disulfide bond with each other or with C245. In addition, the subunits are not linked by disulfide bonds. In HEK293T cells, Shaker B protein is first made as an incompletely glycosylated precursor that is converted to the fully glycosylated mature protein. Glycosylation occurs at two positions in the S1-S2 loop.
Department of Physiology, University of California, Los Angeles School of Medicine 90024-1751.
C T
CT
Schulteis
S A
SA
John
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Y
Huang
C Y
CY
Tang
D M
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Papazian
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D004330:Drosophila / Q000235:genetics; D004594:Electrophysiology; D006031:Glycosylation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D015221:Potassium Channels / Q000737:chemistry* / Q000235:genetics; D014981:Xenopus / Q000235:genetics
D004220:Disulfides; D015221:Potassium Channels; D003545:Cysteine