Pegylation: a method for assessing topological accessibilities in Kv1.3.
Each subunit of a voltage-gated potassium channel (Kv) contains six putative transmembrane segments, S1-S6, and a cytosolic N-terminal recognition domain, T1. Although it is well-established that Kv channels are tetrameric structures, the protein-protein, protein-lipid, and protein-aqueous interfaces are not precisely mapped. The topological accessibility of specific amino acids may help to identify these border residues. Toward this end, a variant of the substituted-cysteine-accessibility method that relies on mass-labeling of accessible SH groups with a large SH reagent, methoxy-polyethylene glycol maleimide, and gel shift assay has been used. Pegylation of full-length Kv1.3, as well as Kv1.3 fragments, integrated into microsomal membranes, allows topological characterization of the 12 native cysteines (C1-C12), as well as cysteines engineered into a T1-T1 interface. Cysteines engineered into the T1-T1 interface had lower rates of pegylation than cytosolic-facing cysteines, namely, C5 in the T1 domain and C10-C12 in the C terminus.
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Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6085, USA.
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D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000502:physiology; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D004594:Electrophysiology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005260:Female; D066298:In Vitro Techniques; D015640:Ion Channel Gating; D051664:Kv1.3 Potassium Channel; D050356:Lipid Metabolism; D008715:Methionine; D008845:Microinjections; D008861:Microsomes / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D010957:Plasmids; D011092:Polyethylene Glycols / Q000378:metabolism; D015221:Potassium Channels / Q000737:chemistry / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated; D014176:Protein Biosynthesis; D012313:RNA / Q000378:metabolism; D013462:Sulfur Radioisotopes; D014982:Xenopus laevis
D004274:DNA, Recombinant; D051664:Kv1.3 Potassium Channel; D010446:Peptide Fragments; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D013462:Sulfur Radioisotopes; D011092:Polyethylene Glycols; D012313:RNA; D008715:Methionine; D003545:Cysteine