Pegylation: a method for assessing topological accessibilities in Kv1.3.

Abstract

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.