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Free keywords:
channel; expression; electron microscopy; structure
Abstract:
Neuronal voltage-dependent K+ channels of the delayed rectifier type consist of multiple Kv α subunit variants, which assemble as hetero- or homotetramers, together with four Kvβ auxiliary subunits. Direct structural information on these proteins has not been forthcoming due to the difficulty in isolating the native K+ channels. We have overexpressed the subunit genes in the yeast Pichia pastoris. The Kv1.2 subunit expressed alone is shown to fold into a native conformation as determined by high-affinity binding of 125I-labelled α-dendrotoxin, while co-expressed Kv1.2 and Kvβ2 subunits co-assembled to form native-like oligomers. Sites of post-translational modifications causing apparent heterogeneity on SDS-PAGE were identified by site-directed mutagenesis. Engineering to include affinity tags and scale-up of production by fermentation allowed routine purification of milligram quantities of homo- and heteroligomeric channels. Single-particle electron microscopy of the purified channels was used to generate a 3D volume to 2.1 nm resolution. Protein domains were assigned by fitting crystal structures of related bacterial proteins. Addition of exogenous lipid followed by detergent dialysis produced well-ordered 2D crystals that exhibited mostly p121 symmetry. Projection maps of negatively stained crystals show the constituent molecules to be 4-fold symmetric, as expected for the octameric K+ channel complex.
