Recreation of Neuronal Kv1 Channel Oligomers by Expression in Mammalian Cells Using Semliki Forest Virus

The multiple roles of voltage-sensitive K+ channels (Kv1 subfamily) in brain are served by subtypes containing pore-forming α (1.1−1.6) and auxiliary β subunits, usually in an (α)4(β)4 stoichiometry. To facilitate structure/activity analysis, combinations that are prevalent in neurones and susceptib...

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Veröffentlicht in:Biochemistry (Easton) 1999-12, Vol.38 (51), p.16766-16776
Hauptverfasser: Shamotienko, Oleg, Akhtar, Sobia, Sidera, Christina, Meunier, Frédéric A, Ink, Barbara, Weir, Malcolm, Dolly, J. Oliver
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Sprache:eng
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Zusammenfassung:The multiple roles of voltage-sensitive K+ channels (Kv1 subfamily) in brain are served by subtypes containing pore-forming α (1.1−1.6) and auxiliary β subunits, usually in an (α)4(β)4 stoichiometry. To facilitate structure/activity analysis, combinations that are prevalent in neurones and susceptible to α-dendrotoxin (αDTX) were reproduced in mammalian cells, using Semliki Forest virus. Infected Chinese hamster ovary cells expressed N-glycosylated Kv1.1 and 1.2 α subunits (M r ∼ 60 and 62 K) that assembled and bound [125I]-αDTX with high affinity; an appreciable proportion appeared on the cell surface, with Kv1.2 showing a 5-fold enrichment in a plasma membrane fraction. To obtain ‘native-like' α/β complexes, β1.1 or 2.1 (M r ∼ 42 and 39 K, respectively) was co-expressed with Kv1.1 or 1.2. This slightly enhanced N-glycosylation and toxin binding, most notable with β2.1 and Kv1.2. Solubilization of membranes from cells infected with Kv.1.2 and β2.1, followed by Ni2+ chromatography, gave a purified α1.2/β2.1 complex with a size of ∼405 K and S 20,W = 15.8 S. Importantly, these values indicate that four α and β subunits co-assembled as in neurones, a conclusion supported by the size (∼260 K) of the homo-tetramer formed by Kv1.2 alone. Thus, an authentic K+ channel octomer has been reconstructed; oligomeric species were also found in plasma membranes. To create ‘authentic-like' hetero-oligomeric channels, Kv1.1 and 1.2 were co-expressed and shown to have assembled by the precipitation of both with IgGs specific for either. Consistently, confocal microscopy of cells labeled with these antibodies showed that the relatively low surface content of Kv1.1 was increased by Kv1.2. [125I]-αDTX binding to these complexes was antagonized by DTXk, a probe selective for Kv1.1, in a manner that mimicks the pattern observed for the Kv1.1/1.2-containing channels in neuronal membranes.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi991039n