title: Recreation of neuronal Kv1 channel oligomers by expression in mammalian cells using Semliki Forest virus.
abstract: The multiple roles of voltage-sensitive K(+) channels (Kv1 subfamily) in brain are served by subtypes containing pore-forming alpha (1.1-1.6) and auxiliary beta subunits, usually in an (alpha)(4)(beta)(4) stoichiometry. To facilitate structure/activity analysis, combinations that are prevalent in neurones and susceptible to alpha-dendrotoxin (alphaDTX) were reproduced in mammalian cells, using Semliki Forest virus. Infected Chinese hamster ovary cells expressed N-glycosylated Kv1.1 and 1.2 alpha subunits (M(r) approximately 60 and 62 K) that assembled and bound [(125)I]-alphaDTX 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' alpha/beta complexes, beta1.1 or 2.1 (M(r) approximately 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 beta2. 1 and Kv1.2. Solubilization of membranes from cells infected with Kv. 1.2 and beta2.1, followed by Ni(2+) chromatography, gave a purified alpha1.2/beta2.1 complex with a size of approximately 405 K and S(20, W) = 15.8 S. Importantly, these values indicate that four alpha and beta subunits co-assembled as in neurones, a conclusion supported by the size ( approximately 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. [(125)I]-alphaDTX binding to these complexes was antagonized by DTX(k), 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.
authors.affiliation: Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, U.K.
authors.forename: O
authors.initials: O
authors.lastname: Shamotienko
authors.forename: S
authors.initials: S
authors.lastname: Akhtar
authors.forename: C
authors.initials: C
authors.lastname: Sidera
authors.forename: F A
authors.initials: FA
authors.lastname: Meunier
authors.forename: B
authors.initials: B
authors.lastname: Ink
authors.forename: M
authors.initials: M
authors.lastname: Weir
authors.forename: J O
authors.initials: JO
authors.lastname: Dolly
mesh_terms: D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D006224:Cricetinae; D004546:Elapid Venoms / Q000378:metabolism; D007457:Iodine Radioisotopes; D051662:Kv1.1 Potassium Channel; D051663:Kv1.2 Potassium Channel; D018716:Molecular Mimicry; D009474:Neurons / Q000737:chemistry / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D024642:Potassium Channels, Voltage-Gated; D011485:Protein Binding / Q000235:genetics; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012672:Semliki forest virus / Q000235:genetics*; D013347:Subcellular Fractions / Q000378:metabolism; D013570:Synaptic Membranes / Q000235:genetics / Q000378:metabolism
chemical_list: D004546:Elapid Venoms; D007457:Iodine Radioisotopes; D051663:Kv1.2 Potassium Channel; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D011994:Recombinant Proteins; D051662:Kv1.1 Potassium Channel; C020273:dendrotoxin