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.

A New Kv1.2 Channelopathy Underlying Cerebellar Ataxia

“…This finding illustrates that CHO cells struggle to incorporate potassium channels containing Kv1.2(I402T) ␣-subunits into the lipid bilayer. It is reasonable to suggest, therefore, that the highly regulated and controlled process of expression of potassium channels at the basket cell terminal would also be significantly affected by the presence of Kv1.2(I402T) ␣-subunits, resulting in perturbations in the location and density of potassium channels (38).…”

Characteristics of Brain Kv1 Channels Tailored to Mimic Native Counterparts by Tandem Linkage of α Subunits

“…Every construct made above was verified by restriction digestion and dideoxy DNA sequencing. For expression in the SFV system, all Kv1 p␤ut2pA constructs were subcloned into pSFV1 vector, employing HindIII and BglII sites to cut out the cDNA fragments that were blunt-ended before ligation with pSFV1 vector (10). cRNAs for each Kv1 construct (in p␤ut2pA and pSFV1) and pS-FVH1 (plasmid encoding viral packaging proteins) were prepared as described previously (10).…”

“…Channels were expressed in BHK cells and analyzed in the native state by gel filtration, or by SDS-PAGE and Western blotting as described previously (10). Saturable binding of 125 I-labeled ␣DTX and DTX k to intact cells expressing the various constructs was measured in triplicate under established conditions (10) by rapid filtration through GF/F glass microfiber filters that had been presoaked in 0.5% (v/v) polyethyleneimine.…”

“…2B), activating at a threshold of Ϫ55 to Ϫ50 mV (Table I); also, its V1 ⁄2 fell between that for the channels made from Kv1. (10), the N-terminal part was replaced with an analogous moiety of Kv1.2 that regulates the efficiency of assembly (see "Introduction"). This construct gave increased surface expression in BHK cells (ϳ2-fold relative to the unmodified Kv1.1), yielding a subunit of the expected M r ϳ60,000 -62,000 on immunoblotting (Fig.…”

“…1A using the antibodies specified. D, another sample was detergent solubilized and chromatographed (10). Fractions were dot-blotted onto polyvinylidene difluoride membrane, with visualization as described in Fig.…”