Characterization of monoclonal antibodies against voltage-dependent potassium channels raised using .alpha.-dendrotoxin acceptors purified from bovine brain

Abstract

Seven monoclonal antibodies raised against alpha-dendrotoxin-sensitive K+ channels, purified from bovine cerebral cortex, recognize these proteins in their native or denatured states, via interaction with the alpha- but not the beta-subunit. This finding, together with a similar observation made with polyclonal antibodies, shows that the latter is a distinct protein and not a proteolytic fragment of the larger subunit. Also, coimmunoprecipitation of alpha- and beta-subunits provides further evidence that both are tightly associated constituents of the K+ channel complexes. At least three isoforms of the K+ channel alpha-subunit are distinguishable by immunoblotting of a detergent extract of synaptic membranes with mAb 5. Likewise, multiple forms are also detectable in the purified protein with mAb 5 although deglycosylation, which does not alter reactivity with any of the mAbs, was required to achieve adequate electrophoretic resolution. These results confirm the proposal that variants of this K+ channel group, known to exist in the nervous system, are heterooligomeric complexes of alpha- and beta-subunits. Although different areas of rat brain contain proteins of similar sizes reactive with mAb 5, these are absent from heart, liver, pancreas, kidney, testes, and spleen, highlighting the selectivity of this antibody.

Primary structure of a beta subunit ofalpha-dendrotoxin-sensitive K+ channels from bovine brain.

“…3A). This yielded a pattern similar to that obtained by silver staining except that an additional band (-56 kDa) was visible; an equivalent minor protein was also detected by autoradiographic analysis of 125I-labeled acceptor (5). The latter may be the bovine equivalent of Kv (28).…”

“…Voltage-sensitive Ca2+ and Na+ channels possess one a subunit of -200 kDa consisting of four repeat domains (2,3). On the other hand, the isolated neuronal K+ channels (see below) contain four a subunits (4); the predominant one has an apparent molecular mass of =78 kDa but different sized minor variants are also present (5). It is well documented that the properties of expressed currents are strongly influenced by 1 subunits in the case of Na+ (6) or by 13, 'y, and a2/8 subunits for Ca2+ channels (2,7,8).…”

Association and colocalization of K+ channel alpha- and beta-subunit polypeptides in rat brain

“…Immunoprecipitation of K+ channel complexes from radioiodinated rat brain membranes using antibodies specific for the Kv2.1 K+ channel o-subunit (nomenclature according to Chandy, 1991) revealed the presence of a 38 kDa P-subunit polypeptide in tight association with this delayed rectifier K+ channel complex (Trimmer, 1991). Similarly, affinity purification and immunoprecipitation of the oc-dendrotoxin (DTX) sensitive K+ channel complex from bovine brain revealed, in addition to o-subunit polypeptides, the presence of 38 kDa and 41 kDa P-subunit polypeptides in association with the DTX acceptor complex (Parcej and Dolly, 1989;Scott et al, 1990;Muniz et al, 1992). A cDNA encoding a DTX acceptor P-subunit was recently isolated from bovine brain by Scott et al (1994b).…”

Structural Characterisation of Neuronal Voltage-sensitive K+ Channels Heterologously Expressed in Pichia pastoris

“…22 This would suggest that the remaining heterogeneity may be due to post-translational modification of the protein. Previously we have shown that Kv1.2 examined in brain membranes 23 exhibited similar behaviour during SDS-PAGE, due partly to N-glycosylation. It has been shown that Kv1.2 and other channels are additionally modified by tyrosine 24 and serine/threonine phosphorylation.…”

“…63 -65 Other methods SDS-PAGE and Western blotting were carried out as described. 23 Rat brain cerebrocortical synaptosomal membranes were prepared as described. 35 …”