Characterization of monoclonal antibodies against voltage-dependent K+ 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.