α Subunit compositions of Kv1.1-containing K+ channel subtypes fractionated from rat brain using dendrotoxins

Abstract

K+ channels from the Kv1 subfamily contain four α-subunits and the combinations (from Kv 1.1-1.6) determine susceptibility to dendrotoxin (DTX) homologues. The subunit composition of certain subtypes in rat brain was investigated using DTX(k) which only interacts with Kv1.1-containing channels and αDTX (and its closely related homologue DTX(i)) that binds preferentially to Kv1.2-possessing homo- or hetero-oligomers. Covalent attachment of [125I]DTX(k) bound to channels in synaptic membranes unveiled subunits of M(r) = 78000 and 96000. Immunoprecipitation of these solubilized and dissociated cross-linked proteins with IgG specific for each of the α-subunits identified Kv1.1, 1.2 and 1.4; this led to assemblies of Kv1.1/1.2 and 1.1/1.4 being established. Kv1.2-enriched channels, purified from rat brain by chromatography on immobilized DTX(i), contained Kv1.1, 1.2 and 1.6 confirming one of the above-noted pairs and indicating an additional Kv1.1-containing oligomer (Kv1.1/1.2/1.6); the notable lack of Kv1.4 excludes a Kv1.1/1.2/1.4 combination. On the other hand, channels with Kv1.1 as a constituent, isolated using DTX(k), possessed Kv1.4 in addition to those found in the DTX(i)-purified oligomers; this provides convergent support for the occurrence of the three combinations established above but adds a possible fourth (Kv1.1/1.4/1.6), though this was not confirmed. Moreover, sequential purification on DTX(i) and DTX(k) resins yielded channels containing only Kv1.1/1.2 but with an apparent predominance of Kv1.1, reaffirming the latter multimer.