Arrangement of Kv1 α subunits dictates sensitivity to tetraethylammonium

Shaker-related Kv1 channels contain four channel-forming α subunits. Subfamily member Kv1.1 often occurs oligomerized with Kv1.2 α subunits in synaptic membranes, and so information was sought on the influence of their positions within tetramers on the channels’ properties. Kv1.1 and 1.2 α genes were tandem linked in various arrangements, followed by expression as single-chain proteins in mammalian cells. As some concatenations reported previously seemed not to reliably position Kv1 subunits in their assemblies, the identity of expressed channels was methodically evaluated. Surface protein, isolated by biotinylation of intact transiently transfected HEK-293 cells, gave Kv1.1/1.2 reactivity on immunoblots with electrophoretic mobilities corresponding to full-length concatenated tetramers. There was no evidence of protein degradation, indicating that concatemers were delivered intact to the plasmalemma. Constructs with like genes adjacent (Kv1.1-1.1-1.2-1.2 or Kv1.2-1.2-1.1-1.1) yielded delayed-rectifying, voltage-dependent K+ currents with activation parameters and inactivation kinetics slightly different from the diagonally positioned genes (Kv1.1-1.2-1.1-1.2 or 1.2–1.1-1.2-1.1). Pore-blocking petidergic toxins, α dendrotoxin, agitoxin-1, tityustoxin-Kα, and kaliotoxin, were unable to distinguish between the adjacent and diagonal concatamers. Unprecedentedly, external application of the pore-blocker tetraethylammonium (TEA) differentially inhibited the adjacent versus diagonal subunit arrangements, with diagonal constructs having enhanced susceptibility. Concatenation did not directly alter the sensitivities of homomeric Kv1.1 or 1.2 channels to TEA or the toxins. TEA inhibition of currents generated by channels made up from dimers (Kv1.1-1.2 and/or Kv1.2-1.1) was similar to the adjacently arranged constructs. These collective findings indicate that assembly of α subunits can be directed by this optimized concatenation, and that subunit arrangement in heteromeric Kv channels affects TEA affinity

Surface biotinylation of heteromeric Kv1 concatemers expressed in CHO cells

Concatenated constructs of Kv1.1-1.2 or Kv1.1-1.2-1.2-1.2 () were expressed CHO cells by electroporation of RNA transcripts from a Semliki Forest virus vector. After 48 h, electroporated cells were harvested in PBS buffer containing 5 mM EDTA. Cell surface biotinylation was performed with sulfo-NHS-LC-biotin (Pierce Chemical Co.), as recommended by the manufacturer. Membrane proteins solubilized with 2% Triton X-100 were precipitated using streptavidin-agarose (Pierce Chemical Co.). Bound proteins were dissolved in lithium dodecyl sulfate sample buffer (Invitrogen) and SDS-PAGE and Western blotting with anti-Kv1.2 antibody were performed, as outlined elsewhere (). Lanes: 1, biotinylated Kv1.1-1.2; 2, total extract from cells expressing Kv1.1-1.2; 3, biotinylated Kv1.1-1.2-1.2-1.2; and 4, total extract from cells expressing Kv1.1-1.2-1.2-1.2.<p><b>Copyright information:</b></p><p>Taken from "How to Validate a Heteromeric Ion Channel Drug Target: Assessing Proper Expression of Concatenated Subunits"</p><p></p><p>The Journal of General Physiology 2008;131(5):415-420.</p><p>Published online Jan 2008</p><p>PMCID:PMC2346572.</p><p></p