Arrangement of Kv1 α subunits dictates sensitivity to tetraethylammonium

Ahmed Al-Sabi, Oleg Shamotienko, Sorcha Ni Dhochartaigh, Nagesh Muniyappa, Marie Le Berre, Hamdy Shaban, Jiafu Wang, Jon T. Sack, J. Oliver Dolly

Research output: Contribution to journalArticlepeer-review

20 Citations (Scopus)

Abstract

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 transiendy 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). Fore-blocking petidergic toxins, ∞ 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 (TFA) 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 adjacendy arranged constructs. These collective findings indicate that assembly of ∞ subunits can be directed by this optimized concatenation, and that subunit arrangement in heteromeric Kv channels affects TEA affinity.

Original languageEnglish
Pages (from-to)273-282
Number of pages10
JournalJournal of General Physiology
Volume136
Issue number3
DOIs
Publication statusPublished - Sep 2010

ASJC Scopus subject areas

  • Physiology

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