Arrangement of Kv1 alpha subunits dictates sensitivity to tetraethylammonium.
Shaker-related Kv1 channels contain four channel-forming alpha subunits. Subfamily member Kv1.1 often occurs oligomerized with Kv1.2 alpha 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 alpha 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, alpha dendrotoxin, agitoxin-1, tityustoxin-Kalpha, 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 alpha subunits can be directed by this optimized concatenation, and that subunit arrangement in heteromeric Kv channels affects TEA affinity.
Al-Sabi
Ahmed
A
International Centre for Neurotherapeutics, Dublin City University, Dublin 9, Ireland.
Shamotienko
Oleg
O
Dhochartaigh
Sorcha Ni
SN
Muniyappa
Nagesh
N
Le Berre
Marie
M
Shaban
Hamdy
H
Wang
Jiafu
J
Sack
Jon T
JT
Dolly
J Oliver
JO
D000595:Amino Acid Sequence; D000818:Animals; D019921:Biotinylation; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D006801:Humans; D051662:Kv1.1 Potassium Channel / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051663:Kv1.2 Potassium Channel / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008564:Membrane Potentials; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D026902:Potassium Channel Blockers / Q000494:pharmacology*; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D021381:Protein Transport; D051381:Rats; D013329:Structure-Activity Relationship; D019789:Tetraethylammonium / Q000494:pharmacology*; D014162:Transfection
C489293:Kcna1 protein, rat; C496972:Kcna2 protein, rat; D051663:Kv1.2 Potassium Channel; D026902:Potassium Channel Blockers; D021122:Protein Subunits; D051662:Kv1.1 Potassium Channel; D019789:Tetraethylammonium
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