Cooperative interactions among subunits of a voltage-dependent potassium channel. Evidence from expression of concatenated cDNAs.
Four copies of the coding sequence for a voltage-dependent potassium channel (RBK1, rat Kv1.1) were ligated contiguously and transcribed in vitro. The resulting RNA encodes four covalently linked subunit domains ([4]RBK1). Injection of this RNA into Xenopus oocytes resulted in the expression of voltage-dependent potassium currents. A single amino acid substitution, Tyr-->Val, located within the outer mouth of the pore, introduced into the equivalent position of any of the four domains, reduced affinity for external tetraethylammonium by approximately the same amount. In constructs containing 0, 1, 2, 3, or 4 Tyr residues the free energy of binding tetraethylammonium was linearly related to the number of Tyr residues. A different amino acid substitution, Leu-->Ile, located in the S4 region, was made in the equivalent position of one, two, three, or four domains. The depolarization required for channel activation increased approximately linearly with the number of Ile residues, whereas models of independent gating of each domain predict marked nonlinearity. Expression of this concatenated channel provides direct evidence that voltage-dependent potassium channels have four subunits positioned symmetrically around a central permeation pathway and that these subunits interact cooperatively during channel activation.
Hurst
R S
RS
Vollum Institute, Oregon Health Sciences University, Portland 97201.
Kavanaugh
M P
MP
Yakel
J
J
Adelman
J P
JP
North
R A
RA
D000818:Animals; D001921:Brain / Q000502:physiology*; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000187:drug effects / Q000502:physiology; D015221:Potassium Channels / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D012313:RNA / Q000235:genetics; D051381:Rats; D015183:Restriction Mapping; D013329:Structure-Activity Relationship; D019789:Tetraethylammonium; D013757:Tetraethylammonium Compounds / Q000494:pharmacology; D014158:Transcription, Genetic; D014443:Tyrosine; D014633:Valine; D014981:Xenopus
D046911:Macromolecular Substances; D015221:Potassium Channels; D013757:Tetraethylammonium Compounds; D014443:Tyrosine; D012313:RNA; D019789:Tetraethylammonium; D004247:DNA; D014633:Valine