Functional role of a conserved aspartate in the external mouth of voltage-gated potassium channels.
Mutation of the glycines in a conserved Gly-Tyr-Gly-Asp sequence in the P-region of voltage-gated K channels has identified determinants of Na/K selectivity. But the function of the negatively charged Asp is not known because mutations at this position are not tolerated, owing to the fourfold replication of mutations in a tetrameric channel. We have successfully mutated Asp378-->Thr in a tandem dimer Kv2.1 construct to yield a twofold neutralization of charge at this site. When expressed in Xenopus oocytes, the mutated channels showed markedly altered ion conduction and blockade. Potassium conduction in the inward direction was selectively reduced, so that the instantaneous current-voltage relationship obtained in isotonic KCl became strongly outwardly rectifying. The relative permeability to Na+, PNa/PK, increased from 0.02 to 0.10 without changing the ion selectivity sequence K > Rb >> Cs >> Na. The IC50 for block by external tetraethylammonium (TEA) increased more than 100-fold without affecting block by internal TEA. We conclude that Asp378 is an essential part of a potassium ion binding site associated with the Na/K selectivity filter at the external mouth of the pore.
Kirsch
G E
GE
Department of Molecular Physiology, Baylor College of Medicine, Houston, Texas 77030, USA.
Pascual
J M
JM
Shieh
C C
CC
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid; D002414:Cations, Monovalent / Q000378:metabolism; D002462:Cell Membrane / Q000502:physiology; D017124:Conserved Sequence; D005260:Female; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000502:physiology; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011188:Potassium / Q000378:metabolism; D015221:Potassium Channels / Q000096:biosynthesis / Q000737:chemistry* / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D013379:Substrate Specificity; D019789:Tetraethylammonium; D013757:Tetraethylammonium Compounds / Q000494:pharmacology; D013912:Threonine; D014982:Xenopus laevis
D002414:Cations, Monovalent; D046911:Macromolecular Substances; D015221:Potassium Channels; D011994:Recombinant Proteins; D013757:Tetraethylammonium Compounds; D013912:Threonine; D001224:Aspartic Acid; D019789:Tetraethylammonium; D011188:Potassium