Functional characterization of a minimal K+ channel expressed from a synthetic gene.
A gene for a slowly activating, voltage-dependent K(+) -selective channel was designed and synthesized on the basis of its known amino acid sequence. The synthetic gene was cloned into a transcription vector, and in vitro transcribed mRNA was injected into Xenopus oocytes for electrophysiological assay of the resulting ionic currents. The currents are voltage-dependent and highly selective for K+ over Na+. The selectivity among monovalent cations follows a familiar K(+)- channel sequence: K+ greater than Rb+ greater than NH4+ greater than Cs+ much greater than Na+, Li+. The currents are inhibited by Ba2+, Cs+, and tetraethylammonium (TEA), common pore blockers of K+ channels. Open-channel blockade by Cs+ (but not by Ba2+ or TEA) depends on applied voltage. The major inhibitory effect of Ba2+ is to alter channel gating by favoring the closed state; this effect is specific for Ba2+ and is relieved by external K+. The results argue that although the polypeptide expressed is very small for a eukaryotic ion channel, 130 amino acid residues in length, the ionic currents observed are indeed mediated by a genuine K(+) -channel protein. This synthetic gene is therefore well suited for a molecular analysis of the basic mechanisms of K(+) -channel function.
Hausdorff
S F
SF
Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, Massachusetts 02254-9110.
Goldstein
S A
SA
Rushin
E E
EE
Miller
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001464:Barium / Q000494:pharmacology; D001483:Base Sequence; D001667:Binding, Competitive; D002586:Cesium / Q000494:pharmacology; D003001:Cloning, Molecular; D005260:Female; D005813:Genes, Synthetic; D005822:Genetic Vectors; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D008969:Molecular Sequence Data; D009865:Oocytes / Q000187:drug effects / Q000502:physiology*; D015221:Potassium Channels / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D015183:Restriction Mapping; D019789:Tetraethylammonium; D013757:Tetraethylammonium Compounds / Q000494:pharmacology; D014158:Transcription, Genetic; D014981:Xenopus
D015221:Potassium Channels; D013757:Tetraethylammonium Compounds; D002586:Cesium; D001464:Barium; D019789:Tetraethylammonium