Inwardly rectifying potassium channels expressed by gene transfection into the green Monkey kidney cell line COS-1.
1. cDNA encoding a functional inwardly rectifying K+ (IRK1) channel was transfected into COS-1 cells (a Green Monkey kidney cell line) using the liposome method, and voltage clamp experiments were done after 48-72 h. 2. Transfected cells showed inward rectification under whole-cell recording. The unitary current-voltage relationships in the inside-out configuration were almost linear in the absence of internal Mg2+ and polyamines, and the channel conductance averaged 34.1 +/- 2.0 pS (n = 15) at 23-26 degrees C. 3. Internal Mg2+ (2-10 microM) induced sublevels in the outward current with one-third and two-thirds of the unitary amplitude as in native channels. 4. To determine the subunit stoichiometry, we constructed tandem multimeric cDNAs consisting of the coding sequences of the IRK1 gene linked in a head-to-tail fashion. Cells transfected with tandem homomultimers up to octamers showed similar inwardly rectifying K+ channels. 5. A mutation (E138Q) eliminated the ionic conductance of the channel. Channels expressed by dimeric constructs containing a single mutant have a conductance ranging between 5 and 35 pS. 6. The E138Q mutant cotransfected with a wild-type dimeric, trimeric or tetrameric construct did not alter the channel conductance. The results do not support the notion that IRK1 channel proteins consist of four subunits.
Omori
K
K
Department of Physiology, Kansai Medical University, Osaka, Japan.
Oishi
K
K
Matsuda
H
H
D000818:Animals; D019556:COS Cells; D018076:DNA, Complementary / Q000737:chemistry; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D016296:Mutagenesis; D015221:Potassium Channels / Q000096:biosynthesis* / Q000235:genetics; D024661:Potassium Channels, Inwardly Rectifying; D011487:Protein Conformation; D014162:Transfection
D018076:DNA, Complementary; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D015246:Deoxyribonuclease EcoRI; D008274:Magnesium