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 COX-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 mu M) 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 in inwardly rectifying Kf 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.