Stable expression of the human large-conductance Ca2+-activated K+ channel alpha- and beta-subunits in HEK293 cells.
We have generated HEK293 cell lines stably expressing high levels of either the human BK channel alpha-subunit alone or the BK channel alpha-subunit and beta-subunit together. For co-expression a plasmid with three expression cassettes was constructed. Patch-clamp recordings on inside-out patches from the transfected cells resulted in macroscopic currents reflecting the expression of 200-800 BK channels per patch. No decrease in channel expression could be detected in cells grown for more than 50 passages. The alpha-subunit when expressed alone conducted currents which were sensitive to intracellular Ca2+ in the physiological range. In the presence of the beta-subunit the steady-state activation curves were shifted by -20 to -30 mV and channel deactivation kinetics were slowed. The BK channel opener NS1608 (10 microM) shifted the steady-state activation curves for the alpha-subunit as well as for the alphabeta-subunits by -40 to -50 mV.
Ahring
P K
PK
NeuroSearch A/S, Glostrup, Denmark. PKA@NeuroSearch.dk
Strøbaek
D
D
Christophersen
P
P
Olesen
S P
SP
Johansen
T E
TE
D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D004594:Electrophysiology; D005786:Gene Expression Regulation; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007700:Kinetics; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051038:Large-Conductance Calcium-Activated Potassium Channel beta Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D018408:Patch-Clamp Techniques; D010671:Phenylurea Compounds / Q000494:pharmacology; D010957:Plasmids; D016133:Polymerase Chain Reaction; D015221:Potassium Channels / Q000235:genetics* / Q000378:metabolism; D024681:Potassium Channels, Calcium-Activated; D011487:Protein Conformation; D014162:Transfection
D007211:Indoles; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051038:Large-Conductance Calcium-Activated Potassium Channel beta Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D010671:Phenylurea Compounds; D015221:Potassium Channels; D024681:Potassium Channels, Calcium-Activated; C103727:NS 1608; C048220:paxilline; D002118:Calcium