Activation of the human intermediate-conductance Ca(2+)-activated K(+) channel by 1-ethyl-2-benzimidazolinone is strongly Ca(2+)-dependent.
Modulation of the cloned human intermediate-conductance Ca(2+)-activated K(+) channel (hIK) by the compound 1-ethyl-2-benzimidazolinone (EBIO) was studied by patch-clamp technique using human embryonic kidney cells (HEK 293) stably expressing the hIK channels. In whole-cell studies, intracellular concentrations of free Ca(2+) were systematically varied, by buffering the pipette solutions. In voltage-clamp, the hIK specific currents increased gradually from 0 to approximately 300 pA/pF without reaching saturation even at the highest Ca(2+) concentration tested (300 nM). In the presence of EBIO (100 microM), the Ca(2+)-activation curve was shifted leftwards, and maximal currents were attained at 100 nM Ca(2+). In current-clamp, steeply Ca(2+)-dependent membrane potentials were recorded and the cells gradually hyperpolarised from -20 to -85 mV when Ca(2+) was augmented from 0 to 300 nM. EBIO strongly hyperpolarised cells buffered at intermediate Ca(2+) concentrations. In contrast, no effects were detected either below 10 nM (no basic channel activation) or at 300 nM Ca(2+) (V(m) close to E(K)). Without Ca(2+), EBIO-induced hyperpolarisations were not obtainable, indicating an obligatory Ca(2+)-dependent mechanism of action. When applied to inside-out patches, EBIO exerted a Ca(2+)-dependent increase in the single-channel open-state probability, showing that the compound modulates hIK channels by a direct action on the alpha-subunit or on a closely associated protein. In conclusion, EBIO activates hIK channels in whole-cell and inside-out patches by a direct mechanism, which requires the presence of internal Ca(2+).
Pedersen
K A
KA
NeuroSearch A/S, Smedeland 26B, DK-2600, Glostrup, Denmark.
Schrøder
R L
RL
Skaaning-Jensen
B
B
Strøbaek
D
D
Olesen
S P
SP
Christophersen
P
P
D001562:Benzimidazoles / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002120:Calcium Channel Agonists / Q000494:pharmacology; D002460:Cell Line; D006801:Humans; D008564:Membrane Potentials; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D001562:Benzimidazoles; D002120:Calcium Channel Agonists; D015221:Potassium Channels; D011994:Recombinant Proteins; C102635:1-ethyl-2-benzimidazolinone; D002118:Calcium