Activation of the human intermediate-conductance Ca2+-activated K+ channelby 1-ethyl-2-benzimidazolinone is strongly Ca2+-dependent

Modulation of the cloned human intermediate-conductance Ca2+-activated K+ c hannel (hIK) by the compound 1-ethyl-2-benzimidazolinone (EBIO) was studied by patch-clamp technique using human embryonic kidney cells (HEK 293) stab ly expressing the hIK channels. In whole-cell studies, intracellular concen trations of free Ca2+ were systematically varied, by buffering the pipette solutions. In voltage-clamp, the hIK specific currents increased gradually from 0 to approximate to 300 pA/pF without reaching saturation even at the highest Ca2+ concentration tested (300 nM). In the presence of EBIO (100 mu M), the Ca2+-activation curve was shifted leftwards, and maximal currents were attained at 100 nM Ca2+. In current-clamp, steeply Ca2+-dependent memb rane potentials were recorded and the cells gradually hyperpolarised from - 20 to -85 mV when Ca2+ was augmented from 0 to 300 nM. EBIO strongly hyperp olarised cells buffered at intermediate Ca2+ concentrations. In contrast, n o effects were detected either below 10 nM (no basic channel activation) or at 300 nM Ca2+ (V-m close to E-K) Without Ca2+, EBIO-induced hyperpolarisa tions were not obtainable, indicating an obligatory Ca2+-dependent mechanis m of action. When applied to inside-out patches, EBIO exerted a Ca2+-depend ent 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 channel s in whole-cell and inside-out patches by a direct mechanism, which require s the presence of internal Ca2+. (C) 1999 Elsevier Science B.V. All rights reserved.