KCNQ4 channels expressed in mammalian cells: functional characteristics and pharmacology.

Human cloned KCNQ4 channels were stably expressed in HEK-293 cells and characterized with respect to function and pharmacology. Patch-clamp measurements showed that the KCNQ4 channels conducted slowly activating currents at potentials more positive than -60 mV. From the Boltzmann function fitted to the activation curve, a half-activation potential of -32 mV and an equivalent gating charge of 1.4 elementary charges was determined. The instantaneous current-voltage relationship revealed strong inward rectification. The KCNQ4 channels were blocked in a voltage-independent manner by the memory-enhancing M current blockers XE-991 and linopirdine with IC(50) values of 5.5 and 14 microM, respectively. The antiarrhythmic KCNQ1 channel blocker bepridil inhibited KCNQ4 with an IC(50) value of 9.4 microM, whereas clofilium was without significant effect at 100 microM. The KCNQ4-expressing cells exhibited average resting membrane potentials of -56 mV in contrast to -12 mV recorded in the nontransfected cells. In conclusion, the activation and pharmacology of KCNQ4 channels resemble those of M currents, and it is likely that the function of the KCNQ4 channel is to regulate the subthreshold electrical activity of excitable cells.

Inhibition of the human intermediate-conductance, Ca2+-activated K+ channel by intracellular acidification.

The effect of changes in pH on the gating properties of the cloned human intermediate-conductance, Ca2+-activated K+ channel (hIK) was studied using the patch-clamp technique. Multi-channel inside-out recordings of patches from HEK-293 cells stably expressing hIK channels revealed that the channel activity is modulated by changes in intracellular pH (pHi). Changes in extracellular pH (pHo) in the range from pH 6.0 to 8.2 did not affect the hIK whole-cell current. Intracellular acidification gradually decreased the activity of the hIK channel, approaching zero activity at pHi 6.0. Decreasing pHi altered neither the conductance nor the inward rectification of hIK channels. The proton-induced inhibition of the multi-channel hIK patch current could not be counteracted by increasing the cytosolic Ca2+ concentration to 30 microM. The molecular sensory mechanism underlying the proton-induced modulation of hIK gating is at present unknown.

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+).