Ya-Jean Wang

Potent stimulation of large-conductance Ca2+-activated K+ channels by rottlerin, an inhibitor of protein kinase C-δ, in pituitary tumor (GH3) cells and in cortical neuronal (HCN-1A) cells

The effects of rottlerin, a known inhibitor of protein kinase C‐δ activation, on ion currents were investigated in pituitary tumor (GH3) cells. Rottlerin (0.3–100 µM) increased the amplitude of Ca2+‐activated K+ current (IK(Ca)) in a concentration‐dependent manner with an EC50 value of 1.7 µM. In intracellular perfusion with rottlerin (1 µM) or staurosporine (10 µM), phorbol 12‐myristate 13‐acetate‐induced inhibition of IK(Ca) in these cells was abolished. In cell‐attached mode, rottlerin applied on the extracellular side of the membrane caused activation of large‐conductance Ca2+‐activated K+ (BKCa) channels, and a further application of BAPTA‐AM (10 µM) to the bath had no effect on rottlerin‐stimulated channel activity. When cells were exposed to rottlerin, the activation curve of these channels was shifted to less positive potential with no change in the slope factor. Rottlerin increased BKCa‐channel activity in outside‐out patches. Its change in kinetic behavior of BKCa channels is primarily due to an increase in mean open time. With the aid of minimal kinetic scheme, a quantitative description of rottlerin stimulation on BKCa channels in GH3 cells was also provided. Under current‐clamp configuration, rottlerin (1 µM) decreased the firing of action potentials. IK(Ca) elicited by simulated action potential waveforms was enhanced by this compound. In human cortical HCN‐1A cells, rottlerin (1 µM) could also interact with the BKCa channel to stimulate IK(Ca). Therefore, rottlerin may directly activate BKCa channels in neurons or endocrine cells. J. Cell. Physiol. 210: 655–666, 2007.

Characterization of aconitine-induced block of delayed rectifier K+ current in differentiated NG108-15 neuronal cells

The effects of aconitine (ACO), a highly toxic alkaloid, on ion currents in differentiated NG108-15 neuronal cells were investigated in this study. ACO (0.3-30 microM) suppressed the amplitude of delayed rectifier K+ current (I K(DR)) in a concentration-dependent manner with an IC50 value of 3.1 microM. The presence of ACO enhanced the rate and extent of I K(DR) inactivation, although it had no effect on the initial activation phase of I K(DR). It could shift the inactivation curve of I K(DR) to a hyperpolarized potential with no change in the slope factor. Cumulative inactivation for I K(DR) was also enhanced by ACO. Orphenadrine (30 microM) or methyllycaconitine (30 microM) slightly suppressed I K(DR) without modifying current decay. ACO (10 microM) had an inhibitory effect on voltage-dependent Na+ current (I Na). Under current-clamp recordings, ACO increased the firing and widening of action potentials in these cells. With the aid of the minimal binding scheme, the ACO actions on I K(DR) was quantitatively provided with a dissociation constant of 0.6 microM. A modeled cell was designed to duplicate its inhibitory effect on spontaneous pacemaking. ACO also blocked I K(DR) in neuroblastoma SH-SY5Y cells. Taken together, the experimental data and simulations show that ACO can block delayed rectifier K+ channels of neurons in a concentration- and state-dependent manner. Changes in action potentials induced by ACO in neurons in vivo can be explained mainly by its blocking actions on I K(DR) and I Na.

Time-Dependent Block of Ultrarapid-Delayed Rectifier K+ Currents by Aconitine, a Potent Cardiotoxin, in Heart-Derived H9c2 Myoblasts and in Neonatal Rat Ventricular Myocytes

Aconitine (ACO), a highly toxic diterpenoid alkaloid, is recognized to have effects on cardiac voltage-gated Na(+) channels. However, it remains unknown whether it has any effects on K(+) currents. The effects of ACO on ion currents in differentiated clonal cardiac (H9c2) cells and in cultured neonatal rat ventricular myocytes were investigated in this study. In H9c2 cells, ACO suppressed ultrarapid-delayed rectifier K(+) current (I(Kur)) in a time- and concentration-dependent fashion. The IC(50) value for ACO-induced inhibition of I(Kur) was 1.4 microM. ACO could accelerate the inactivation of I(Kur) with no change in the activation time constant of this current. Steady-state inactivation curve of I(Kur) during exposure to ACO could be demonstrated. Recovery from block by ACO was fitted by a single-exponential function. The inhibition of I(Kur) by ACO could still be observed in H9c2 cells preincubated with ruthenium red (30 microM). Intracellular dialysis with ACO (30 microM) had no effects on I(Kur). I(Kur) elicited by simulated action potential (AP) waveforms was sensitive to block by ACO. Single-cell Ca(2+) imaging revealed that ACO (10 microM) alone did not affect intracellular Ca(2+) in H9c2 cells. In cultured neonatal rat ventricular myocytes, ACO also blocked I(Kur) and prolonged AP along with appearance of early afterdepolarizations. Multielectrode recordings on neonatal rat ventricular tissues also suggested that ACO-induced electrocardiographic changes could be associated with inhibition of I(Kur). This study provides the evidence that ACO can produce a depressant action on I(Kur) in cardiac myocytes.

Potent Activation of Large-Conductance Ca2+-Activated K+ Channels by the Diphenylurea 1,3-Bis-[2-hydroxy-5-(trifluoromethyl)phenyl]urea (NS1643) in Pituitary Tumor (GH3) Cells

1,3-Bis-[2-hydroxy-5-(trifluoromethyl)phenyl]urea (NS1643) is reported to be an activator of human ether-à-go-go-related gene current. However, it remains unknown whether it has any effects on other types of ion channels. The effects of NS1643 on ion currents and membrane potential were investigated in this study. NS1643 stimulated Ca2+-activated K+ current [IK(Ca)] in a concentration-dependent manner with an EC50 value of 1.8 μM in pituitary tumor (GH3) cells. In inside-out recordings, this compound applied to the intracellular side of the detached channels stimulated large-conductance Ca2+-activated K+ (BKCa) channels with no change in single-channel conductance. It shifted the activation curve of BKCa channels to less depolarized voltages without altering the gating charge of the channels. NS1643-stimulated channel activity depended on intracellular Ca2+, and mean closed time during exposure to NS1643 was reduced. NS1643 (3 μM) had little or no effect on peak amplitude of ether-à-go-go-related gene-mediated K+ current evoked by membrane hyperpolarization, although it increased the amplitude of late-sustained components of K+ inward current, which was suppressed by paxilline but not by azimilide. NS1643 (3 μM) had no effect on L-type Ca2+ current. This compound reduced repetitive firing of action potentials, and further application of paxilline attenuated its decrease in firing rate. In addition, NS1643 enhanced BKCa-channel activity in human embryonic kidney 293T cells expressing α-hSlo. In summary, we clearly show that NS1643 interacts directly with the BKCa channel to increase the amplitude of IK(Ca) in pituitary tumor (GH3) cells. The α-subunit of the channel may be a target for the action of this small compound.