Ming-Wei Lin

Contribution of BKCa-Channel Activity in Human Cardiac Fibroblasts to Electrical Coupling of Cardiomyocytes-Fibroblasts

Cardiac fibroblasts are involved in the maintenance of myocardial tissue structure. However, little is known about ion currents in human cardiac fibroblasts. It has been recently reported that cardiac fibroblasts can interact electrically with cardiomyocytes through gap junctions. Ca2+-activated K+ currents (IK[Ca]) of cultured human cardiac fibroblasts were characterized in this study. In whole-cell configuration, depolarizing pulses evoked IK(Ca) in an outward rectification in these cells, the amplitude of which was suppressed by paxilline (1xa0μM) or iberiotoxin (200 nM). A large-conductance, Ca2+-activated K+ (BKCa) channel with single-channel conductance of 162 ± 8 pS was also observed in human cardiac fibroblasts. Western blot analysis revealed the presence of α-subunit of BKCa channels. The dynamic Luo-Rudy model was applied to predict cell behavior during direct electrical coupling of cardiomyocytes and cardiac fibroblasts. In the simulation, electrically coupled cardiac fibroblasts also exhibited action potential; however, they were electrically inert with no gap-junctional coupling. The simulation predicts that changes in gap junction coupling conductance can influence the configuration of cardiac action potential and cardiomyocyte excitability. Ik(Ca) can be elicited by simulated action potential waveforms of cardiac fibroblasts when they are electrically coupled to cardiomyocytes. This study demonstrates that a BKCa channel is functionally expressed in human cardiac fibroblasts. The activity of these BKCa channels present in human cardiac fibroblasts may contribute to the functional activities of heart cells through transfer of electrical signals between these two cell types.

Pharmacological roles of the large-conductance calcium-activated potassium channel.

The gating of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel is primarily controlled by intracellular Ca(2+) and/or membrane depolarization. These channels play a role in the coupling of excitation-contraction and stimulus-secretion. A variety of structurally distinct compounds may influence the activity of these channels. Squamocin, an Annonaceous acetogenin, could interact with the BK(Ca) channel to increase the amplitude of Ca(2+)-activated K(+) current in coronary smooth muscle cells. Its stimulatory effect is related to intracellular Ca(2+) concentrations. In inside-out patches, application of ceramide to the bath suppressed the activity of BK(Ca) channels recorded from pituitary GH(3) cells and from retinal pigment epithelial cells. ICI-182,780, an estrogen receptor antagonist, was found to modulate BK(Ca)-channel activity in cultured endothelial cells and smooth muscle cells in a mechanism unlinked to the inhibition of estrogen receptors. Caffeic acid phenethyl ester (CAPE) and its analogy, cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate, could directly increase the activity of BK(Ca) channels in GH(3) cells. CAPE also reduced the frequency and amplitude of intracellular Ca(2+) oscillations in these cells. The CAPE-stimulated activity in BK(Ca) channels is thought to be unassociated with its inhibition of NF-kappaB activation. Cilostazol, an inhibitor of cyclic nucleotide phosphodiesterase, could stimulate BK(Ca) channel-activity and reduce the firing of action currents simultaneously in GH(3) cells. Therefore, the regulation by these compounds of BK(Ca) channels may in part be responsible for their regulatory actions on cell functions.

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.

Stimulatory actions of di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (di-8-ANEPPS), voltage-sensitive dye, on the BKCa channel in pituitary tumor (GH3) cells

Di-8-ANEPPS (4-{2-[6-(dibutylamino)-2-naphthalenyl]-ethenyl}-1-(3-sulfopropyl)pyridinium inner salt) has been used as a fast-response voltage-sensitive styrylpyridinium probe. However, little is known regarding the mechanism of di-8-ANEPPS actions on ion currents. In this study, the effects of this dye on ion currents were investigated in pituitary GH3 cells. In whole-cell configuration, di-8-ANEPPS (10xa0μM) reversibly increased the amplitude of Ca2+-activated K+ current. In inside-out configuration, di-8-ANEPPS (10xa0μM) applied to the intracellular surface of the membrane caused no change in single-channel conductance; however, it did enhance the activity of large-conductance Ca2+-activated K+ (BKCa) channels with an EC50 value of 7.5xa0μM. This compound caused a left shift in the activation curve of BKCa channels with no change in the gating charge of these channels. A decrease in mean closed time of the channels was seen in the presence of this dye. In the cell-attached mode, di-8-ANEPPS applied on the extracellular side of the membrane also activated BKCa channels. However, neither voltage-gated K+ nor ether-à-go-go-related gene (erg)-mediated K+ currents in GH3 cells were affected by di-8-APPNES. Under current-clamp configuration, di-8-ANEPPS (10xa0μM) decreased the firing of action potentials in GH3 cells. In pancreatic βTC-6 cells, di-8-APPNES (10xa0μM) also increased BKCa-channel activity. Taken together, this study suggests that during the exposure to di-8-ANEPPS, the stimulatory effects on BKCa channels could be one of potential mechanisms through which it may affect cell excitability.