ATP-Dependent inhibition of Ca2+-activated K+ channels in vascular smooth muscle cells by neuropeptide Y

Neuropeptide Y(NPY) inhibits Ca²⁺-activated K⁺ channels reversibly in vascular smooth muscle cells from the rat tail artery. NPY (200 M) had no effect in the absence of intracellular adenosine 5triphosphate (ATP) and when the metabolic poison cyanide-M-chlorophenyl hydrozone (10 M) was included in the intracellular pipette solution. NPY was also not effective when ATP was substituted by the non-hydrolysable ATP analogue adenosine 5-[, -methylene]-triphosphate (AMP-PCP). NPY inhibited Ca²⁺-activated K⁺ channel activity when ATP was replaced by adenosine 5-O-(3-thiotriphosphate) (ATP [-S]) and the inhibition was not readily reversed upon washing. Protein kinase inhibitor (1 M), a specific inhibitor of adenosine 3, 5-cyclic monophosphatedependent protein kinase, had no significant effect on the inhibitory action of NPY. The effect of NPY on single-channel activity was inhibited by the tyrosine kinase inhibitor genistein (10 M) but not by daidzein, an inactive analogue of genistein. These observations suggest that the inhibition by NPY of Ca²⁺-activated K⁺ channels is mediated by ATP-dependent phosphorylation. The inhibitory effect of NPY was antagonized by the tyrosine kinase inhibitor genistein.     

Ca2+-activated K+ channels in airway smooth muscle are inhibited by cytoplasmic adenosine triphosphate

Large-conductance Ca²⁺-activated K⁺ channels were studied in membranes of cultured rabbit airway smooth muscle cells, using the patch-clamp technique. In cell-attached recordings, channel openings were rare and occurred only at very positive potentials. Bradykinin (10 M), an agonist which releases Ca²⁺ from the sarcoplasmic reticulum, transiently increased channel activity. The metabolic blocker 2,4-dinitrophenol (20 M), which lowers cellular adenosine triphosphate (ATP) levels, induced a sustained increase of channel activity in cell-attached patches. In excised patches, these channels had a slope conductance of 155 pS at 0 mV, were activated by depolarization and by increasing the Ca²⁺ concentration at the cytoplasmic side above 10^–7 mol/l. ATP, applied to the cytoplasmic side of the patches, dose-dependently decreased the channel's open-state probability. An inhibition constant (K _i) of 0.2 mmol/l was found for the ATP-induced inhibition. ATP reduced the Ca²⁺ sensitivity of the channel, shifting the Ca²⁺ activation curve to the right and additionally reducing its steepness. Our results demonstrate that cytoplasmic ATP inhibits a large-conductance Ca²⁺-activated K⁺ channel in airway smooth muscle. This ATP modulation of Ca²⁺-activated K⁺ channels might serve as an important mechanism linking energy status and the contractile state of the cells.     

Small-conductance Ca2+-activated K+ channels in bovine chromaffin cells

Simultaneous whole-cell patch-clamp and fura-2 fluorescence [Ca²⁺]_i measurements were used to characterize Ca²⁺-activated K⁺ currents in cultured bovine chromaffin cells. Extracellular application of histamine (10 M) induced a rise of [Ca²⁺]_i concomitantly with an outward current at holding potentials positive to –80 mV. The activation of the current reflected an increase in conductance, which did not depend on membrane potential in the range –80 mV to –40 mV. Increasing the extracellular K⁺ concentration to 20 mM at the holding potential of –78 mV was associated with inwardly directed currents during the [Ca²⁺]_i elevations induced either by histamine (10 M) or short voltage-clamp depolarizations. The current reversal potential was close to the K⁺ equilibrium potential, being a function of external K⁺ concentration. Current fluctuation analysis suggested a unit conductance of 3–5 pS for the channel that underlies this K⁺ current. The current could be blocked by apamin (1 M). Whole-cell current-clamp recordings snowed that histamine (10 M) application caused a transient hyperpolarization, which evolved in parallel with the [Ca²⁺]_i changes. It is proposed that a small-conductance Ca²⁺-activated K⁺ channel is present in the membrane of bovine chromaffin cells and may be involved in regulating catecholamine secretion by the adrenal glands of various species.     

Caffeine induces periodic oscillations of Ca2+-activated K+ current in pulmonary arterial smooth muscle cells

The periodic oscillations of outward currents were studied in smooth muscle cells of the rabbit pulmonary artery. The combined stimuli of superfusion with 1 mM caffeine and depolarization of the membrane potential to 0 mV evoked periodic oscillations of outward currents with fairly uniform amplitudes and intervals. The oscillating outward currents induced by caffeine were dependent on intracellular Ca²⁺ concentration ([Ca²⁺]_i) and had a reversal potential near to the equilibrium potential for K⁺. So the oscillating outward currents are carried by K⁺ through Ca²⁺-dependent K⁺ channels (I _K(Ca)), and may reflect the oscillations of [Ca²⁺]_i. The oscillating outward currents were abolished, or their frequency reduced, by lowering external [Ca²⁺], Ca²⁺ channel blockers, or by 1 M ryanodine, indicating that: (1) there is a continuous influx of Ca²⁺ through the plasma membrane at a holding potential of 0 mV; (2) the periodic transient increases of [Ca²⁺]_i are ascribed to the rhythmic release of Ca²⁺ from ryanodinesensitive intracellular store by the mechanism of Ca²⁺-induced Ca²⁺ release (CICR). On the basis of the above results, we simulated the oscillation of [Ca²⁺]_i induced by caffeine, which is known to lower the threshold of CICR. The patterns of peak amplitude histograms of spontaneous transient outward currents (STOC) in the oscillating cells were different from those in non-oscillating cells. The amplitudes of STOC in the latter were more variable than those in the former. The oscillating outward currents were modulated by 1 M forskolin and 1 M sodium nitroprusside, but STOC were little affected. The above differences between STOC and oscillating outward currents suggest that the two currents are activated by the Ca²⁺ originating from different intracellular Ca²⁺ stores which are functionally heterogeneous.     

Neuropeptide Y inhibits Ca2+-activated K+ channels in vascular smooth muscle cells from the rat tail artery

The effects of neuropeptide Y (NPY) on the Ca²⁺-activated K⁺ channel in smooth muscle cells from the rat tail artery were studied by whole-cell and single-channel patch-clamp recording techniques. In the presence of nifedipine (1 M), whole-cell outward currents through Ca²⁺-activated K⁺ channels were inhibited by NPY in a dose-dependent manner from 20 to 200 nM. A maximum inhibition to about 48% of the control current could be achieved. Recordings from outside-out patches showed that the open probability of Ca²⁺-activated K⁺ channels were similarly inhibited by NPY. At 200 nM NPY, the open probability was reduced to about 36% of the control value. NPY did not affect the open times or current amplitude, but increased significantly the short (from 0.49 to 0.58 ms) and long (from 441 to 728 ms) closed times. Inhibition of Ca²⁺-activated K⁺ channels by NPY may contribute to its excitatory action on vascular smooth muscle cells.     

Tedisamil blocks single large-conductance Ca2+-activated K+ channels in membrane patches from smooth muscle cells of the guinea-pig portal vein

Enzymatically dispersed smooth muscle cells of the guinea-pig portal vein were studied by the patch-clamp technique. They were found to have Ca²⁺-dependent K⁺ channels with the typical properties of the BK channel, i.e. a reversal potential at the calculated equilibrium potential for K⁺ ions, a striking voltage dependence, and a conductance of approximately 200 pS ([K⁺]₀ 50 mM, [K⁺]_i 150 mM, positive patch potentials). Tedisamil, a new bradycardic agent with an inhibitory action on K⁺ currents in heart muscle, reduced the open probability of the BK channels concentration-dependently (1–100 M) when applied at the cytosolic side of membrane inside-out patches. At 100 M [Ca²⁺]_i, the IC₅₀ of tedisamil was 13.8 M (¯x, n=5). Tedisamil increased the frequency of channel closures, and reduced the mean duration of openings from 8 ms to < 1 ms, while the mean duration of closures within bursts (1–2 ms) was not altered. Tedisamil did not affect long closures (> 160 ms) between bursts, either. The mean time of residence of tedisamil at the BK channel was estimated to be 1–2ms. Hence, tedisamil, in comparison to the slow blocker Ba²⁺ and the fast blocker tetraethylammonium, holds the position of an intermediate K⁺ channel blocker.Supported by the Deutsche Forschungsgemeinschaft     

ATP suppresses activity of Ca2+ -activated K+ channels by Ca2+ chelation

Ca²⁺-activated maxi K⁺ channels were studied in inside-out patches from smooth muscle cells isolated from either porcine coronary arteries or guinea-pig urinary bladder. As described by Groschner et al. (Pflügers Arch 417:517, 1990), channel activity (NP _o) was stimulated by 3 M [Ca²⁺]_c (1 mM Ca-EGTA adjusted to a calculated pCa of 5.5) and was suppressed by the addition of 1 mM Na₂ATP. The following results suggest that suppression of NP _o by Na₂ATP is due to Ca²⁺ chelation and hence reduction of [Ca²⁺]_c and reduced Ca²⁺ activation of the channel. The effect was absent when Mg ATP was used instead of Na₂ATP. The effect was diminished by increasing the [EGTA] from 1 to 10 mM. The effect was absent when [Ca²⁺]_c was buffered with 10 mM HDTA (apparent pK _Ca 5.58) instead of EGTA (pK _Ca 6.8). A Ca²⁺-sensitive electrode system indicated that 1 mM Na₂ATP reduced [Ca²⁺]_c in 1 mM Ca-EGTA from 3 M to 1.4 M. Na₂ATP, Na₂GTP, Li₄AMP-PNP and NaADP reduced measured [Ca²⁺]_c in parallel with their suppression of NP _o. After the Na₂ATP-induced reduction of [Ca²⁺]_c was re-adjusted by adding either CaCl₂ or MgCl₂, the effect of Na₂ATP on NP _o disappeared. In vivo, intracellular [Mg²⁺] exceeds free [ATP^4–], hence ATP modulation of maxi K⁺ channels due to Ca²⁺ chelation is without biological relevance.     

Comparison of Ca2+-dependent K+ channels in the membrane of smooth muscle cells isolated from adult and foetal human aorta

Ca²⁺-activated K⁺ ionic currents in the membrane of cultured smooth muscle cells isolated from foetal and adult human aorta were studied using whole cell and single-channel patch-clamp techniques. Whole cell currents in adult smooth muscle cells were 3–8 times larger than in foetal cells of similar sizes. The elementary conductance and ionic selectivity of single Ca²⁺-activated K⁺ were identical for both types of cells. Channel openings occurred in burst, the duration of which was 3–5-fold longer in adult than in foetal cells. The voltage dependency of the channel activating mechanism and the dependency of the mean open time on the Ca²⁺ concentration on the inner side of the membrane were similar for both types of cells. These results suggest that the main reason for the increase in potassium conductance during development is an alteration in the open time of the Ca²⁺-activated K⁺ channels.     

Blocking actions of Ca2+ antagonists on the Ca2+ channels in the smooth muscle cell membrane of rabbit small intestine

Actions of Ca²⁺ antagonists, verapamil, nicardipine and diltiazem, were investigated on the Ca²⁺ inward current in the fragmented smooth muscle cell membrane (smooth muscle ball; SMB) obtained from the longitudinal muscle layer of the rabbit ileum, by enzymatic dispersion. All Ca²⁺ antagonists inhibited the inward current, in a dose-dependent manner. The ID₅₀ value on the maximum amplitude of the inward current of nicardipine was 24 nM, and this value was roughly 50 times lower than values obtained with verapamil and diltiazem, when the inward current was provoked by 0 mV command pulse from the holding potential of –60 mV. Lowering the holding potential to –80 mV shifted the dose-response curve to the right. When depolarizing pulses (100 ms, stepped up to 0 mV from –60 mV or –80 mV) were applied every 20 s, the peak amplitude of the inward current remained unchanged, but nicardipine immediately, and diltiazem and verapamil slowly reduced the peak amplitude. These slow inhibitions by the latter two drugs depended on the frequency or number of stimulations. Nicardipine but not diltiazem and verapamil shifted the voltage-dependent inactivation curve to the left (3 s duration of the conditioning pulse). However, with a longer conditioning pulse (10 s) verapamil and diltiazem shifted the voltage-dependent inactivation curves to the left. Therefore, the inhibitory actions of these Ca²⁺ antagonists differ. Namely, diltiazem and verapamil inhibit the Ca²⁺ channels, mainly in a frequency-or use-dependent manner while nicardipine does so in a voltage-dependent manner.     

Fluoxetine protects against big endothelin-1 induced anti-apoptosis by rescuing Kv1.5 channels in human pulmonary arterial smooth muscle cells

Purpose

Pulmonary Kv channels are thought to play a crucial role in the regulation of cell proliferation and apoptosis. Previous studies have shown that fluoxetine upregulated the expression of Kv1.5 and prevented pulmonary arterial hypertension in monocrotaline-induced or hypoxia-induced rats and mice. The current study was designed to test how fluoxetine regulates Kv1.5 channels, subsequently promoting apoptosis in human PASMCs cultured in vitro.

Materials and Methods

Human PASMCs were incubated with low-serum DMEM, ET-1, and fluoxetine with and without ET-1 separately for 72 h. Then the proliferation, apoptosis, and expression of TRPC1 and Kv1.5 were detected.

Results

In the ET-1 induced group, the upregulation of TRPC1 and down regulation of Kv1.5 enhanced proliferation and anti-apoptosis, which was reversed when treated with fluoxetine. The decreased expression of TRPC1 increased the expression of Kv1.5, subsequently inhibiting proliferation while promoting apoptosis.

Conclusion

The results from the present study suggested that fluoxetine protects against big endothelin-1 induced anti-apoptosis and rescues Kv1.5 channels in human pulmonary arterial smooth muscle cells, potentially by decreasing intracellular concentrations of Ca²⁺.     

L-alanine evokes opening of single Ca2+-activated K+ channels in rat liver cells

Cellular uptake of neutral amino acids via Na⁺ cotransporters is known to be associated with an increased membrane K⁺ conductance mediated by an unknown mechanism that is essential for avoiding excessive cell swelling. We now demonstrate by patch-clamp single-channel current recording that exposure of rat liver cells to L-alanine, but not the poorly transported D-stereoisomer, evokes opening of single K⁺ channels and that this effect is reversible upon removal of the amino acid. The nature of the conductance pathways opened in the intact cell by L-alanine has been investigated in cell-free excised membrane patches where it can be shown that the K⁺-selective channels are opened by Ca²⁺ acting from the inside of the membrane at a concentration as low as 0.1 M.     

Different inhibitions of the voltage-dependent K+ current by Ca2+ antagonists in the smooth muscle cell membrane of rabbit small intestine

Actions of Ca²⁺ antagonists (verapamil, nicardipine and diltiazem) on the voltage-dependent K⁺ current, obtained from the fragmented smooth muscle cell membrane (smooth muscle ball; SMB) of the rabbit small intestine, were investigated using voltage clamp techniques. To eliminate the influence of the Ca²⁺-dependent K⁺ current, the voltage-dependent K⁺ current was recorded in 2.5 mM Mn²⁺ (Ca²⁺ omitted) solution. These three Ca²⁺ antagonists inhibited the peak amplitude of the K⁺ current, in a dose-dependent manner. During application of a long command pulse (duration, 3 s), the amplitude of the voltage-dependent K⁺ current decreased slowly with time. Diltiazem inhibited the K⁺ current with a slight prolongation of the 20% decay time, while TEA (tetraethyl-ammonium), a K⁺ channel blocker, inhibited the current, without affecting the decay. By contrast, verapamil and nicardipine accelerated inactivation. In the control, the voltage-dependent inactivation was also seen in the K⁺ current. This inactivation curve below 0 mV was not modified by 10 M diltiazem, 5 M verapamil nor 3 M nicardipine. These results indicate that inhibition of the voltage-dependent K⁺ current by verapamil or nicardipine differed from that by diltiazem.An abstract of this work was reported at the 59th general meeting of Japan Pharmacological Society (Terada et al. 1986).     

Ca2+ dependence of small Ca2+-activated K+ channels in cultured N1E-115 mouse neuroblastoma cells

Single-channel properties of Ca²⁺-activated K⁺ channels have been investigated in excised membrane patches of N1E-115 mouse neuroblastoma cells under asymmetric K⁺ concentrations at 0 mV. The SK channels are blocked by 3 nM external apamin, are unaffected by 20 mM external tetraethylammonium (TEA) and have a single-channel conductance of 5.4 pS. The half-maximum open probability and opening frequency of SK channels are observed at 1 M internal Ca²⁺. Concentration/effect curves of these parameters are very steep with exponential slope factors between 7 and 13. Opentime distributions demonstrate the existence of at least two open states. The mean short open time increases with [Ca²⁺]_i, whereas the mean long open time is independent of [Ca²⁺]_i. At low [Ca²⁺]_i the short-lived open state predominates. At saturating [Ca²⁺]_i the number of longlived openings is more enhanced than the number of short-lived openings and both open states occur equally frequently. The opening frequency as well as the open times of SK channels are independent of the membrane potential in the range of –16 to +40 mV. The results indicate that activation of K⁺ current through SK channels is mainly determined by the Ca²⁺-dependent single-channel opening frequency. BK channels in N1E-115 cells are insensitive to 100 nM external apamin, are sensitive to external TEA in the millimolar range and have a single-channel conductance of 98 pS. Half-maximum open probability and opening frequency of the BK channel are observed at 7.5–21 M internal Ca²⁺. The slope factors of concentration/effect curves range between 1.7 and 2.9. As the BK channel open time is markedly enhanced at raised [Ca²⁺]_i, the Ca²⁺ dependence of the current through BK channels is determined by the single-channel opening frequency as well as the open time. SK as well as BK channels appear to be clustered and interact in a negative cooperative manner in multiple channel patches. The differences in Ca²⁺ dependence suggest that BK channels are activated by a local high [Ca²⁺]_i associated with Ca²⁺ influx, whereas SK channels may be activated by Ca²⁺ released from internal stores as well.     

Biophysical properties of Ca2+- and Mg-ATP-activated K+ channels in pulmonary arterial smooth muscle cells isolated from the rat

A novel class of Ca²⁺-activated K⁺ channel, also activated by Mg-ATP, exists in the main pulmonary artery of the rat. In view of the sensitivity of these K_Ca,ATP channels to such charged intermediates it is possible that they may be involved in regulating cellular responses to hypoxia. However, their electrophysiological profile is at present unknown. We have therefore characterised the sensitivity of K_Ca,ATP channels to voltage, intracellular Ca²⁺ ([Ca²⁺]_i) and Mg-ATP. They have a conductance of 245 pS in symmetrical K⁺ and are approximately 20 times more selective for K⁺ ions than Na⁺ ions, with a K⁺ permeability (P _K) of 4.6×10^–13cm s^–1. Ca²⁺ ions applied to the intracellular membrane surface of K_Ca,ATP channels causes a marked enhancement of their activity. This activation is probably the result of simultaneous binding of at least two Ca²⁺ ions, determined using Hill analysis, to the channel or some closely associated protein. This results in a shift of the voltage activation threshold to more hyperpolarized membrane potentials. The activation of K_Ca,ATP channels by Mg-ATP has an EC₅₀ of approximately 50 M. Although the EC₅₀ is unaffected by [Ca²⁺]_i, channel activation by Mg-ATP is enhanced by increasing [Ca²⁺]_i. One possible interpretation of these data is that Mg-ATP increases the sensitivity of K_Ca,ATP channels to Ca²⁺. It is therefore possible that under hypoxic conditions, where lower levels of Mg-ATP may be encountered, the sensitivity of K_Ca,ATP channels to Ca²⁺ and therefore voltage is reduced. This would tend to induce a depolarising influence, which would favour the influx of Ca²⁺ through voltage-activated Ca²⁺ channels, ultimately leading to increased vascular tone.     

The mechanism of action of Ba2+ and TEA on single Ca2+-activated K+-channels in arterial and intestinal smooth muscle cell membranes

The interaction of Ba²⁺ and TEA with Ca²⁺-activated K⁺ channels was studied in isolated membrane patches of cells from longitudinal jejunal smooth muscle of rabbit and from guinea-pig small mesenteric artery (100 m external diameter). Ba²⁺ applied from the inside of the membrane did not reduce unit current, except at high concentrations, but channels failed to open for long periods (s). This effect became much stronger when the potential gradient was in a direction driving Ba²⁺ into the channel and was reduced by increasing K⁺ ion concentration on the outside of the membrane. These results are consistent with Ba²⁺ entering the open channel and blocking at a site most of the way through the channel bore. In contrast, TEA and procaine dose-dependently reduced unit current amplitude at all patch potentials and slightly increased mean open time. Their effects were not detectably voltage-dependent and could be explained by TEA and procaine blocking the open channel with a timecourse that was faster than the frequency response of the recording system. The lack of appreciable voltage-dependence suggests that TEA and procaine bind to a site near to the inner mouth of the channel.     

慢性吸烟大鼠肺动脉平滑肌细胞钾通道Kv1.5、BKCa表达的变化

目的：观察吸烟对大鼠肺动脉平滑肌大电导的钙激活的钾通道（BK_Ca）和电压依赖性延迟整流钾通道Kv1.5蛋白和mRNA表达的影响，以阐明吸烟引起的肺血管反应性改变中钾通道表达的变化。方法：复制大鼠的慢性吸烟模型，采用HE染色、免疫组织化学染色、原位杂交等方法。结果：（1）慢性吸烟可降低大鼠肺动脉平滑肌 BK_Ca 蛋白和mRNA表达；（2）慢性吸烟可降低大鼠肺动脉平滑肌Kv1.5蛋白和mRNA表达；（3）大动脉 BK_Ca的降低程度大于Kv1.5，小动脉 BK_Ca和Kv1.5的降低程度无明显差异。结论：慢性吸烟可下调大鼠肺动脉平滑肌钾通道 BK_Ca和Kv1.5的表达水平，是导致肺血管反应性增高的机制之一。     

Variations of membrane cholesterol alter the kinetics of Ca2+-dependent K+ channels and membrane fluidity in vascular smooth muscle cells

The patch-clamp technique and fluorescence polarization analysis were used to study the dependence of Ca²⁺-dependent K⁺ channel kinetics and membrane fluidity on cholesterol (CHS) levels in the plasma membranes of cultured smooth muscle rabbit aortic cells. Mevinolin (MEV), a potent inhibitor of endogenous CHS biosynthesis was used to deplete the CHS content. Elevation of CHS concentration in the membrane was achieved using a CHS-enriching medium. Treatment of smooth muscle cells with MEV led to a nearly twofold increase in the rotational diffusion coefficient of DPH (D) and to about a ninefold elevation of probability of the channels being open (P _o). The addition of CHS to the cells membrane resulted in a nearly twofold decrease in D and about a twofold decrease in P _o. Elementary conductance of the channels did not change under these conditions. These data suggest that variations of the CHS content in the plasma membrane of smooth muscle cells affect the kinetic properties of Ca²⁺-dependent K⁺ channels presumably due to changes in plasma membrane fluidity. Our results give a possible explanation for the reported variability of Ca²⁺-dependent K⁺ channels kinetics in different preparations.     

Ca2+ and K+ current in cultured vascular smooth muscle cells from rat aorta

Ca²⁺ (I_Ca) and K⁺ (I_K) currents were recorded in single cultured cells from rat aorta using the whole cell clamp technique with patch electrodes. I_Ca was detected at–30 mV, and at 20 mV it reached a peak in about 10 ms and decayed with a t_1/2=50 ms. The mean maximum slope conductance (G_Ca) was 30 S/cm². I_K was detected at–10 mV and at 20 mV reached its maximum with a t_1/2=12 ms. For I_K, G_K=200 S/cm². These channels can be activated during action potentials and play a role in the excitation and contraction of vascular smooth muscle cells.Doctoral training program UAM-1     

Intracellular Na+ modulates large conductance Ca2+-activated K+ currents in human umbilical vein endothelial cells

We studied the effects of Na⁺ influx on large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels in cultured human umbilical vein endothelial cells (HUVECs) by means of patch clamp and SBFI microfluorescence measurements. In current-clamped HUVECs, extracellular Na⁺ replacement by NMDG⁺ or mannitol hyperpolarized cells. In voltage-clamped HUVECs, changing membrane potential from 0 mV to negative potentials increased intracellular Na⁺ concentration ([Na⁺]_i) and vice versa. In addition, extracellular Na⁺ depletion decreased [Na⁺]_i. In voltage-clamped cells, BK_Ca currents were markedly increased by extracellular Na⁺ depletion. In inside-out patches, increasing [Na⁺]_i from 0 to 20 or 40 mM reduced single channel conductance but not open probability (NPo) of BK_Ca channels and decreasing intracellular K⁺ concentration ([K⁺]_i) gradually from 140 to 70 mM reduced both single channel conductance and NPo. Furthermore, increasing [Na⁺]_i gradually from 0 to 70 mM, by replacing K⁺, markedly reduced single channel conductance and NPo. The Na⁺–Ca²⁺ exchange blocker Ni²⁺ or KB-R7943 decreased [Na⁺]_i and increased BK_Ca currents simultaneously, and the Na⁺ ionophore monensin completely inhibited BK_Ca currents. BK_Ca currents were significantly augmented by increasing extracellular K⁺ concentration ([K⁺]_o) from 6 to 12 mM and significantly reduced by decreasing [K⁺]_o from 12 or 6 to 0 mM or applying the Na⁺–K⁺ pump inhibitor ouabain. These results suggest that intracellular Na⁺ inhibit single channel conductance of BK_Ca channels and that intracellular K⁺ increases single channel conductance and NPo. GH Liang and MY Kim contributed equally to this publication and therefore share the first authorship.     

Different modulation of Ca-activated K channels by the intracellular redox potential in pulmonary and ear arterial smooth muscle cells of the rabbit

Astract We investigated the electrical responses of Ca-activated K (KCa) currents induced by hypoxia and reduction or oxidation of the channel protein in pulmonary (PASMC) and ear (EASMC) arterial smooth muscle cells using the patch-clamp technique. In cell-attached patches, in the presence of a high K solution (containing 0.316 (M Ca²⁺), the activity of K_Ca channels from PASMC was decreased (by 49±7% compared to control, pipette potential = –70 mV) by changing to a hypoxic solution (1 mM Na₂S₂O₄, aeration with 100% N₂ gas). EASMC channels did not respond to hypoxia. In order to investigate the possible mechanisms involved, using inside-out patches bathed symmetrically in 150 mM KCl, we applied redox couples to the intracellular side. Reducing agents, such as dithiothreitol (DDT, 5 mM), reduced glutathione, (GSH, 5 mM), and nicotinamide adenine dinucleotide reduced (NADH, 2 mM) decreased PASMC, but not EASMC, K_Ca channel activity. However, oxidizing agents such as 5,5-dithio-bis(2-nitrobenzoic acid) (DTNB, 1 mM), oxidized glutathione (GSSG, 5 mM) and NAD (2 mM) increased K_Ca channel activity in both PASMC and EASMC. The increased activity due to oxidizing agents was restored by applying reducing agents. From these results, we could suggest that the basal redox state of the EASMC K_Ca channel is more reduced than that of the PASMC channel, since the response of K_Ca channels of the EASMC to intracellular reducing agents differs from that of the PASMC. This difference may be related to the different responses of PASMC and EASMC K_Ca channels to hypoxia.