Nitric oxide directly activates large conductance Ca2+-activated K+ channels (rSlo)

We investigated whether nitric oxide (NO) directly activates the cloned alpha-subunit of large conductance Ca2+-activated K+ (Maxi-K) channels from rat brain (rSlo), expressed either in HEK293 cells or Xenopus oocytes. In inside-out patches, the application of S-nitroso-N-acetylpenicillamine (SNAP), a NO-releasing compound, reversibly activated the channel shifting the voltage dependent activation curve of the macroscopic Maxi-K current to the left by about 15 mV. Pretreatment of the patches with N-ethylmaleimide to alkylate free sulfhydryl groups did not prevent the effect of SNAP, suggesting that NO may directly interact with the channels. These results suggest that Maxi-K channels might be one of the physiological targets of NO in the brain.     

Gamma-dendrotoxin blocks large conductance Ca2+-activated K+ channels in neuroblastoma cells

In N1E 115 neuroblastoma cells, gamma-dendrotoxin (DTX, 200 nM) blocked the outward K(+) current by 31.1 +/- 3.5% (n = 4) with approximately 500 nM Ca(2+) in the pipet solution, but had no effect on the outward K(+) current when internal Ca(2+) was reduced. Using a ramp protocol, iberiotoxin (IbTX, 100 nM) inhibited a component of the whole cell current, but in the presence of 200 nM gamma-DTX, no further inhibition by IbTX was observed. Two types of single channels were seen using outside-out patches when the pipette free Ca(2+) concentration was approximately 500 nM; a 63 pS and a 187 pS channel. The 63 pS channel was TEA-, IbTX- and gamma-DTX-insensitive, while the 187 pS channel was blocked by 1 mM TEA, 100 nM IbTX or 200 nM gamma-DTX. Both channels were activated by external application of ionomycin, when the pipet calcium concentration was reduced. gamma-DTX (200 nM) reduced the probability of openings of the 187 pS channel, with an IC(50) of 8.5 nM. In GH(3) cells gamma-DTX (200 nM) also blocked an IbTX-sensitive component of whole-cell K(+) currents. These results suggest that gamma-DTX blocks a large conductance Ca(2+) activated K(+) current in N1E 115 cells. This is the first indication that any of the dendrotoxins, which have classically been known to block voltage-gated (Kv) channels, can also block Ca(2+) activated K(+) channels.     

Natural bile acids and synthetic analogues modulate large conductance Ca2+-activated K+ (BKCa) channel activity in smooth muscle cells

Bile acids have been reported to produce relaxation of smooth muscle both in vitro and in vivo. The cellular mechanisms underlying bile acid-induced relaxation are largely unknown. Here we demonstrate, using patch-clamp techniques, that natural bile acids and synthetic analogues reversibly increase BK(Ca) channel activity in rabbit mesenteric artery smooth muscle cells. In excised inside-out patches bile acid-induced increases in channel activity are characterized by a parallel leftward shift in the activity-voltage relationship. This increase in BK(Ca) channel activity is not due to Ca(2+)-dependent mechanism(s) or changes in freely diffusible messengers, but to a direct action of the bile acid on the channel protein itself or some closely associated component in the cell membrane. For naturally occurring bile acids, the magnitude of bile acid-induced increase in BK(Ca) channel activity is inversely related to the number of hydroxyl groups in the bile acid molecule. By using synthetic analogues, we demonstrate that such increase in activity is not affected by several chemical modifications in the lateral chain of the molecule, but is markedly favored by polar groups in the side of the steroid rings opposite to the side where the methyl groups are located, which stresses the importance of the planar polarity of the molecule. Bile acid-induced increases in BK(Ca) channel activity are also observed in smooth muscle cells freshly dissociated from rabbit main pulmonary artery and gallbladder, raising the possibility that a direct activation of BK(Ca) channels by these planar steroids is a widespread phenomenon in many smooth muscle cell types. Bile acid concentrations that increase BK(Ca) channel activity in mesenteric artery smooth muscle cells are found in the systemic circulation under a variety of human pathophysiological conditions, and their ability to enhance BK(Ca) channel activity may explain their relaxing effect on smooth muscle.     

Large-conductance Ca2+-activated K+ channels in freshly dissociated smooth muscle cells

Freshly dissociated cells from the stomach muscularis of the toad Bufo marinus have been employed to carry out a systematic set of electrophysiological studies on the membrane properties of smooth muscle. The existence of Ca2+-activated K+ channels became apparent during the first studies under current clamp. In subsequent studies under voltage clamp, a Ca2+-activated. TEA-sensitive outward current was evident, and it was more than an order of magnitude larger than any other current observed in the cells. The channel responsible, at least in part, for this large outward current has been identified on the basis of single-channel records, and some of its main characteristics have been studied. It is similar in many respects to the large-conductance, Ca2+-activated K+ channel seen in other preparations. This channel has now been found in a considerable diversity of smooth muscle types.     

Functional and molecular consequences of ionizing irradiation on large conductance Ca2+-activated K+ channels in rat aortic smooth muscle cells

AimsThe goal of this study was to evaluate the influence of γ-irradiation on Ca²⁺-activated K⁺ channel (BK_Ca) function and expression in rat thoracic aorta.Main methodsAortic cells or tissues were studied by the measurement of force versus [Ca²⁺]_i, patch-clamp technique, and RT-PCR.Key findingsStimulation of smooth muscle cells with depolarizing voltage steps showed expression of outward K⁺ currents. Paxilline, an inhibitor of BK_Ca channels, decreased outward K⁺ current density. Outward currents in smooth muscle cells obtained from irradiated animals 9 and 30 days following radiation exposure demonstrated a significant decrease in K⁺ current density. Paxilline decreased K⁺ current in cells obtained 9 days, but was without effect 30 days after irradiation suggesting the absence of BK_Ca channels. Aortic tissue from irradiated animals showed progressively enhanced contractile responses to phenylephrine in the post-irradiation period of 9 and 30 days. The concomitant Ca²⁺ transients were significantly smaller, as compared to tissues from control animals, 9 days following irradiation but were increased above control levels 30 days following irradiation. Irradiation produced a decrease in BK_Ca α- and β₁-subunit mRNA levels in aortic smooth muscle cells suggesting that the vasorelaxant effect of these channels may be diminished.SignificanceThese results suggest that the enhanced contractility of vascular tissue from animals exposed to radiation may result from an increase in myofilament Ca²⁺ sensitivity in the early post-irradiation period and a decrease in BK_Ca channel expression in the late post-irradiation period.     

A lineage-specific Ca(2+)-activated K+ conductance in HL-60 cells.

Cells of the human promyelocytic cell line HL-60 can be controllably induced to terminally differentiate into either granulocytes or monocyte/macrophages. HL-60 promyelocytes and terminally differentiated macrophages express a K(+)-selective ion channel which is activated by intracellular free Ca2+ concentrations above 10(-7) M. Because of its voltage independence, this channel can be distinguished from the voltage- and Ca(2+)-activated family of outward-rectifying channels. The channel is selective for K+ against Na+ and is blocked by Ba2+, thus it may be similar to the Ca(2+)-activated K+ channel previously described in human macrophages. In its sensitivity to block by charybdotoxin, this channel also resembles a Ca(2+)-activated K+ channel of lymphocytes, which plays a role in activation-dependent hyperpolarization. In contrast to promyelocytes and macrophages, functional expression of the Ca(2+)-activated K+ channel is suppressed to nearly undetectable levels in granulocytes derived from HL-60 cells by retinoic acid-induced differentiation. These data suggest that signals which produce elevation of intracellular Ca2+ will hyperpolarize promyelocytes and differentiated macrophages by activating this conductance; however, signals which elevate free Ca2+ in granulocytes must act on other effectors, which may produce a different final influence on membrane potential.     

ATP inhibits smooth muscle Ca2(+)-activated K+ channels

There has been much recent interest in the roles played by smooth-muscle K+ channels in protecting cells against ischemic and anoxic insults and in therapeutic vaso- and bronchodilation (Buckingham 1990; Longmore & Weston 1990). A K+ channel, which is uniquely sensitive to cytoplasmic ATP (KATP), has been identified as a likely candidate for mediating these important functions (Standen et al. 1989). We now show, by using electrophysiological techniques in three different types of smooth muscle, that a large-conductance voltage and Ca2(+)-sensitive channel, otherwise indistinguishable from the the large-conductance Ca2(+)-activated K+ channel (BK channel), is also sensitive to cytoplasmic ATP and cromakalim. ATP, in a dose-dependent manner, decreased the probability of channel opening (Po) of rabbit aortic, rabbit tracheal and pig coronary artery BK channels with a Ki of 0.2-0.6 mM. Cromakalim, 10 microM, partially reversed the ATP induced inhibition and increased Po. Our observations raise the possibility that the ubiquitous BK channel may play a role during pathophysiological events.     

pH effects on high conductance Ca2+-activated K+ channels (BK(Ca)) in human internal mammary artery smooth muscle cells

INTRODUCTION: In vascular smooth muscle cells, different types of K+ channels participate in the regulation of membrane potential and consequently in the contractile behavior of the vessel. There is little information about the properties and role of K+ channels in human internal mammary artery (HIMA), the vessel of choice for coronary revascularization. METHODS: Patch-clamp technique on isolated HIMA smooth muscle cells was used. RESULTS: This work presents for the first time single-channel properties of the high conductance Ca2+-activated K+ channel (BK(Ca)) of HIMA. It presents a single-channel conductance of 228+/-4 pS (n=44, 8 cells), is sensitive to 100 nM iberiotoxin, and its open probability is Ca2+- and voltage-dependent. Inside-out results show that BK(Ca) channels in HIMA are directly activated by increasing the pH of intracellular media (NPo=0.096+/-0.032 at pH 7.4 and NPo=0.459+/-0.111 at pH 7.6, n=12 cells, p<0.05) and inhibited by lowering this pH (NPo=0.175+/-0.067 at pH 7.4 and NPo=0.051+/-0.019 at pH 6.8, n=13 cells, p<0.05). CONCLUSIONS: The evidences presented about single-channel properties and intracellular pH sensitivity of BK(Ca) from HIMA smooth muscle cells provide useful information to elucidate physiological or pathological mechanisms in this vessel, as well as for future studies where drugs could have BK(Ca) channels as targets for pharmacological therapies.     

Ca(2+)-activated K+ channels in human leukemic T cells

Using the patch-clamp technique, we have identified two types of Ca(2+)-activated K+ (K(Ca)) channels in the human leukemic T cell line. Jurkat. Substances that elevate the intracellular Ca2+ concentration ([Ca2+]i), such as ionomycin or the mitogenic lectin phytohemagglutinin (PHA), as well as whole-cell dialysis with pipette solutions containing elevated [Ca2+]i, activate a voltage-independent K+ conductance. Unlike the voltage-gated (type n) K+ channels in these cells, the majority of K(Ca) channels are insensitive to block by charybdotoxin (CTX) or 4-aminopyridine (4-AP), but are highly sensitive to block by apamin (Kd less than 1 nM). Channel activity is strongly dependent on [Ca2+]i, suggesting that multiple Ca2+ binding sites may be involved in channel opening. The Ca2+ concentration at which half of the channels are activated is 400 nM. These channels show little voltage dependence over a potential range of -100 to 0 mV and have a unitary conductance of 4-7 pS in symmetrical 170 mM K+. In the presence of 10 nM apamin, a less prevalent type of K(Ca) channel with a unitary conductance of 40-60 pS can be observed. These larger-conductance channels are sensitive to block by CTX. Pharmacological blockade of K(Ca) channels and voltage-gated type n channels inhibits oscillatory Ca2+ signaling triggered by PHA. These results suggest that K(Ca) channels play a supporting role during T cell activation by sustaining dynamic patterns of Ca2+ signaling.     

Suppression of human detrusor smooth muscle excitability and contractility via pharmacological activation of large conductance Ca2+-activated K+ channels

Overactive bladder syndrome is frequently associated with increased detrusor smooth muscle (DSM) contractility. We tested the hypothesis that pharmacological activation of the large-conductance voltage- and Ca(2+)-activated K(+) (BK) channel with NS-1619, a selective BK channel opener, reduces the excitability and contractility of human DSM. We used the amphotericin-perforated whole cell patch-clamp technique on freshly isolated human DSM cells, live-cell Ca(2+) imaging, and isometric DSM tension recordings of human DSM strips obtained from open bladder surgeries. NS-1619 (30 μM) significantly increased the amplitude of the voltage step-induced whole cell BK currents, and this effect was abolished by pretreatment with 200 nM iberiotoxin (IBTX), a selective BK channel inhibitor. In current-clamp mode, NS-1619 (30 μM) significantly hyperpolarized the resting membrane potential, and the hyperpolarization was reversed by IBTX (200 nM). NS-1619 (30 μM) significantly decreased the intracellular Ca(2+) level in isolated human DSM cells. BK channel activation with NS-1619 (30 μM) significantly inhibited the amplitude, muscle force, frequency, duration, and tone of the spontaneous phasic and pharmacologically induced DSM contractions from human DSM isolated strips. IBTX (200 nM) suppressed the inhibitory effects of NS-1619 on spontaneous contractions. The amplitude of electrical field stimulation (0.5-50 Hz)-induced contractions was significantly reduced by NS-1619 (30 μM). Our data suggest that pharmacological activation of BK channels could represent a novel treatment option to control bladder dysfunction in humans.     

Ketamine blocks Ca2+-activated K+ channels in rabbit cerebral arterial smooth muscle cells

Although ketamine and Ca2+-activated K+ (KCa) channels have been implicated in the contractile activity regulation of cerebral arteries, no studies have addressed the specific interactions between ketamine and the KCa channels in cerebral arteries. The purpose of this study was to examine the direct effects of ketamine on KCa channel activities using the patch-clamp technique in single-cell preparations of rabbit middle cerebral arterial smooth muscle. We tested the hypothesis that ketamine modulates the KCa channel activity of the cerebral arterial smooth muscle cells of the rabbit. Vascular myocytes were isolated from rabbit middle cerebral arteries using enzymatic dissociation. Single KCa channel activities of smooth muscle cells from rabbit cerebral arteries were recorded using the patch-clamp technique. In the inside-out patches, ketamine in the micromolar range inhibited channel activity with a half-maximal inhibition of the ketamine concentration value of 83.8 +/- 12.9 microM. The Hill coefficient was 1.2 +/- 0.3. The slope conductance of the current-voltage relationship was 320.1 +/- 2.0 pS between 0 and +60 mV in the presence of ketamine and symmetrical 145 mM K+. Ketamine had little effect on either the voltage-dependency or open- and closed-time histograms of KCa channel. The present study clearly demonstrates that ketamine inhibits KCa channel activities in rabbit middle cerebral arterial smooth muscle cells. This inhibition of KCa channels may represent a mechanism for ketamine-induced cerebral vasoconstriction.     

Peroxynitrite reversibly inhibits Ca(2+)-activated K(+) channels in rat cerebral artery smooth muscle cells

Peroxynitrite (ONOO(-)) is a contractile agonist of rat middle cerebral arteries. To determine the mechanism responsible for this component of ONOO(-) bioactivity, the present study examined the effect of ONOO(-) on ionic current and channel activity in rat cerebral arteries. Whole cell recordings of voltage-clamped cells were made under conditions designed to optimize K(+) current. The effects of iberiotoxin, a selective inhibitor of large-conductance Ca(2+)-activated K(+) (BK) channels, and ONOO(-) (10-100 microM) were determined. At a pipette potential of +50 mV, ONOO(-) inhibited 39% of iberiotoxin-sensitive current. ONOO(-) was selective for iberiotoxin-sensitive current, whereas decomposed ONOO(-) had no effect. In excised, inside-out membrane patches, channel activity was recorded using symmetrical K(+) solutions. Unitary currents were sensitive to increases in internal Ca(2+) concentration, consistent with activity due to BK channels. Internal ONOO(-) dose dependently inhibited channel activity by decreasing open probability and mean open times. The inhibitory effect of ONOO(-) could be overcome by reduced glutathione. Glutathione, added after ONOO(-), restored whole cell current amplitude to control levels and reverted single-channel gating to control behavior. The inhibitory effect of ONOO(-) on membrane K(+) current is consistent with its contractile effects in isolated cerebral arteries and single myocytes. Taken together, our data suggest that ONOO(-) has the potential to alter cerebral vascular tone by inhibiting BK channel activity.     

Big-conductance Ca2+-activated K+ channels in physiological and pathophysiological urinary bladder smooth muscle cells

Contraction and relaxation of urinary bladder smooth muscle cells (UBSMCs) represent the important physiological functions of the bladder. Contractile responses in UBSMCs are regulated by a number of ion channels including big-conductance Ca²⁺- activated K⁺ (BK) channels. Great progress has been made in studies of BK channels in UBSMCs. The intent of this review is to summarize recent exciting findings with respect to the functional interactions of BK channels with muscarinic receptors, ryanodine receptors (RyRs) and inositol triphosphate receptors (IP₃Rs) as well as their functional importance under normal and pathophysiological conditions. BK channels are highly expressed in UBSMCs. Activation of muscarinic M₃ receptors inhibits the BK channel activity, facilitates opening of voltage-dependent Ca²⁺ (Ca_V) channels, and thereby enhances excitability and contractility of UBSMCs. Signaling molecules and regulatory mechanisms involving RyRs and IP₃Rs have a significant effect on functions of BK channels and thereby regulate cellular responses in UBSMCs under normal and pathophysiological conditions including overactive bladders. Moreover, BK channels may represent a novel target for the treatment of bladder dysfunctions.     

In situ characterization of the Ca2+ sensitivity of large conductance Ca2+-activated K+ channels: implications for their use as near-membrane Ca2+ indicators in smooth muscle cells.

The Ca2+ sensitivity of large conductance Ca2+- and voltage-activated K+ channels (BKV,Ca) has been determined in situ in freshly isolated myocytes from the guinea pig urinary bladder. In this study, in situ denotes that BKV,Ca channel activity was recorded without removing the channels from the cell. By combining patch clamp recording in the cell-attached configuration and microfluorometry of fura-2, we were able to correlate BKV,Ca channel activity with changes in cytoplasmic intracellular [Ca2+] ([Ca2+]i). The latter were induced by ionomycin, an electroneutral Ca2+ ionophore. At 0 mV, the Hill coefficient (nH) and the [Ca2+]i to attain half of the maximal BKV,Ca channel activity (Ca50) were 8 and 1 microM, respectively. The data suggest that this large Hill number was not a consequence of the difference between the near-membrane [Ca2+] ([Ca2+]s) and the bulk [Ca2+]i, indicated by fura-2. High Hill numbers in the activation by Ca2+ of BKV,Ca channels have been seen by different groups (e.g., filled squares in Fig. 4 of Silberberg, S. D., A. Lagrutta, J. P. Adelman, and K. L. Magleby. 1996. Biophys. J. 70:2640-2651). However, such high nH has always been considered a peculiarity rather than the rule. This work shows that a high Ca2+ cooperativity is the normal situation for BKV,Ca channels in myocytes from guinea pig urinary bladder. Furthermore, the Ca50 did not display any significant variation among different channels or cells. It was also evident that BKV,Ca channel activity could decrease in elevated [Ca2+]i, either partially or completely. This work implies that the complete activation of BKV,Ca channels occurs with a smaller increment in [Ca2+]s than previously expected from in vitro characterization of the Ca2+ sensitivity of these channels. Additionally, it appears that the activity of BKV,Ca channels in situ does not strictly follow changes in near-membrane [Ca2+].     

High extracellular Ca2+ hyperpolarizes human parathyroid cells via Ca(2+)-activated K+ channels

Membrane potential has a major influence on stimulus-secretion coupling in various excitable cells. The role of membrane potential in the regulation of parathyroid hormone secretion is not known. High K+-induced depolarization increases secretion from parathyroid cells. The paradox is that increased extracellular Ca2+, which inhibits secretion, has also been postulated to have a depolarizing effect. In this study, human parathyroid cells from parathyroid adenomas were used in patch clamp studies of K+ channels and membrane potential. Detailed characterization revealed two K+ channels that were strictly dependent of intracellular Ca2+ concentration. At high extracellular Ca2+, a large K+ current was seen, and the cells were hyperpolarized (-50.4 +/- 13.4 mV), whereas lowering of extracellular Ca2+ resulted in a dramatic decrease in K+ current and depolarization of the cells (-0.1 +/- 8.8 mV, p < 0.001). Changes in extracellular Ca2+ did not alter K+ currents when intracellular Ca2+ was clamped, indicating that K+ channels are activated by intracellular Ca2+. The results were concordant in cell-attached, perforated patch, whole-cell and excised membrane patch configurations. These results suggest that [Ca2+]o regulates membrane potential of human parathyroid cells via Ca2+-activated K+ channels and that the membrane potential may be of greater importance for the stimulus-secretion coupling than recognized previously.     

MCI-154 activates the Ca2+-activated K+ channel of vascular smooth muscle cells

The aim of this study was to examine the effects of MCI-154, a new positive inotropic agent with vasodilating properties, on the Ca²⁺-activated K⁺ channel (K_Ca channel) of vascular smooth muscle cells. Cultured smooth muscle cells from a porcine coronary artery were studied using the patch-clamp technique. Extracellular application of 100 μM MCI-154 activated the K_Ca channel in intact cell-attached patch configurations. In excised inside-out patch configurations, application of 100μM MCI-154 to the cytosolic side activated the K_Ca channel directly, suggesting that the Ca₂₊ sensitivity of the K_Ca channel itself is modulated. Though extracellular application of 100 μM amrinone, a phosphodiesterase inhibitor, activated the K_Ca channel in the cell-attached patch configurations, application of 100 μm amrinone to the cytosolic side could not activate the K_Ca channel in inside-out patch configurations. These results indicate that different from amrinone, MCI-154 can modulate Ca²⁺ sensitivity of the K_Ca channel in vascular smooth muscle cells.