Calcium-independent cell volume regulation in human lymphocytes. Inhibition by charybdotoxin

The properties of the K+ pathway underlying regulatory volume decrease (RVD) in human blood lymphocytes were investigated. Evidence is presented for the existence of three types of K+ conductance in these cells. Ionomycin, a Ca2+ ionophore, induced a K(+)-dependent hyperpolarization, indicating the presence of Ca2(+)-activated K+ channels, which were blocked by charybdotoxin (CTX). CTX also induced a depolarization of the resting membrane potential, even at subphysiological cytosolic [Ca2+]([Ca2+]i), which suggests the existence of a second CTX-sensitive, but Ca2(+)-independent conductance. A CTX-resistant K+ conductance was also detected. RVD in blood lymphocytes was partially (approximately 75%) blocked by CTX. However, volume regulation was not accompanied by detectable changes in [Ca2+]i, nor was it prevented by removal of extracellular Ca2+ and depletion or buffering of intracellular Ca2+. These observations suggest that K+ loss during RVD is mediated by Ca2(+)-independent, CTX-sensitive channels or that Ca2(+)-dependent channels can be activated by cell swelling at normal or subnormal [Ca2+]i. The former interpretation is supported by findings in rat thymic lymphocytes. These cells also displayed a CTX-sensitive Ca2(+)-dependent hyperpolarization. However, CTX did not significantly alter the resting potential, suggesting the absence of functional Ca2(+)-independent, toxin-sensitive channels. Volume regulation in thymic lymphocytes was less efficient than in human blood cells. In contrast to blood lymphocytes, RVD in thymocytes was not affected by CTX. These observations indicate that, though present in lymphocytes, Ca2(+)-activated K+ channels do not play an important role in volume regulation. Instead, RVD seems to be mediated by Ca2(+)-independent K+ channels. We propose that two types of channels, one CTX sensitive and the other CTX insensitive, mediate RVD in human blood lymphocytes, whereas only the latter type is involved in rat thymocytes.     

BKCa channels activating at resting potential without calcium in LNCaP prostate cancer cells

Large-conductance Ca2+-dependent K+ (BK(Ca)) channels are activated by intracellular Ca2+ and membrane depolarization in an allosteric manner. We investigated the pharmacological and biophysical characteristics of a BK(Ca)-type K+ channel in androgen-dependent LNCaP (lymph node carcinoma of the prostate) cells with novel functional properties, here termed BK(L). K+ selectivity, high conductance, activation by Mg2+ or NS1619, and inhibition by paxilline and penitrem A largely resembled the properties of recombinant BK(Ca) channels. However, unlike conventional BK(Ca) channels, BK(L) channels activated in the absence of free cytosolic Ca2+ at physiological membrane potentials; the half-maximal activation voltage was shifted by about -100 mV compared with BK(Ca) channels. Half-maximal Ca2+-dependent activation was observed at 0.4 microM: for BK(L) (at -20 mV) and at 4.1 microM: for BK(Ca) channels (at +50 mV). Heterologous expression of hSlo1 in LNCaP cells increased the BK(L) conductance. Expression of hSlo-beta1 in LNCaP cells shifted voltage-dependent activation to values between that of BK(L) and BK(Ca) channels and reduced the slope of the P (open) (open probability)-voltage curve. We propose that LNCaP cells harbor a so far unknown type of BK(Ca) subunit, which is responsible for the BK(L) phenotype in a dominant manner. BK(L)-like channels are also expressed in the human breast cancer cell line T47D. In addition, functional expression of BK(L) in LNCaP cells is regulated by serum-derived factors, however not by androgens.     

Properties of Mg(2+)-dependent cation channels in human leukemia K562 cells

The endogenous Mg(2+)-inhibited cation (MIC) current was recently described in different cells of hematopoietic lineage and was implicated in the regulation of Mg2+ homeostasis. Here we present a single channel study of endogenously expressed Mg(2+)-dependent cation channels in the human myeloid leukemia K562 cells. Inwardly directed unitary currents were activated in cell-attached experiments in the absence of Ca2+ and Mg2+ in the pipette solution. The current-voltage (I-V) relationships displayed strong inward rectification and yielded a single channel slope conductance of approximately 30 pS at negative potentials. The I-V relationships were not altered by patch excision into divalent-free solution. Channel open probability (P(o)) and mean closed time constant (tau(C)) were strongly voltage-dependent, indicating that gating mechanisms may underlie current inward rectification. Millimolar concentrations of Ca2+ or Mg2+ applied to the cytoplasmic side of the membrane produced slow irreversible inhibition of channel activity. The Mg(2+)-dependent cation channels described in this study differ from the MIC channels described in human T-cells, Jurkat, and rat basophilic leukemia (RBL) cells in their I-V relationships, kinetic parameters and dependence on intracellular divalent cations. Our results suggested that endogenously expressed Mg(2+)-dependent cation channels in K562 cells and the MIC channels in other hematopoietic cells might be formed by different channel proteins.     

A peptide derived from the Shaker B K+ channel produces short and long blocks of reconstituted Ca(2+)-dependent K+ channels.

A 20 amino acid synthetic peptide, corresponding to the amino-terminal region of the Shaker B (ShB) K+ channel and responsible for its fast inactivation, can block large conductance Ca(2+)-dependent K+ channels from rat brain and muscle. The ShB inactivation peptide produces two kinetically distinct blocking events in these channels. At lower concentrations, it produces short blocks, and at higher concentrations long-lived blocks also appear. The L7E mutant peptide produces only infrequent short blocks (no long-lived blocks) at a much higher concentration. Internal tetraethylammonium competes with the peptide for the short block, which is also relieved by K+ influx. These results suggest that the peptide induces the short block by binding within the pore of Ca(2+)-dependent K+ channels. The long block is not affected by increased K+ influx, indicating that the binding site mediating this block may be different from that involved in the short block. The short block of Ca(2+)-dependent K+ channels and the inactivation of Shaker exhibit similar characteristics with respect to blocking affinity and open pore blockade. This suggests a conserved binding region for the peptide in the pore regions of these very different classes of K+ channel.     

Ghrelin suppression of potassium currents in smooth muscle cells of human mesenteric artery

Ghrelin is a 28-amino acid peptide hormone which modulates many physiological functions including cardiovascular homeostasis. Here we report some novel findings about the action of ghrelin on smooth muscle cells (SMC) freshly isolated from human mesenteric arteries. Ghrelin (10(-7) mol/l) significantly suppressed the iberiotoxin-blockable component of potassium currents (I(K)) and depolarized the cell membrane, while having no effect on Ca(2+) currents. Inhibition of inositol-trisphosphate (IP(3))-activated Ca(2+) release channels, depletion of sarcoplasmic reticulum (SR) Ca(2+) stores, blockade of phospholipase D (PLD) or protein kinase C (PKC) each abolished the effect of ghrelin on I(K), while the inhibition of phospholipase C (PLC) did not. These data imply that in human mesenteric artery SMC ghrelin suppresses I(K) via PLD, PKC and SR Ca(2+)-dependent signaling pathway.     

Calcium-dependent,swelling-activated K+ conductance in human neuroblastoma cells

In most mammalian cells, regulatory volume decrease (RVD) is mediated by swelling-activated Cl(-) and K(+) channels. Previous studies in the human neuroblastoma cell line CHP-100 have demonstrated that exposure to hypoosmotic solutions activates Cl(-) channels which are sensitive to Ca(2+). Whether a Ca(2+)-dependent K(+) conductance is activated after cell swelling was investigated in the present studies. Reducing the extracellular osmolarity from 290 to 190 mOsm/kg H(2)O rapidly activated 86Rb effluxes. Hypoosmotic stress also increased cytosolic Ca(2+) in fura-2 loaded cells. Pretreatment with 2.5 mM EGTA and nominally Ca(2+) free extracellular solution significantly decreased the hypoosmotically induced rise in cytosolic Ca(2+) and the swelling-activated 86Rb efflux. In cell-attached patch-clamp studies, decreasing the extracellular osmolarity activated a K(+) conductance that was blocked by Ba(2+). In addition, the swelling-activated K(+) channels were significantly inhibited in the presence of nominally free extracellular Ca(2+) and 2.5mM EGTA. These results suggest that in response to hypoosmotic stress, a Ca(2+)-dependent K(+) conductance is activated in the human neuroblastoma cell line CHP-100.     

Calcium-activated chloride conductance of lactotrophs: Comparison of activation in normal and tumoral cells during thyrotropin-releasing-hormone stimulation

We studied a chloride (Cl-) conductance activated by calcium (Ca2+) in normal rat lactotrophs and compared its activation during TRH stimulation in normal rat lactotrophs and in GH3 tumoral lactosomatotrophs cells, using the whole-cell configuration of the patch-clamp technique. The Cl- specificity of the conductance was assessed by manipulation of internal and external Cl- concentrations. The reversal potentials were in agreement with those predicted by the Nernst equation. Ca2+ ionophore A23187 and membrane depolarizations activated the Cl- conductance. However, a feedback effect of Cl- gradient modifications on Ca2+ movements was also observed in normal lactotrophs. In the latter, TRH (100 nM) mobilization of intracellular Ca2+ activated this Cl- conductance together with the potassium (K+) conductance when both ions were present in the intracellular medium (IM) or alone when K+ was absent. Chloride conductance was not activated in the GH3 cells, where mobilization of intracellular Ca2+ by TRH (100 nM) activated only Ca2(+)-dependent K+ conductance. It seems likely that the activation of Cl- conductance in these two different cell types involves different mechanisms.     

槲皮素填充的脂质体对电离辐射处理的大鼠胸主动脉平滑肌细胞上BK_(Ca)的作用(英文)

本文旨在研究槲皮素填充的卵磷脂脂质体(quercetin-filled phosphatidylcholine liposomes,PCL-Q)对非致死性全身电离辐射处理的大鼠胸主动脉平滑肌细胞(smooth muscle cells,SMCs)上的大电导钙离子依赖性钾通道(large conductance Ca2+-dependent K+channels,BKCa)的作用。我们使用膜片钳技术,观察到去极化阶跃脉冲刺激的SMCs上出现外向K+电流,该电流对paxilline(BKCa的一种特异性抑制剂)敏感,因此认为该电流主体为BKCa电流。相对正常对照大鼠,辐照处理大鼠SMCs的BKCa电流幅度明显减小。这种通道抑制效应在辐照后第9天表现显著,且在整个30天的实验期间是逐渐增强的。辐照处理大鼠的SMCs的血管舒张能力可能因此而减弱。在本实验中,PCL-Q有效地恢复了辐照后SMCs的BKCa功能。而PCL-Q的组分,单一的槲皮素或空白的脂质体(PCL)恢复BKCa功能的能力较二者的组合体有明显的减弱。上述结果表明,PCL-Q能恢复辐照处理后SMCs的BKCa正常功能。PCL-Q的保护能力可能来源于其抗氧化...     

K+ channel cAMP activated in guinea pig gallbladder epithelial cells

In guinea pig gallbladder epithelial cells, an increase in intracellular cAMP levels elicits the rise of anion channel activity. We investigated by patch-clamp techniques whether K(+) channels were also activated. In a cell-attached configuration and in the presence of theophylline and forskolin or 8-Br-cAMP in the cellular incubation bath, an increase of the open probability (P(o)) values for Ca(2+)-activated K(+) channels with a single-channel conductance of about 160 pS, for inward current, was observed. The increase in P(o) of these channels was also seen in an inside-out configuration and in the presence of PKA, ATP, and cAMP, but not with cAMP alone; phosphorylation did not influence single-channel conductance. In the inside-out configuration, the opioid loperamide (10(-5) M) was able to reduce P(o) when it was present either in the microelectrode filling solution or on the cytoplasmic side. Detection in the epithelial cells by RT-PCR of the mRNA corresponding to the alpha subunit of large-conductance Ca(2+)-activated K(+) channels (BK(Ca)) indicates that this gallbladder channel could belong to the BK family. Immunohistochemistry experiments confirm that these cells express the BK alpha subunit, which is located on the apical membrane. Other K(+) channels with lower conductance (40 pS) were not activated either by 8-Br-cAMP (cell-attached) or by PKA + ATP + cAMP (inside-out). These channels were insensitive to TEA(+) and loperamide. The data demonstrate that under conditions that induce secretion, phosphorylation activates anion channels as well as Ca(2+)-dependent, loperamide-sensitive K(+) channels present on the apical membrane.     

Swelling-activated Ca2+ entry via TRPV4 channel is defective in cystic fibrosis airway epithelia

The vertebrate transient receptor potential cationic channel TRPV4 has been proposed as an osmo- and mechanosensor channel. Studies using knock-out animal models have further emphasized the relevance of the TRPV4 channel in the maintenance of the internal osmotic equilibrium and mechanosensation. However, at the cellular level, there is still one important question to answer: does the TRPV4 channel generate the Ca(2+) signal in those cells undergoing a Ca(2+)-dependent regulatory volume decrease (RVD) response? RVD in human airway epithelia requires the generation of a Ca(2+) signal to activate Ca(2+)-dependent K(+) channels. The RVD response is lost in airway epithelia affected with cystic fibrosis (CF), a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator channel. We have previously shown that the defective RVD in CF epithelia is linked to the lack of swelling-dependent activation of Ca(2+)-dependent K(+) channels. In the present study, we show the expression of TRPV4 in normal human airway epithelia, where it functions as the Ca(2+) entry pathway that triggers the RVD response after hypotonic stress, as demonstrated by TRPV4 antisense experiments. However, cell swelling failed to trigger Ca(2+) entry via TRPV4 channels in CF airway epithelia, although the channel's response to a specific synthetic activator, 4 alpha-phorbol 12,13-didecanoate, was maintained. Furthermore, RVD was recovered in CF airway epithelia treated with 4 alpha-phorbol 12,13-didecanoate. Together, these results suggest that defective RVD in CF airway epithelia might be caused by the absence of a TRPV4-mediated Ca(2+) signal and the subsequent activation of Ca(2+)-dependent K(+) channels.     

Fatty acid modifies Ca2+-dependent potassium channel activity in smooth muscle cells from the human aorta

By using the patch-clamp technique the effect of 2-decenoic acid (DA) on Ca2+-activated potassium (K+) channels in the membrane of smooth muscle cells from the human aorta was studied. In the presence of 0.5 microM Ca2+ and 2 mM Mg2+ on the cytoplasmic side of the membrane, a more than tenfold elevation in the probability of the channels being open (po) was observed under the effect of DA. With divalent cation concentrations of less than 1 nM DA caused a more than twofold elevation in po. In the DA-treated membranes Mg2+ ions, which normally fail to activate the channels, brought about a nearly threefold increase in the channel activity when applied to the inner membrane surface. Channel sensitivity to the activating effect of cytoplasmic Ca2+ ions did not increase with the application of DA. Single-channel conductance was unchanged by DA exposure. We suggest that DA alters the Ca2+-binding mechanism of the channel, increasing its sensitivity to Mg2+ ions, presumably owing to membrane fluidization.     

Differential regulation of voltage- and calcium-activated potassium channels in human B lymphocytes.

The expression and characteristics of K+ channels of human B lymphocytes were studied by using single and whole-cell patch-clamp recordings. They were gated by depolarization (voltage-gated potassium current, IKv, 11-20 pS) and by an increase in intracellular Ca2+ concentration (calcium-activated potassium current, IKCa, 26 pS), respectively. The level of expression of these channels was correlated with the activational status of the cell. Both conductances are blocked by tetraethylammonium, verapamil, and charybdotoxin, and are insensitive to apamin; 4-aminopyridine blocks IK, preferentially. We used a protein kinase C activator (PMA) or antibodies to membrane Ig (anti-mu) to activate resting splenocytes in culture. Although IKv was recorded in the majority of the resting lymphocytic population, less than 20% of the activated cells expressed this conductance. However, in this subset the magnitude of IKv was 20-fold larger than in resting cells. On the other hand, IKCa was detected in nearly one half of the resting cells, whereas all activated cells expressed this current. The magnitude of IKCa was, on average, 30 times larger in activated than in nonactivated cells. These results probably reflect that during the course of activation 1) the number of voltage-dependent K+ channels per cell decreases and increases in a small subset and 2) the number of Ca(2+)-dependent K+ channels per cell increases in all cells. We suggest that the expression of functional Ca(2+)- and voltage-activated K+ channels are under the control of different regulatory signals.     

Properties of a native cation channel activated by Ca2+ store depletion in vascular smooth muscle cells

Depletion of intracellular Ca(2+) stores activates capacitative Ca(2+) influx in smooth muscle cells, but the native store-operated channels that mediate such influx remain unidentified. Recently we demonstrated that calcium influx factor produced by yeast and human platelets with depleted Ca(2+) stores activates small conductance cation channels in excised membrane patches from vascular smooth muscle cells (SMC). Here we characterize these channels in intact cells and present evidence that they belong to the class of store-operated channels, which are activated upon passive depletion of Ca(2+) stores. Application of thapsigargin (TG), an inhibitor of sarco-endoplasmic reticulum Ca(2+) ATPase, to individual SMC activated single 3-pS cation channels in cell-attached membrane patches. Channels remained active when inside-out membrane patches were excised from the cells. Excision of membrane patches from resting SMC did not by itself activate the channels. Loading SMC with BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), which slowly depletes Ca(2+) stores without a rise in intracellular Ca(2+), activated the same 3-pS channels in cell-attached membrane patches as well as whole cell nonselective cation currents in SMC. TG- and BAPTA-activated 3-pS channels were cation-selective but poorly discriminated among Ca(2+), Sr(2+), Ba(2+), Na(+), K(+), and Cs(+). Open channel probability did not change at negative membrane potentials but increased significantly at high positive potentials. Activation of 3-pS channels did not depend on intracellular Ca(2+) concentration. Neither TG nor a variety of second messengers (including Ca(2+), InsP3, InsP4, GTPgammaS, cyclic AMP, cyclic GMP, ATP, and ADP) activated 3-pS channels in inside-out membrane patches. Thus, 3-pS nonselective cation channels are present and activated by TG or BAPTA-induced depletion of intracellular Ca(2+) stores in intact SMC. These native store-operated cation channels can account for capacitative Ca(2+) influx in SMC and can play an important role in regulation of vascular tone.     

Distinct metal ion binding sites on Ca(2+)-activated K+ channels in inside-out patches of human erythrocytes.

Effects of Cd2+, Co2+, Pb2+, Fe2+ and Mg2+ (1-100 microM) on single-channel properties of the intermediate conductance Ca(2+)-activated K+ (CaK) channels were investigated in inside-out patches of human erythrocytes in a physiological K+ gradient. Cd2+, Co2+ and Pb2+, but not Fe2+ and Mg2+, were able to induce CaK channel openings. The potency of the metals to open CaK channels in human erythrocytes follows the sequence Pb2+, Cd2+ > Ca2+ > or = Co2+ > Mg2+, Fe2+. At higher concentrations Pb2+, Cd2+ and Co2+ block the CaK channel by reducing the opening frequency and the single-channel current amplitude. The potency of the metals to reduce CaK channel opening frequency follows the sequence Pb2+ > Cd2+, Co2+ > Ca2+, which differs from the potency sequence Cd2+ > Pb2+, Co2+ > Ca2+ to reduce the unitary single-channel current amplitude. Fe2+ reduced the channel opening frequency and enhanced the two open times of CaK channels activated by Ca2+, whereas up to 100 microM Mg2+ had no effect on any of the measured single-channel parameters. It is concluded that the activation of CaK channels of human erythrocytes by various metal ions occurs through an interaction with the same regulatory site at which Ca2+ activates these channels. The different potency orders for the activating and blocking effects suggest the presence of at least one activation and two blocking sites. A modulatory binding site for Fe2+ exists as well. In addition, the CaK channels in human erythrocytes are distinct from other subtypes of Ca(2+)-activated K+ channels in their sensitivity to the metal ions.     

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.     

Low-conductance states of K+ channels in adult mouse skeletal muscle

Single-channel currents were recorded from Ca2+-activated or ATP-sensitive K+ channels in inside-out membrane patches excised from isolated mouse toe muscles. In addition to the closed and fully open configurations, both types of channels may exhibit several intermediate low-conductance states which are clustered near multiples of elementary conductance units. The units are 1/8 or 1/6 of the channel conductance for Ca2+-activated channels and 1/4 or 1/3 for ATP-sensitive channels. Normally, low-conductance states are rare, but they occur more frequently directly after patch excision. An increased probability of low-conductance states of ATP-sensitive K+ channels was also observed in the presence and during washout of the internal channel blocker adenine. The results suggest that Ca2+-activated and ATP-sensitive K+ channels are composed of several membrane pores with strong positive cooperativity among the elementary conductance units.     

Potassium channel activity recorded from the apical membrane of freshly isolated epithelial cells in rat caudal epididymis.

K+ channels were recorded in excised, inside-out patches from the apical membrane of the freshly isolated tubule of the caudal portion of the rat epididymis. With asymmetric K+ concentrations in bath and pipette (140 mM K+in/6 mM K+out), the channels had a slope conductance of 54.2 pS at 0 mV. The relative permeability of K+ over Na+ was about 171 to 1. The channels were activated by intracellular Ca2+ and by membrane depolarization. These channels belong to a class defined as "intermediate-conductance Ca2+-activated K+ channel. " External tetraethylammonium ions (TEA+) caused a flickery block of the channel with reduction in single-channel current amplitude measured at a range of holding membrane potentials (-40 to 60 mV). Activity of the K+ channels was inhibited by intracellular ATP (KD =1.188 mM). The channel activity was detected only occasionally in patches from the apical membrane (about 1 in 17 patches containing active channels). The presence of the intermediate-conductance Ca2+-activated K+ channels indicates that they could provide a route for K+ secretion in a Ca2+-dependent process responsible for a high luminal K+ concentration found in the epididymal duct of the rat.     

A calmodulin dependent Ca2+-activated K+ channel in the adipocyte plasma membrane

Increased membrane permeability (conductance) that is specific for K+ and directly activated by Ca2+ ions, has been identified in isolated adipocyte plasma membranes using the K+ analogue, 86Rb+. Activation of these K+ conductance pathways (channels) by free Ca2+ was concentration dependent with a half-maximal effect occurring at 32 +/- 4 nM free Ca2+ (n = 7). Addition of calmodulin further enhanced the Ca2+ activating effect on 86Rb+ uptake (K+ channel activity). Ca2+-dependent 86Rb+ uptake was inhibited by tetraethylammonium ion and low pH. It is concluded that the adipocyte plasma membrane possesses K+ channels that are activated by Ca2+ and amplified by calmodulin.     

Involvement of K(+)-channel opening in endothelin-1 induced suppression of spontaneous contractions in the guinea pig taenia coli.

Effects of porcine-human endothelin-1 on mechanical as well as electrical activities and on intracellular free Ca2+ levels in the guinea pig taenia coli were compared with those of nifedipine, a voltage-dependent Ca2+ channel blocker. Endothelin-1 (0.1-100 nM) caused a concentration-dependent suppression of spontaneous contractions but did not significantly affect the sustained contraction evoked by 40 mM KCl. However, nifedipine (0.1-100 nM) inhibited both types of contractions in a concentration-dependent manner. In electrophysiological studies, endothelin-1 (30 nM) or nifedipine (30 nM) eliminated spontaneous spike discharges. Endothelin-1 produced hyperpolarization, while nifedipine did not change the resting membrane potential. The endothelin-1 induced suppression of spontaneous contractions was dose-dependently antagonized by apamin (0.01-10 nM), an inhibitor of a small conductance Ca(2+)-dependent K+ channel, and D-tubocurarine (10-100 microM), an inhibitor of Ca(2+)-dependent K+ channel, but was unaffected by 4-aminopyridine (0.01-1 mM), an inhibitor of a voltage-dependent K+ channel. In the study with fura 2 excited at 340 nm, endothelin-1 abolished, from the tissue, the fluorescence signals that were coupled with spontaneous contraction. It is suggested that the inhibitory action of endothelin-1 on spontaneous contraction may be caused by hyperpolarization of the membrane that reduces the spontaneous generation of spike discharge coupled normally to an increase in the intracellular free Ca2+ levels in the guinea pig taenia coli. The hyperpolarization may be caused by activating apamin-sensitive Ca(2+)-dependent K+ channels.