Extracellular ATP activates Ca2(+)-dependent K+ conductance via Ca2+ influx in mouse macrophages

1. Using the perforated patch recording, the effects of ATP on membrane current were investigated in mouse peritoneal macrophages. 2. Extracellularly applied ATP induced a biphasic current consisting of a initial inward current [Ii(ATP)] followed by an outward current [Io(ATP)]. These currents were associated with a marked increase in conductance at their peaks. 3. Ii(ATP) reversed close to 0 mV and was attenuated by removal of external Na+. 4. Io(ATP) reversed near -80 mV and was increased by decreasing the external concentration of K+. 5. Io(ATP) was completely abolished by removal of external Ca2+, treatment with an intracellular Ca2+ chelator, the acetoxymethyl ester of 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetra acetic acid (BAPTA-AM) and bath applied quinidine but not tetraethylammonium (TEA) or apamin. 6. These results suggest that Ii(ATP) and Io(ATP) are due to an activation of nonspecific cationic and Ca2(+)-dependent K+ conductances, respectively, and raise the possibility that the putative ATP receptor may be important in regulating macrophage functions, motility, phagocytosis and cytokines secretion.     

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+].     

Identification of a protein component of the Ca2+-dependent K+ channel by affinity labelling with apamin

The red blood cell membrane proteins and plasma proteins of normal and spontaneously hypertensive rats were studied by uni- and bidimensional polyacrylamide gel electrophoresis. The amount of band 3, the major intrinsic protein of the erythrocyte membrane, was observed to be significantly reduced in spontaneously hypertensive rats. Plasma from these rats contained two additional heat stable proteins, characterized by a molecular weight of 16,000 daltons and isoelectric points of 4.7 and 5.1, respectively. These proteins were not detected in normotensive control Wistar Kyoto rats, in normal Wistar rats, or in Wistar rats with experimentally induced hypertension.     

An amino acid outside the pore region influences apamin sensitivity in small conductance Ca2+-activated K+ channels

Small conductance calcium-activated potassium channels (SK, K(Ca)) are a family of voltage-independent K+ channels with a distinct physiology and pharmacology. The bee venom toxin apamin inhibits exclusively the three cloned SK channel subtypes (SK1, SK2, and SK3) with different affinity, highest for SK2, lowest for SK1, and intermediate for SK3 channels. The high selectivity of apamin made it a valuable tool to study the molecular makeup and function of native SK channels. Three amino acids located in the outer vestibule of the pore are of particular importance for the different apamin sensitivities of SK channels. Chimeric SK1 channels, enabling the homomeric expression of the rat SK1 (rSK1) subunit and containing the core domain (S1-S6) of rSK1, are apamin-insensitive. By contrast, channels formed by the human orthologue human SK1 (hSK1) are sensitive to apamin. This finding hinted at the involvement of regions beyond the pore as determinants of apamin sensitivity, because hSK1 and rSK1 have an identical amino acid sequence in the pore region. Here we investigated which parts of the channels outside the pore region are important for apamin sensitivity by constructing chimeras between apamin-insensitive and -sensitive SK channel subunits and by introducing point mutations. We demonstrate that a single amino acid situated in the extracellular loop between the transmembrane segments S3 and S4 has a major impact on apamin sensitivity. Our findings enabled us to convert the hSK1 channel into a channel that was as sensitive for apamin as SK2, the SK channel with the highest sensitivity.     

Ca2+- and voltage-dependent K+ conductance in dispersed parathyroid cells

The membrane ionic conductances of dispersed parathyroid cells kept in primary culture were studied using the "whole-cell" and "inside-out excised patch" variants of the patch-clamp technique. The major component of the total current was a voltage-dependent outward K+ current without an appreciable inward current. The amplitude of the K+ current was markedly reduced when free internal Ca2+ was buffered by addition of 10 mM EGTA. Recordings of single-channel current in excised membrane patches revealed the presence of K+ channels with large unitary conductance (200 pS in symmetrical 130 mM K+ solutions) which were also activated by depolarization when internal Ca2+ concentration was about 10(-5)-10(-6) M. At any membrane voltage these channels were closed most of the time at internal Ca2+ concentrations lower than 10(-10) M. These results demonstrate the existence of a Ca2+- and voltage-dependent K+ permeability in parathyroid cells which may participate in the unusual membrane potential changes induced by alterations of external Ca2+ and, possibly, in the regulation of parathormone secretion.     

The coexistence in rat muscle cells of two distinct classes of Ca2+-dependent K+ channels with different pharmacological properties and different physiological functions

An Arthrobacter sp. has been shown to dehalogenate 4-chlorobenzoate yielding 4-hydroxybenzoate. Experiments with 18O indicate that, in the presence of cell-free extracts, the hydroxyl group which is substituted onto the aromatic nucleus during dehalogenation is derived from water and not from molecular oxygen. Dehalogenation therefore is not catalysed by a mixed-function oxidase; instead a novel aromatic hydroxylase is implicated in the reaction.     

Ca2+-dependent K+ transport in lymphocytes

The treatment of rat thymocytes with A23187 + Ca2+, ascorbate-phenazine methosulphate or propranolol induced quinine-sensitive fluxes of K+ (Rb+) suggesting the presence in the cell membrane of Ca2+-dependent K+ channels. Concanavalin A induced K+ channel activation only at very high doses (13 micrograms/ml). Neither quinine nor the increase of the K+ concentration in the medium to 30 mM prevented the stimulation of amino acid transport induced by concanavalin A, suggesting that the Ca2+-dependent K+ channel is not involved in the early phenomena of lymphocyte activation.     

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.     

尼古丁调节大鼠冠状动脉平滑肌细胞大电导钙激活钾通道

目的:研究尼古丁对Wistar大鼠冠状动脉平滑肌大电导钙激活钾通道(BKca)活性的抑制作用及其细胞信号转导机制。方法:8周雄性Wistar大鼠随机分为两组:生理盐水组和尼古丁组;分别予以生理盐水和尼古丁2mg/(kg.d)注射21 d,蛋白酶法分离冠状动脉血管平滑肌细胞,将两组平滑肌细胞分别以对氯苯硫基环腺苷酸(CPT-cAMP,100μmol/L)和佛司可林(forskolin,10μmol/L)干预,单通道膜片钳记录干预前后平滑肌细胞单通道电流的平均开放时间(To)、平均关闭时间(Tc)、平均开放概率(Po)。结果:CPT-cAMP和Forskolin均能显著延长生理盐水组大鼠BKca的平均开放时间,缩短平均关闭时间,增加通道开放概率(P均<0.01)。对尼古丁组BKca的To、Tc、Po均无明显影响。结论:尼古丁促使冠状动脉血管收缩的生理机制是通过抑制cAMP/PKA途径诱导的大电导钙激活钾通道活性增加实现的。     

Coassembly of big conductance Ca2+-activated K+ channels and L-type voltage-gated Ca2+ channels in rat brain

Based on electrophysiological studies, Ca(2+)-activated K(+) channels and voltage-gated Ca(2+) channels appear to be located in close proximity in neurons. Such colocalization would ensure selective and rapid activation of K(+) channels by local increases in the cytosolic calcium concentration. The nature of the apparent coupling is not known. In the present study we report a direct coassembly of big conductance Ca(2+)-activated K(+) channels (BK) and L-type voltage-gated Ca(2+) channels in rat brain. Saturation immunoprecipitation studies were performed on membranes labeled for BK channels and precipitated with antibodies against alpha(1C) and alpha(1D) L-type Ca(2+) channels. To confirm the specificity of the interaction, precipitation experiments were carried out also in reverse order. Also, additive precipitation was performed because alpha(1C) and alpha(1D) L-type Ca(2+) channels always refer to separate ion channel complexes. Finally, immunochemical studies showed a distinct but overlapping expression pattern of the two types of ion channels investigated. BK and L-type Ca(2+) channels were colocalized in various compartments throughout the rat brain. Taken together, these results demonstrate a direct coassembly of BK channels and L-type Ca(2+) channels in certain areas of the brain.     

Effects of phalloidin on K+-dependent, Ca2+-independent neurotransmitter efflux and K+-facilitated, Ca2+-dependent neurotransmitter release.

Phalloidin tightly binds to actin and converts soluble actin into depolymerization-resistant actin filaments. Phalloidin promotes the potassium-dependent, calcium-independent efflux of γ-amino butyric acid and nore-pinephrine from synaptosomes but inhibits the potassium-facilitated, calcium-dependent release of these neurotransmitters. This suggests that an actomyosin system is involved in synaptic transmission.     

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.     

Acid secretagogues induce Ca2+ mobilization coupled to K+ conductance activation in rat parietal cells in tissue culture

Intracellular recordings from cultured parietal cells of the rat gastric fundus showed that carbachol, pentagastrin, histamine (in the presence of isobutylmethylxanthine; IBMX) and dibutyryl cyclic AMP induced hyperpolarizing responses which were sensitive to a K+ channel blocker, quinine. The Ca2+ ionophore, ionomycin, also induced a quinine-sensitive hyperpolarization. Deprivation of extracellular Ca2+ preferentially inhibited the hyperpolarizing responses to histamine (plus IBMX) and to dibutyryl cyclic AMP. Caffeine, oxalate and dantrolene sodium, which are known to affect Ca2+ transport in the endoplasmic reticulum, selectively inhibited the carbachol response. Mitochondrial inhibitors (KCN and carbonylcyanide p-trifluoromethoxyphenylhydrazone) preferentially suppressed the gastrin response. Cytosolic Ca2+ measurements with fura-2 indicated that significant increases in the intracellular concentration of free Ca2+ were induced not only by Ca2+-mediated acid secretagogues (carbachol and gastrin), but also by a cyclic AMP-mediated secretagogue (histamine plus IBMX). Dibutyryl cyclic AMP also increased cytosolic Ca2+ ions. It is concluded that stimulation of receptors to histamine, carbachol and gastrin gives rise to mobilization of Ca2+ ions into the cytoplasm from the different sources, thereby stimulating Ca2+-activated K+ channels in cultured rat parietal cells.     

Ca2+-dependent membrane currents in vascular smooth muscle cells of the rabbit.

The membrane potential in vascular smooth muscle cells contributes to the regulation of cytosolic [Ca2+], which in turn regulates membrane potential by means of Ca2+i-dependent ionic currents. We investigated the characteristics of Ca2+i-dependent currents in rabbit coronary and pulmonary arterial smooth muscle cells. Ca2+i-dependent currents were recorded using the whole-cell patch-clamp technique while cytosolic [Ca2+] was increased by caffeine. The reversal potentials of caffeine-induced currents were between -80 and -10 mV under normal ionic conditions, whereas they were about 0 mV when K+-free NaCl solutions were used both in pipette and bath. The total substitution of extracellular Na+ with membrane-impermeable cation N-Methyl-D-glucamine did not affect caffeine-induced currents, implying no significant contribution of Na+ as a permeant ion to the currents. The substitution of extracellular NaCl with sucrose reduced outward component of the currents and shifted the reversal potentials according to the change in Cl- equilibrium potential. Upon application of the niflumic acid under K+-free conditions, most of the current induced by caffeine was inhibited. Taken together, the results of the present study indicate that K+ and Cl- currents are major components of Ca2+i-dependent currents in vascular smooth muscles isolated from coronary and pulmonary arteries of the rabbit, and the relative contribution of each type of current to total currents are not different between the two arteries.     

Contryphan-Vn: a modulator of Ca2+-dependent K+ channels

Contryphan-Vn is a D-tryptophan-containing disulfide-constrained nonapeptide isolated from the venom of Conus ventricosus, the single Mediterranean cone snail species. The structure of the synthetic Contryphan-Vn has been determined by NMR spectroscopy. Unique among Contryphans, Contryphan-Vn displays the peculiar presence of a Lys-Trp dyad, reminiscent of that observed in several voltage-gated K(+) channel blockers. Electrophysiological experiments carried out on dorsal unpaired median neurons isolated from the cockroach (Periplaneta americana) nerve cord on rat fetal chromaffin cells indicate that Contryphan-Vn affects both voltage-gated and Ca(2+)-dependent K(+) channel activities, with composite and diversified effects in invertebrate and vertebrate systems. Voltage-gated and Ca(2+)-dependent K(+) channels represent the first functional target identified for a conopeptide of the Contryphan family. Furthermore, Contryphan-Vn is the first conopeptide known to modulate the activity of Ca(2+)-dependent K(+) channels.     

Impaired Ca2+-dependent activation of large-conductance Ca2+-activated K+ channels in the coronary artery smooth muscle cells of Zucker Diabetic Fatty rats

The large-conductance Ca²⁺-activated K⁺ (BK) channels play an important role in the regulation of cellular excitability in response to changes in intracellular metabolic state and Ca²⁺ homeostasis. In vascular smooth muscle, BK channels are key determinants of vasoreactivity and vital-organ perfusion. Vascular BK channel functions are impaired in diabetes mellitus, but the mechanisms underlying such changes have not been examined in detail. We examined and compared the activities and kinetics of BK channels in coronary arterial smooth muscle cells from Lean control and Zucker Diabetic Fatty (ZDF) rats, using single-channel recording techniques. We found that BK channels in ZDF rats have impaired Ca²⁺ sensitivity, including an increased free Ca²⁺ concentration at half-maximal effect on channel activation, a reduced steepness of Ca²⁺ dose-dependent curve, altered Ca²⁺-dependent gating properties with decreased maximal open probability, and a shortened mean open-time and prolonged mean closed-time durations. In addition, the BK channel β-subunit-mediated activation by dehydrosoyasaponin-1 (DHS-1) was lost in cells from ZDF rats. Immunoblotting analysis confirmed a 2.1-fold decrease in BK channel β₁-subunit expression in ZDF rats, compared with that of Lean rats. These abnormalities in BK channel gating lead to an increase in the energy barrier for channel activation, and may contribute to the development of vascular dysfunction and complications in type 2 diabetes mellitus.     

Ca2+-conductances in cultured rat retinal pigment epithelial cells

Membrane conductances for Ca²⁺ in cultured rat pigment epithelial cells were studied in the whole-cell configuration of the patch-clamp technique using barium (10 mM) as a charge carrier. Two types of voltage-dependent and verapamiland diltiazem-sensitive Ba²⁺ currents were observed. First, a nearly sustained current was activated by depolarization to potentials more positive than — 30mV and blocked by nifedipine (1 μM). This current was observed in cells of primary cultures less than 13 days old. Second, a transient nifedipine (1 μM) insensitive current was activated by depolarization to potentials more positive than — 55mV in cultures which were more than 13 days old. This current was not carried by sodium and blocked by 1 μM tetrodotoxin (TTX). In summary, cultured rat retinal pigment epithelial cells in younger primary cultures express Ba²⁺ currents indicating the presence of L-type Ca²⁺ channels. In order primary cultures a low-voltage activated channel was observed with properties different from T-type calcium channels or TTX-sensitive calcium conducting sodium channels. © 1994 Wiley-Liss, Inc.     

ATP-inhibited and Ca2+-dependent K+ channels in the soma membrane of cultured leech Retzius neurons

The properties of one ATP-inhibited and one Ca²⁺-dependent K⁺ channel were investigated by the patch-clamp technique in the soma membrane of leech Retzius neurons in primary culture. Both channels rectify at negative potentials. The ATP-inhibited K⁺ channel with a mean conductance of 112 pS is reversibly blocked by ATP (K _i = 100 m), TEA (K _i =0.8 mm) and 10 mm Ba²⁺ and irreversibly blocked by 10 nm glibenclamide and 10 m tolbutamide. It is Ca²⁺ and voltage independent. Its open state probability (P _o) decreases significantly when the pH at the cytoplasmic face of inside-out patches is altered from physiological to acid pH values. The Ca²⁺-dependent K⁺ channel with a mean conductance of 114 pS shows a bell-shaped Ca²⁺ dependence of P _o with a maximum at pCa 7–8 at the cytoplasmic face of the membrane. The P _o is voltage independent at the physiologically relevant V range. Ba²⁺ (10 mm) reduces the single channel amplitude by around 25% (ATP, TEA, glibenclamide, tolbutamide, and Ba²⁺ were applied to the cytoplasmic face of the membrane).We conclude that the ATP-dependent K⁺ channel may play a role in maintaining the membrane potential constant—independently from the energy state of the cell. The Ca²⁺-dependent K⁺ channel may play a role in generating the resting membrane potential of leech Retzius neurons as it shows maximum activity at the physiological intracellular Ca²⁺ concentration.This study was supported by the Deutsche Forschungsgemeinschaft (W.-R. Schlue) and by a fellowship of the Konrad-Adenauer-Stiftung (G. Frey). We thank Dr. Draeger (Hoechst AG) for the gift of glibenclamide. The data are part of a future Ph.D. thesis of G. Frey.     

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.