Cloned Ca(2+)-dependent K+ channel modulated by a functionally associated protein kinase

Calcium-dependent potassium (KCa) channels carry ionic currents that regulate important cellular functions. Like some other ion channels, KCa channels are modulated by protein phosphorylation. The recent cloning of complementary DNAs encoding Slo KCa channels has enabled KCa channel modulation to be investigated. We report here that protein phosphorylation modulates the activity of Drosophila Slo KCa channels expressed in Xenopus oocytes. Application of ATP-gamma S to detached membrane patches increases Slo channel activity by shifting channel voltage sensitivity. This modulation is blocked by a specific inhibitor of cyclic AMP-dependent protein kinase (PKA). Mutation of a single serine residue in the channel protein also blocks modulation by ATP-gamma S, demonstrating that phosphorylation of the Slo channel protein itself modulates channel activity. The results also indicate that KCa channels in oocyte membrane patches can be modulated by an endogenous PKA-like protein kinase which remains functionally associated with the channels in the detached patch.     

Ca2+-dependent inactivation of a store-operated Ca2+ current in human submandibular gland cells. Role of a staurosporine-sensitive protein kinase and the intracellular Ca2+ pump

Stimulation of human submandibular gland cells with carbachol, inositol trisphosphate (IP3), thapsigargin, or tert-butylhydroxyquinone induced an inward current that was sensitive to external Ca2+ concentration ([Ca2+]e) and was also carried by external Na+ or Ba2+ (in a Ca2+-free medium) with amplitudes in the order Ca2+ > Ba2+ > Na+. All cation currents were blocked by La3+ and Gd3+ but not by Zn2+. The IP3-stimulated current with 10 microM 3-deoxy-3-fluoro-D-myo-inositol 1,4,5-triphosphate and 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid in the pipette solution, showed 50% inactivation in <5 min and >5 min with 10 and 1 mM [Ca2+]e, respectively. The Na+ current was not inactivated, whereas the Ba2+ current inactivated at a slower rate. The protein kinase inhibitor, staurosporine, delayed the inactivation and increased the amplitude of the current, whereas the protein Ser/Thr phosphatase inhibitor, calyculin A, reduced the current. Thapsigargin- and tert-butylhydroxyquinone-stimulated Ca2+ currents inactivated faster. Importantly, these agents accelerated the inactivation of the IP3-stimulated current. The data demonstrate that internal Ca2+ store depletion-activated Ca2+ current (ISOC) in this salivary cell line is regulated by a Ca2+-dependent feedback mechanism involving a staurosporine-sensitive protein kinase and the intracellular Ca2+ pump. We suggest that the Ca2+ pump modulates ISOC by regulating [Ca2+]i in the region of Ca2+ influx.     

Ca(2+)-dependent kinase and phosphatase control inositol 1,4,5-trisphosphate-mediated Ca2+ release. Modification by agonist stimulation

A permeable cell system in which Ca2+ release can be evoked by inositol 1,4,5-trisphosphate (IP3) or agonist stimulation was used to study the regulation of Ca2+ release by Ca2+ itself. At low concentrations, Ca2+ activated IP3-mediated Ca2+ release (IMCR) with half-maximal effect at about 15 nM. At high concentrations, Ca2+ inhibited IMCR giving rise to a biphasic [Ca2+] dependence of IMCR. The activation of IMCR by Ca2+ appears to be mediated by a kinase, probably the Ca(2+)-and calmodulin-dependent protein kinase (CaMKII). Thus, the activation required MgATP, completely blocked at 0 degrees C, required Ca2+, and was inhibited by the CaMKII inhibitors KT5926 and KN62. The inhibition of IMCR seems to be mediated by a protein phosphatase, probably the Ca(2+)-dependent protein phosphatase 2B. Hence, the inhibition required Ca2+, was prevented by the general protein phosphatase inhibitor pyrophosphate and by the immunosuppressants cyclosporin A and FK506, but not by okadaic acid or VO4(2-), and was modified by chelating agents such as EGTA. Stimulation with agonists modified the activities of the kinase and phosphatase to make the release independent of [Ca2+]. This appears to be due to an increase in the apparent affinity for Ca2+ in stimulating IMCR and inhibition of the phosphatase. We suggest that agonist-dependent modification of the kinase/phosphatase activity ratio can be the biochemical pathway responsible for regulation of Ca2+ release and in turn [Ca2+]i oscillations.     

Wanderlust kinetics and variable Ca(2+)-sensitivity of Drosophila, a large conductance Ca(2+)-activated K+ channel, expressed in oocytes

Cloned large conductance Ca(2+)-activated K+ channels (BK or maxi-K+ channels) from Drosophila (dSlo) were expressed in Xenopus oocytes and studied in excised membrane patches with the patch-clamp technique. Both a natural variant and a mutant that eliminated a putative cyclic AMP-dependent protein kinase phosphorylation site exhibited large, slow fluctuations in open probability with time. These fluctuations, termed "wanderlust kinetics," occurred with a time course of tens of seconds to minutes and had kinetic properties inconsistent with simple gating models. Wanderlust kinetics was still observed in the presence of 5 mM caffeine or 50 nM thapsigargin, or when the Ca2+ buffering capacity of the solution was increased by the addition of 5 mM HEDTA, suggesting that the wanderlust kinetics did not arise from Ca2+ release from caffeine and thapsigargin sensitive internal stores in the excised patch. The slow changes in kinetics associated with wanderlust kinetics could be generated with a discrete-state Markov model with transitions among three or more kinetic modes with different levels of open probability. To average out the wanderlust kinetics, large amounts of data were analyzed and demonstrated up to a threefold difference in the [Ca2+]i required for an open probability of 0.5 among channels expressed from the same injected mRNA. These findings indicate that cloned dSlo channels in excised patches from Xenopus oocytes can exhibit large variability in gating properties, both within a single channel and among channels.     

Regulation and possible physiological role of the Ca(2+)-dependent K+ channel of cortical collecting ducts of the rat

In the luminal membrane of rat cortical collecting duct (CCD) a big Ca(2+)-dependent and a small Ca(2+)-independent K+ channel have been described. Whereas the latter most likely is responsible for the K+ secretion in this nephron segment, the function of the large-conductance K+ channel is unknown. The regulation of this channel and its possible physiological role were examined with the conventional cell-free and the cell-attached nystatin patch-clamp techniques. Patch-clamp recordings were obtained from the luminal membrane of isolated perfused CCD segments and from freshly isolated CCD cells. Intracellular calcium was measured using the calcium-sensitive dye fura-2. The large-conductance K+ channel was strongly voltage- and calcium-dependent. At 3 mumol/l cytosolic Ca2+ activity it was half-maximally activated. At 1 mmol/l it was neither regulated by cytosolic pH nor by ATP. At 1 mumol/l Ca2+ activity the open probability (Po) of this channel was pH-dependent. At pH 7.0 Po was decreased to 4 +/- 2% (n = 9) and at pH 8.5 it was increased to 425 +/- 52% (n = 9) of the control. At this low Ca2+ activity the Po of the channel was reduced by 1 mmol/l ATP to 8 +/- 4% (n = 6). Cell swelling activated the large-conductance K+ channel (n = 14) and hyperpolarized the membrane potential of the cells by 9 +/- 1 mV (n = 23). Intracellular Ca2+ activity increased after hypotonic stress. This increase depended on the extracellular Ca2+ activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+ channels, ryanodine receptors and Ca(2+)-activated K+ channels: a functional unit for regulating arterial tone

Local calcium transients ('Ca2+ sparks') are thought to be elementary Ca2+ signals in heart, skeletal and smooth muscle cells. Ca2+ sparks result from the opening of a single, or the coordinated opening of many, tightly clustered ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR). In arterial smooth muscle, Ca2+ sparks appear to be involved in opposing the tonic contraction of the blood vessel. Intravascular pressure causes a graded membrane potential depolarization to approximately -40 mV, an elevation of arterial wall [Ca2+]i and contraction ('myogenic tone') of arteries. Ca2+ sparks activate calcium-sensitive K+ (KCa) channels in the sarcolemmal membrane to cause membrane hyperpolarization, which opposes the pressure induced depolarization. Thus, inhibition of Ca2+ sparks by ryanodine, or of KCa channels by iberiotoxin, leads to membrane depolarization, activation of L-type voltage-gated Ca2+ channels, and vasoconstriction. Conversely, activation of Ca2+ sparks can lead to vasodilation through activation of KCa channels. Our recent work is aimed at studying the properties and roles of Ca2+ sparks in the regulation of arterial smooth muscle function. The modulation of Ca2+ spark frequency and amplitude by membrane potential, cyclic nucleotides and protein kinase C will be explored. The role of local Ca2+ entry through voltage-dependent Ca2+ channels in the regulation of Ca2+ spark properties will also be examined. Finally, using functional evidence from cardiac myocytes, and histological evidence from smooth muscle, we shall explore whether Ca2+ channels, RyR channels, and KCa channels function as a coupled unit, through Ca2+ and voltage, to regulate arterial smooth muscle membrane potential and vascular tone.     

Activation of Ca2+-sensitive Cl- current by reverse mode Na+/Ca2+ exchange in rabbit ventricular myocytes

We investigated how Ca2+-sensitive transient outward current, Ito(Ca), is activated in rabbit ventricular myocytes in the presence of intracellular Na+ (Na+i) using the whole-cell patch-clamp technique at 36 degreesC. In cells dialysed with Na+-free solutions, the application of nicardipine (5 microM) to block L-type Ca2+ current (ICa) completely inhibited Ito(Ca). In cells dialysed with a [Na+]i>/=5 mM, however, Ito(Ca) could be observed after blockade of ICa, indicating the activity of an ICa-independent component. The amplitude of ICa-independent Ito(Ca) increased with voltage in a [Na+]i-dependent manner. The block of Ca2+ release from the sarcoplasmic reticulum by caffeine, ryanodine or thapsigargin blocked ICa-independent Ito(Ca). In Ca2+-free bath solution Ito(Ca) was completely abolished. The application of 2 mM Ni2+ or the newly synthesized compound KBR7943, a selective blocker of the reverse mode of Na+/Ca2+ exchange, or perfusion with pipette solution containing XIP (10 microM), a selective blocker of the exchanger, blocked ICa-independent Ito(Ca). From these results we conclude that, in the presence of Na+i, Ito(Ca) can be activated via Ca2+-induced Ca2+ release triggered by Na+/Ca2+ exchange operating in the reverse mode after blockade of ICa.     

Electrophysiological effects of ketamine on Ca(2+)-activated K+ channel in single rabbit portal vein cell

The effects of ketamine on Ca(2+)-activated K+ channel currents were studied in dispersed single smooth muscle cells from rabbit portal vein using inside-out patch clamp technique. In a near physiological K+ and Ca2+ gradient, three populations of outward rectangular single currents were recorded in isolated cell membrane of rabbit portal vein at +60 mV membrane potential. These currents were judged as Ca(2+)-activated K+ channel currents since application of EGTA or Apamin in the internal solution inhibited these currents. Application of 10(-5)M or 10(-4)M ketamine inhibited the number of occurrences of channel opening and decreased open times, but did not reduce the amplitudes. When the 10(-3)M ketamine was applied, the Ca(2+)-activated K+ channel currents were abolished. We suggest that the depression of Ca(2+)-activated K+ channel currents may explain the continuous contraction observed in rabbit portal vein at a clinical concentration of ketamine from a point of electrophysiological K+ current study.     

Ca(2+)-activated K+ channels of human and rabbit erythrocytes display distinctive patterns of inhibition by venom peptide toxins

Despite recent progress in the molecular characterization of high-conductance Ca(2+)-activated K+ (maxi-K) channels, the molecular identities of intermediate conductance Ca(2+)-activated K+ channels, including that of mature erythrocytes, remains unknown. We have used various peptide toxins to characterize the intermediate conductance Ca(2+)-activated K+ channels (Gardos pathway) of human and rabbit red cells. With studies on K+ transport and on binding of 125I-charybdotoxin (ChTX) and 125I-kaliotoxin (KTX) binding in red cells, we provide evidence for the distinct nature of the red cell Gardos channel among described Ca(2+)-activated K+ channels based on (i) the characteristic inhibition and binding patterns produced by ChTX analogues, iberiotoxin (IbTX) and IbTX-like ChTX mutants, and KTX (1-37 and 1-38 variants); (ii) the presence of some properties heretofore attributed only to voltage-gated channels, including inhibition of K transport by margatoxin (MgTX) and by stichodactyla toxin (StK); (iii) and the ability of scyllatoxin (ScyTX) and apamin to displace bound 125I-charybdotoxin, a novel property for K+ channels. These unusual pharmacological characteristics suggest a unique structure for the red cell Gardos channel.     

Interaction between alpha- and beta-adrenergic receptor agonists modulating the slow Ca(2+)-activated K+ current IAHP in hippocampal neurons

Noradrenaline inhibits the Ca(2+)-activated K+ current IAHP, which underlies the slow afterhyperpolarization and spike frequency adaptation in hippocampal and neocortical neurons. The resulting increase in excitability probably contributes to the state control of the forebrain during arousal and attention. The modulation of IAHP by noradrenaline has previously been shown to be mediated by beta 1 receptors, cyclic AMP and protein kinase A, but not by alpha receptors. We have now tested the possibility that alpha receptors also contribute to IAHP modulation through interaction with beta receptors, by the use of whole-cell recordings in CA1 pyramidal cells of rat hippocampal slices. The alpha-receptor agonist 6-fluoro-noradrenaline strongly potentiated the effect of isoproterenol on IAHP. The synergistic effect of 6-fluoro-noradrenaline and isoproterenol was blocked by the beta-receptor antagonist timolol, but the receptor type mediating the effect of 6-fluoro-noradrenaline could not be unequivocally identified by using alpha-receptor antagonists. The effect of high concentrations of noradrenaline on IAHP was only partly blocked by the beta-receptor antagonist timolol, and was further reduced by blocking alpha receptors, again suggesting a contribution from alpha receptors. In contrast, the effect of low concentrations of noradrenaline seemed to be potentiated by the alpha-receptor antagonist phentolamine in 57% of the cells, suggesting concentration-dependent antagonistic interaction between alpha and beta receptors. Further tests indicated that the cross-talk between 6-fluoro-noradrenaline and isoproterenol occurs upstream from cyclic AMP production, and that protein kinase A serves as a final common path for the modulation of IAHP by noradrenaline, and by the combination of 6-fluoro-noradrenaline and isoproterenol.     

P2u purinoceptor modulation of intracellular Ca2+ in a human lung adenocarcinoma cell line: down-regulation of Ca2+ influx by protein kinase C

The aim of the present investigation was to study the functional alterations in the stomatognathic system following orthodontic-surgical management of skeletal vertical excess problems. The sample comprised 43 patients who received combined orthodontic-surgical treatment including bilateral vertical ramus osteotomy for posterior repositioning and counterclockwise rotation of the mandible (n = 26) or Le Fort I osteotomy for maxillary impaction (n = 17). All subjects were examined within 1 week before operation and 6 months postsurgery. Methods of examination included: (a) evaluation of dysfunction by means of a clinical index, (b) measurement of mandibular range of motion, (c) assessment of the number and intensity of occlusal contacts, and (d) tomographic evaluation of condyle-fossa relationships. The results of the study indicated that postoperatively (a) there was an increase of patients with dysfunction in the mandibular osteotomy group and a decrease of patients with dysfunction in the maxillary osteotomy group; (b) the maximum interincisal opening decreased significantly in the mandibular osteotomy group; (c) there was a significant increase in the number and intensity of occlusal contacts in both groups; and (d) the shortest posterior and anterior interarticular distances increased significantly in the mandibular osteotomy group.     

Ca(2+)-permeable, outwardly-rectifying K+ channels in mesophyll cells of Arabidopsis thaliana

Advances in cannulation techniques and instruments have helped in difficult bile duct cannulation and thus stone extraction. For small common bile duct (CBD) stones, endoscopic papillary balloon dilatation has been proposed as an alternative to endoscopic papillotomy (EPT). The technique must undergo further evaluation before recommending its routine use. For most patients with bile duct stones, EPT remains the method of choice. Out of 8204 patients treated in three surgical endoscopy centers (Chile, Germany, and India), 86% to 91% of all CBD stones could be extracted subsequently after EPT using a Dormia basket; 4% to 7% required mechanical lithotripsy (ML) before removal and 3% to 10% of the patients needed other sophisticated techniques, such as electrohydraulic lithotripsy (EHL), laser-induced shock-wave lithotripsy (LISL), or extracorporeal shock-wave lithotripsy (ESWL). The local expertise and availability of equipment determines the choice of method used. In general, EHL or LISL is used for impacted CBD stones including stones in Mirizzi syndrome refractory to ML. ESWL is best suited for intrahepatic stones. Permanent stenting can be offered to poor risk patients instead of extensive procedures to clear the bile duct. Using currently available nonsurgical techniques, fewer than 1% of all patients with bile duct stones still require surgical intervention.     

Ca2+-dependent K+ currents induced by muscarinic receptor activation in guinea pig adrenal chromaffin cells

The neuronal effects of the metabotropic glutamate receptor agonist (1S,3R)-aminocyclopentane-1,3-dicarboxylic acid have been studied in cultured rat cerebellar granule cells, and compared with those of the endogenous excitotoxin glutamate, and the dietary excitotoxin beta-N-methylamino-L-alanine. Glutamate, beta-N-methylamino-L-alanine, and (1S,3R)-aminocyclopentane-1,3-dicarboxylic acid all caused concentration-dependent cerebellar granule cell death over a 24-h exposure period. The metabotropic antagonist (RS)-alpha-methyl-4-carboxyphenylglycine reduced glutamate-, beta-N-methylamino-L-alanine-, and (1S,3R)-aminocyclopentane-1,3-dicarboxylic acid-induced death by 50, 37, and 90%, respectively. (1S,3R)-Aminocyclopentane-1,3-dicarboxylic acid-induced death was unaffected by the group I antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid, increased by the group II antagonist ethylglutamic acid, and markedly decreased by the group III antagonist (RS)-alpha-methylserine-O-phosphate. Neither (1S,3R)-aminocyclopentane-1,3-dicarboxylic acid nor the group I agonist (RS)-3,5-dihydroxyphenylglycine caused an increase in intracellular free calcium levels. The group III agonist L-(+)-2-amino-4-phosphonobutyric acid also induced concentration-dependent cerebellar granule cell death, and so it was suggested that the group III metabotropic glutamate receptors were responsible for (1S,3R)-aminocyclopentane-1,3-dicarboxylic acid-induced death. Blocking these receptors with (RS)-alpha-methylserine-O-phosphate also prevented a proportion of glutamate- and beta-N-methylamino-L-alanine-induced death.     

Immunocytochemical localization of Ca(2+)-dependent protease from Allomyces arbuscula

The Ca(2+)-dependent protease antisera and the purified specific antibodies from Allomyces arbuscula have shown very specific recognition when blotted against the total protein extract or the purified 43-40 kDa Ca(2+)-dependent protease from this aquatic fungus. By immunoblotting and immunofluorescence techniques using specific antibodies, we have shown that the enzyme activity is developmentally regulated and is related to the presence of antigen and not to any specific inhibitor. The immunofluorescence was absent in zoospores but appeared in polarized forms in germinating spores. In elongating hyphae the protease was mainly localized along the cytoplasmic membrane and in the cytoplasm, with predominance at the apex.     

Apamin-sensitive Ca(2+)-activated K+ channels regulate pacemaker activity in nigral dopamine neurons

Mesencephalic dopamine-containing neurons exhibit a Ca(2+)-dependent oscillation in membrane potential believed to underlie the ability of these cells to maintain spontaneous activity in the absence of afferent synaptic drive. In the present series of experiments, sharp electrode intracellular recording techniques were used in conjunction with an in vitro brain slice preparation to explore the ionic mechanisms underlying rhythmogenesis in nigral dopamine neurons in the rat. Our results indicate that the K+ channel producing the prolonged post-spike afterhyperpolarization exhibited by these neurons is also principally responsible for generating the falling phase of the autogenous pacemaker oscillation. Alterations in the expression of this conductance are associated with marked changes in neuronal firing pattern, indicating that modulation of ligand-gated Ca(2+)-activated K+ channels may constitute a functional means of altering temporal coding among the major mesotelencephalic dopamine systems.     

Different effects of endothelin-3 on the Ca2+ discharge induced by agonists and Ca(2+)-ATPase inhibitors in human platelets

1. The present study demonstrates that endothelin-3 (ET-3), previously shown to attenuate thrombin-evoked aggregation of human platelets, delayed the dose-dependent aggregatory response to thapsigargin (Tg). As this Ca(2+)-ATPase inhibitor induces platelet activation in part through the depletion of internal Ca(2+)-stores, we examined the influence of ET-3 on Ca2+ discharge from internal pools. 2. Cytosolic Ca2+ concentration was evaluated with Fura-2 in the absence of Ca2+ influx. Platelet preincubation for 15 min with 5 x 10(-7) M ET-3 decreased the Ca2+ release evoked by thrombin and U46619, a thromboxane-mimetic. However, ET-3 did not affect Ca2+ movements induced by 1 microM ADP. Addition of Tg (0.5 to 5 microM) to resting platelets induced a cytosolic [Ca2+] rise with concentration-dependent increase of the initial rate and decrease of the time to reach the peak. ET-3 slowed down these dose-dependent effects with a more marked influence on the responses induced by low concentrations of Tg. 3. ET-3 did not modify the Ca2+ response to another Ca(2+)-ATPase inhibitor, 2,5-di-(tert-butyl)-1,4-benzohydroquinone(tBuBHQ). The thromboxane A2 receptor antagonist, SQ 29548, reduced by 53% the calcium signal evoked by 1 microM Tg, which became similar to that induced by 15 microM tBuBHQ. Under these conditions, the ET-3 effects were suppressed. A subsequent addition of thrombin induced a substantial further Ca2+ increase which was again sensitive to ET-3. 4. ET-3 attenuates Ca2+ mobilization from an internal pool dependent on the stimulation of thrombin and thromboxane A2 receptors and insensitive to the direct effect of Ca2+-ATPase inhibitors. The small but significant inhibitory effect of ET-3 leads us to propose that endothelin-3 acts as a modulator of platelet activation.