Activation Of Large-conductance Research Articles

Transient forebrain ischemia induces persistent hyperactivity of large conductance Ca2+-activated potassium channels via oxidation modulation in rat hippocampal CA1 pyramidal neurons.

The present study examined temporal changes in activity of large conductance, Ca2+-activated potassium (BKCa) channels in postischemic CA1 pyramidal neurons at 2, 6, 24 and 48 h after reperfusion. These changes in activity and possible cellular mechanisms were examined using the inside--out configuration of patch clamp. The unitary conductance of postischemic BKCa channels increased transiently to 119% of the control at 2 h after reperfusion, and recovered to the control level thereafter. A persistent increase in [Ca2+]i sensitivity of BKCa channels was observed in postischemic CA1 neurons with the maximal sensitivity to [Ca2+]i at 6 h after reperfusion while channel voltage- dependence showed no obvious changes. Kinetic analyses showed that the postischemic enhancement of BKCa channel activity was due to longer open times and shorter closed times as there was no significant changes in opening frequency after ischemia. Glutathione disulphide markedly increased BKCa channel activity in normal CA1 neurons, while reducing glutathione caused a decrease in BKCa channel activity by reducing the sensitivity of this channel to [Ca2+]i in postischemic CA1 neurons. Similar modulatory effects on postischemic BKCa channels were also observed with another redox couple, DTNB and DTT, suggesting an oxidation modulation of BKCa channel function after ischemia. The present results indicate that a persistent enhancement in activity of BKCa channels, probably via oxidation of channels, in postischemic CA1 pyramidal neurons may account for the decrease in neuronal excitability and increase in fAHP after ischemia. The ischemia-induced augmentation in BKCa channel activity may be also associated with the postischemic neuronal injury.

A Catalytically Inactive Mutant of Type I cGMP-dependent Protein Kinase Prevents Enhancement of Large Conductance, Calcium-sensitive K+ Channels by Sodium Nitroprusside and cGMP

The activation of large conductance, calcium-sensitive K(+) (BK(Ca)) channels by the nitric oxide (NO)/cyclic GMP (cGMP) signaling pathway appears to be an important cellular mechanism contributing to the relaxation of smooth muscle. In HEK 293 cells transiently transfected with BK(Ca) channels, we observed that the NO donor sodium nitroprusside and the membrane-permeable analog of cGMP, dibutyryl cGMP, were both able to enhance BK(Ca) channel activity 4-5-fold in cell-attached membrane patches. This enhancement correlated with an endogenous cGMP-dependent protein kinase activity and the presence of the alpha isoform of type I cGMP-dependent protein kinase (cGKI). We observed that co-transfection of cells with BK(Ca) channels and a catalytically inactive ("dead") mutant of human cGKIalpha prevented enhancement of BK(Ca) channel in response to either sodium nitroprusside or dibutyryl cGMP in a dominant negative fashion. In contrast, expression of wild-type cGKIalpha supported enhancement of channel activity by these two agents. Importantly, both endogenous and expressed forms of cGKIalpha were found to associate with BK(Ca) channel protein, as demonstrated by a reciprocal co-immunoprecipitation strategy. In vitro, cGKIalpha was able to directly phosphorylate immunoprecipitated BK(Ca) channels, suggesting that cGKIalpha-dependent phosphorylation of BK(Ca) channels in situ may be responsible for the observed enhancement of channel activity. In summary, our data demonstrate that cGKIalpha alone is sufficient to promote the enhancement of BK(Ca) channels in situ after activation of the NO/cGMP signaling pathway.

Fatty acid-activated K+ channels in autonomic neurons: activation by an endogenous source of free fatty acids.

Application of arachidonic acid evoked robust activation of large-conductance K+ channels in cell-attached and excised inside-out patches from acutely isolated chick ciliary ganglion neurons. A similar effect was produced by 5,8,11,14-eicosatetraynoic acid, a nonmetabolizable analogue of arachidonic acid. The unitary conductance of fatty acid-activated channels was 35-40 pS at +20 mV with physiological gradients of K+ and 165 pS at +20 mV with an extracellular K+ concentration of 37.5 mM and an intracellular K+ concentration of 150 mM. Gating of these channels in cell-attached patches was potentiated by membrane stretch. Channel gating evoked by both lipids was concentration-dependent, with detectable activation apparent at 4 microM in the majority of patches and maximal activation occurring between 32 and 64 microM. Gating was relatively voltage-independent. Large-conductance K+ channels were also activated in inside-out patches by the monounsaturated fatty acid 11-cis-eicosenoic acid but not by the fully saturated fatty acid arachidic acid. Application of 100 microM H2O2, an agent that activates cytosolic phospholipase A2, also caused activation of large-conductance K+ channels in intact neurons. The stimulatory effects of H2O2 were blocked by pretreatment with 20 microM 4-bromophenacyl bromide, an irreversible inhibitor of phospholipase A2. Therefore, mobilization of endogenous fatty acids can cause activation of large-conductance K+ channels in autonomic neurons.

Calcium sparks in smooth muscle.

Local intracellular Ca(2+) transients, termed Ca(2+) sparks, are caused by the coordinated opening of a cluster of ryanodine-sensitive Ca(2+) release channels in the sarcoplasmic reticulum of smooth muscle cells. Ca(2+) sparks are activated by Ca(2+) entry through dihydropyridine-sensitive voltage-dependent Ca(2+) channels, although the precise mechanisms of communication of Ca(2+) entry to Ca(2+) spark activation are not clear in smooth muscle. Ca(2+) sparks act as a positive-feedback element to increase smooth muscle contractility, directly by contributing to the global cytoplasmic Ca(2+) concentration ([Ca(2+)]) and indirectly by increasing Ca(2+) entry through membrane potential depolarization, caused by activation of Ca(2+) spark-activated Cl(-) channels. Ca(2+) sparks also have a profound negative-feedback effect on contractility by decreasing Ca(2+) entry through membrane potential hyperpolarization, caused by activation of large-conductance, Ca(2+)-sensitive K(+) channels. In this review, the roles of Ca(2+) sparks in positive- and negative-feedback regulation of smooth muscle function are explored. We also propose that frequency and amplitude modulation of Ca(2+) sparks by contractile and relaxant agents is an important mechanism to regulate smooth muscle function.

Substance P and bradykinin activate different types of KCa currents to hyperpolarize cultured porcine coronary artery endothelial cells.

1. Substance P and bradykinin, endothelium-dependent vasodilators of pig coronary artery, trigger in endothelial cells a rise in cytosolic Ca2+ concentration ([Ca2+]i) and membrane hyperpolarization. The aim of the present study was to determine the type of Ca2+-dependent K+ (KCa) currents underlying the endothelial cell hyperpolarization. 2. The substance P-induced increase in [Ca2+]i was 30 % smaller than that induced by bradykinin, although the two peptides triggered a membrane hyperpolarization of the same amplitude. The two agonists evoked a large outward K+ current of the same conductance at maximal stimulation. Agonists applied together produced the same maximal current amplitude as either one applied alone. 3. Iberiotoxin (50 nM) reduced by about 40 % the K+ current activated by bradykinin without modifying the substance P response. Conversely, apamin (1 microM) inhibited the substance P-induced K+ current by about 65 %, without affecting the bradykinin response. Similar results were obtained on peptide-induced membrane hyperpolarization. 4. Bradykinin-induced, but not substance P-induced, endothelium-dependent relaxation resistant to NG-nitro-L-arginine and indomethacin was partly inhibited by 3 microM 17-octadecynoic acid (17-ODYA), an inhibitor of cytochrome P450 epoxygenase. Similarly, the bradykinin-induced K+ current was reduced by 17-ODYA. 5. Our results show that responses to substance P and bradykinin result in a hyperpolarization due to activation of different KCa currents. A current consistent with the activation of large conductance (BKCa) channels was activated only by bradykinin, whereas a current consistent with the activation of small conductance (SKCa) channels was stimulated only by substance P. The observation that a similar electrical response is produced by different pools of channels implies distinct intracellular pathways leading to KCa current activation.

Expression of potassium channels during postnatal differentiation of rabbit Müller glial cells.

The postnatal maturation of Müller glial cells from immature radial glial cells is accompanied by specific changes in the activity of distinct types of K+ channels, as shown by whole-cell and cell-attached records on freshly isolated cells from retinae of young (postnatal days 1-30, P1-P30) and adult rabbits. (i) The density of inwardly rectifying currents, providing the main K+ conductance in adult Müller cells, was very low (0.8 pA/pF) from P1 to P6 but increased rapidly thereafter until a relatively stable level of 11.0 pA/pF was established at P17. (ii) Transient (A-type) K+ currents were expressed in all immature cells at a high density (9.6 pA/pF). After P12, both the percentage of cells with A-type currents and the peak amplitudes of the currents (2.8 pA/pF) declined. (iii) Delayed rectifying K+ currents developed slowly until after P30. (iv) The postnatal maturation of radial glial cells was accompanied by a strong decrease in the activity of large-conductance, Ca2+-activated K+ channels, the open probability of which (measured at the resting membrane potential) decreased from 0.69 at P2-4 to 0.06 at P13-14. The developmental decrease of the activity of Ca2+-activated K+ channels is assumed to be mainly caused by alteration of the resting membrane potential which developed from low values (-49 mV) at P1-6 to high adult values (-84 mV) after P13. The activity of each distinct type of K+ channel investigated is differently modulated by developmental regulation. This may reflect different functional requirements of immature and mature Müller cells.

Potassium secretion in rat distal colon during dietary potassium loading: role of pH regulated apical potassium channels

BackgroundChronic dietary K+ loading increases the abundance of large conductance (210 pS) apical K+ channels in surface cells of rat distal colon, resulting in enhanced K+ secretion in this epithelium....

Internally applied endotoxin and the activation of BK channels in cerebral artery smooth muscle via a nitric oxide-like pathway.

1. In this study the role of nitric oxide synthase (NOS) in the acute activation of large conductance, Ca2+-activated K+ channels (BK channels) by internally applied E. coli lipopolysaccharide (LPS, endotoxin) was examined in vascular smooth muscle cells. 2. Cerebrovascular smooth muscle cells (CVSMCs) were enzymatically dispersed from the middle, posterior communicating and posterior cerebral arteries of adult Wistar rats and maintained at 4 degrees C for 2-4 days before recording with standard patch-clamp techniques. 3. Acute application of LPS (100 microg ml(-1)) to inside-out patches of CVSMC membrane isolated in a cell-free environment rapidly and reversibly increased the open probability, Po of BK channels in these patches by 3.3+/-0.30 fold. 4. Acute application of the nitric oxide (NO) donor sodium nitroprusside (SNP, 100 microM) to inside-out patches of CVSMC membrane, studied in the presence of intact cells, also reversibly increased Po, by some 1.8+/-0.2 fold over control. 5. Kinetic analysis showed that both LPS and SNP increased Po by accelerating the rate of BK channel reopening, rather than by retarding the closure of open channels. 6. Neither LPS nor SNP altered the reversal potential or conductance of BK channels. 7. The NOS substrate L-arginine (1 microM) potentiated the acute activation of BK channels by LPS, while the synthetic enantiomer D-arginine (1 microM) inhibited the action of LPS on BK channels. 8. The acute activation of BK channels by LPS was suppressed by pre-incubation of cells with N(omega)-nitro-L-arginine (50 microM) or N(omega)-nitro-L-arginine methyl ester (1 mM), two competitive antagonists of nitric oxide synthases. N(omega)-nitro-D-arginine (50 microM), a poor inhibitor of NOS in in vitro assays, had no effect on BK channel activation by LPS. 9. These results indicate that excised, inside-out patches of CVSMC membrane exhibit a NOS-like activity which is acutely activated when LPS is present at the cytoplasmic membrane surface. Possible relationships between this novel mechanism and the properties of known isoforms of nitric oxide synthase are discussed.

Relaxation of porcine tracheal smooth muscle by parathyroid hormone-related protein

Parathyroid hormone-related protein (PTHrp) has been shown to relax uterine and gastrointestinal smooth muscles, but the mechanisms underlying its effects have not been characterized. Furthermore, its effect on pulmonary smooth muscle is unknown. Therefore we designed the present study to determine the PTHrp dose-response; the interaction of PTHrp and PTH; and the role of cyclic nucleotides and potassium channels in the PTHrp response in porcine tracheal smooth muscle (TSM). Our results indicate that, (1-34)PTHrp causes dose-dependent relaxation of TSM; that (1-34)PTHrp and (1-34)PTH demonstrate cross-tachyphylaxis to one another; that phosphodiesterase inhibition augments and phosphodiesterase stimulation attenuates the relaxation response while guanylate cyclase blockade has little effect, and that charybdotoxin and iberiotoxin, inhibitors of large conductance, Ca2+-activated, K+ channels, diminish the relaxation response. These findings suggest that (1-34)PTHrp-induced relaxation of TSM is mediated through a common PTHrp/PTH pathway or receptor, stimulation of cAMP and activation of large conductance, Ca2+-activated, K+ channels.

Cyclic AMP enhances agonist-induced Ca2+ entry into endothelial cells by activation of potassium channels and membrane hyperpolarization.

The mechanism underlying cyclic AMP (cAMP)-mediated amplification of agonist-induced Ca2+ responses in endothelial cells was investigated in pig endothelial cells. Forskolin, adenosine and isoprenaline, as well as the membrane-permeant cAMP analogue dibutyryl cAMP, enhanced bradykinin-induced rises in intracellular free Ca2+ as well as bradykinin-induced Mn2+ entry. These agents were also found to hyperpolarize endothelial cells without increasing intracellular Ca2+ by itself, i.e. in the absence of bradykinin. Both amplification of bradykinin effects and the hyperpolarizing action was blocked by the protein kinase inhibitor H-8. The involvement of K+ channels in the hyperpolarizing effects of forskolin was consequently studied in perforated outside-out vesicles. Two different types of K+ channels were recorded, one of which had a large conductance (170 pS) and was activated by forskolin. We suggest that stimulation of endothelial adenylate cyclase results in activation of large-conductance K+ channels and consequently in membrane hyperpolarization, which in turn enhances bradykinin-induced entry of Ca2+ by increasing its electrochemical gradient.

Guinea‐pig isolated trachealis: the effects of charybdotoxin on mechanical activity, membrane potential changes and the activity of plasmalemmal K+‐channels

1. A study has been made, in guinea-pig isolated trachealis, of the effects of charybdotoxin in modulating (a) the activity of large conductance K(+)-channels, (b) the spontaneous electrical activity of intact cells and (c) the mechanical effects of some bronchodilator drugs. 2. Single smooth muscle cells were isolated from guinea-pig trachealis by enzymic digestion and were studied by the patch clamp recording technique. Recordings were made from outside-out plasmalemmal patches when the medium bathing the external surface of the patches contained 1.2 mM Ca2+ and 6 mM K+ while that bathing the cytosolic surface contained 0.1 microM Ca2+ and 140 mM K+. Charybdotoxin (100 nM), applied to the external surface of patches held at 0 mV, abolished the unitary currents associated with the opening of large conductance K(+)-channels. 3. Opened segments of guinea-pig trachea were used for the simultaneous recording of membrane potential and tension changes. In these experiments charybdotoxin (100 nM) caused the conversion of spontaneous electrical slow waves into spike-like action potentials. This effect was accompanied by a very small reduction in resting membrane potential. 4. Tissue bath recording showed that charybdotoxin (100 nM) increased the spontaneous mechanical tone of the tissue, antagonized (2.8 fold in each case) the relaxant actions of isoprenaline and theophylline but did not antagonize the relaxant actions of cromakalim or RP 49356. 5. It is concluded that charybdotoxin is an effective inhibitor of large conductance K(+)-channels in guinea-pig trachealis cells. The ability of charybdotoxin to convert spontaneous slow waves into spike-like action potentials suggests that the large, charybdotoxin-sensitive, K+-channels play an important role in determining the strong outward rectifying behaviour of the cells. The ability of charybdotoxin to antagonize isoprenaline and theophylline, but not to antagonize cromakalim and RP 49356, suggests that opening of the large conductance, charybdotoxin-sensitive K+-channel is implicated in the action of the former but not the latter pair of bronchodilator drugs.