Dual regulation of the ATP-sensitive potassium channel by activation of cGMP-dependent protein kinase

Adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channels couple cellular metabolic status to membrane electrical activity. In this study, we performed patch-clamp recordings to investigate how cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) regulates the function of K(ATP) channels, using both transfected human SH-SY5Y neuroblastoma cells and embryonic kidney (HEK) 293 cells. In intact SH-SY5Y cells, the single-channel currents of Kir6.2/sulfonylurea receptor (SUR) 1 channels, a neuronal-type K(ATP) isoform, were enhanced by zaprinast, a cGMP-specific phosphodiesterase inhibitor; this enhancement was abolished by inhibition of PKG, suggesting a stimulatory role of cGMP/PKG signaling in regulating the function of neuronal K(ATP) channels. Similar effects of cGMP accumulation were confirmed in intact HEK293 cells expressing Kir6.2/SUR1 channels. In contrast, direct application of purified PKG suppressed rather than activated Kir6.2/SUR1 channels in excised, inside-out patches, while tetrameric Kir6.2LRKR368/369/370/371AAAA channels expressed without the SUR subunit were not modulated by zaprinast or purified PKG. Lastly, reconstitution of the soluble guanylyl cyclase/cGMP/PKG signaling pathway by generation of nitric oxide led to Kir6.2/SUR1 channel activation in both cell types. Taken together, here, we report novel findings that PKG exerts dual functional regulation of neuronal K(ATP) channels in a SUR subunit-dependent manner, which may provide new means of therapeutic intervention for manipulating neuronal excitability and/or survival.     

Functional linkage of the cardiac ATP-sensitive K+ channel to the actin cytoskeleton

The role of the cytoskeleton in the rundown and reactivation of adenosine triphosphate (ATP) sensitive K⁺ channels (KATP channels) was examined by perturbing selectively the intracellular surface of inside-out membrane patches excised from guinea-pig ventricular myocytes. Actin filament-depolymerizing agents (cytochalasins and desoxyribonuclease I) accelerated channel rundown, while actin filament stabilizer (phalloidin) or phosphatidylinositol biphosphate (PIP₂; inhibitor of F-actin-severing proteins) inhibited spontaneous and/or Ca²⁺-induced rundown. When rundown was induced by cytochalasin D or by long exposure to high Ca²⁺, channel activity could not be restored by exposure to MgATP, but application of F-actin with MgATP could reinstitute channel activity. The processes of rundown and reactivation of cardiac K_ATP channels may thus be influenced by the assembly and disassembly of the actin cytoskeletal network, which provides a novel regulatory mechanism of this channel.     

Nucleotide diphosphates activate the ATP-sensitive potassium channel in mouse skeletal muscle

Patch-clamp techniques were used to study the effects of internal nucleotide diphosphates on the K_ATP channel in mouse skeletal muscle. In inside-out patches, application of GDP (100 M) and ADP (100 M) reversibly increased the channel activity. In the presence of internal Mg²⁺ (1 mM), low concentrations of ADP (<300 M) enhanced channel activity and high concentrations of ADP (>300 M) limited channel opening while GDP activated the channel at all concentrations tested. In the absence of internal Mg²⁺, ADP decreased channel activity at all concentrations tested while GDP had no noticeable effect at submillimolar concentrations and inhibited channel activity at millimolar concentrations. GDP [S] (100 M), which behaved as a weak GDP agonist in the presence of Mg²⁺, stimulated ADP-evoked activation whereas it inhibited GDP-evoked activation. The K⁺ channel opener pinacidil was found to activate the K_ATP channel but only in the presence of internal GDP, ADP and GDP [S]. The results are discussed in terms of the existence of multiple nucleotide binding sites, in charge of the regulation of the K_ATP channel.     

Intracellular and extracellular amino acids that influence C-type inactivation and its modulation in a voltage-dependent potassium channel

The rate of C-type inactivation of the cloned voltage-gated potassium channel, Kv1.3, measured in membrane patches from Xenopus oocytes, increases when the patch is detached from the cell; the structural basis for this on-cell/off-cell change was examined. First, four serine and threonine residues, that are putative sites for phosphorylation by protein kinases A and C, were mutated to alanines. Mutating any one of these residues, or two or three of them simultaneously, does not eliminate the change in C-type inactivation. However, the basal rate of C-type inactivation in the cell-attached patch is markedly slower in the triple phosphorylation site mutant. Second, a homologous potassium channel, Kv 1.6, does not exhibit the on-cell/off-cell change. When an extracellular histidine at position 401 of Kv1.3 is replaced with tyrosine, the residue at the equivalent position (430) in Kv1.6, the resulting Kv1.3 H401Y mutant channel does not undergo the on-cell/off-cell change. The results indicate that several potentially phosphorylatable intracellular amino acids influence the basal rate of C-type inactivation, but are not essential for the on-cell/off-cell change in inactivation kinetics. In contrast, an extracellular amino acid is critical for this on-cell/off-cell change.     

Identification of an ATP-sensitive K+ channel in rat cultured cortical neurons

To determine whether membranes of mammalian central neurons contain an ATP-sensitive K⁺ (K_ATP) channel similar to that present in pancreatic cells, the patch-clamp technique was applied to cultured neurons prepared from the neonatal rat cerebral cortex and hippocampus. In whole-cell experiments with hippocampal neurons, extracellular application of 0.5 mM diazoxide (a K_ATP channel activator) elicited a hyperpolarization concomitant with an increase in membrane conductance, whereas application of 0.5 mM tolbutamide (a K_ATP channel blocker) induced a depolarization with a decrease in conductance. Similar results were obtained with cortical neurons. In outside-out patch experiments with cortical neurons, a K⁺ channel sensitive to these drugs was found. The channel was completely blocked by 0.5 mM tolbutamide and activated by 0.5 mM diazoxide. The single-channel conductance was 65 pS under symmetrical 145 mM K⁺ conditions and 24 pS in a physiological K⁺ gradient. In inside-out patch experiments, this channel was demonstrated to be inhibited by an application of 0.2–1 mM ATP to the cytoplasmic surface of the patch membrane. These results indicate that the membranes of rat cortical neurons contain a K_ATP channel that is quite similar to that found in pancreatic cells. It is also suggested that the same or a similar K⁺ channel may exist in membranes of hippocampal neurons.     

Hydrogen sulfide protects neurons against hypoxic injury via stimulation of ATP-sensitive potassium channel/protein kinase C/extracellular signal-regulated kinase/heat shock protein90 pathway

Cerebral hypoxia is one of the main causes of cerebral injury. This study was conducted to investigate the potential protective effect of H₂S in in vitro hypoxic models by subjecting SH-SY5Y cells to either oxygen–glucose deprivation or Na₂S₂O₄ (an oxygen scavenger) treatment. We found that treatment with NaHS (an H₂S donor, 10–100 μM) 15 min prior to hypoxia increased cell viability in a concentration-dependent manner. Time-course study showed that NaHS was able to exert its protective effect even when added 8 h before or less than 4 h after hypoxia induction. Interestingly, endogenous H₂S level was markedly reduced by hypoxia induction. Over-expression of cystathionine-β-synthase prevented hypoxia induced cell apoptosis. Blockade of ATP-sensitive K⁺ (K_ATP) channels with glibenclamide and HMR-1098, protein kinase C (PKC) with its three specific inhibitors (chelerythrine, bisindolylmaleide I and calphostin C), extracellular signal-regulated kinase 1/2 (ERK1/2) with PD98059 and heat shock protein 90 (Hsp90) with geldanamycin and radicicol significantly attenuated the protective effects of NaHS. Western blots showed that NaHS significantly stimulated ERK1/2 activation and Hsp90 expression. In conclusion, H₂S exerts a protective effect against cerebral hypoxia induced neuronal cell death via K_ATP/PKC/ERK1/2/Hsp90 pathway. Our findings emphasize the important neuroprotective role of H₂S in the brain during cerebral hypoxia.     

ATP-sensitive potassium channels counteract anoxia in neurones of the substantia nigra

Summary The ATP-sensitive potassium channel (K_ATP channel) is a unique ionophore in that it appears to reflect cell metabolism. In the brain, the highest density of binding sites for the K_ATP channel is the substantia nigra. To evaluate the role of the K_ATP channel in this key brain area for motor control, we used exposure to cyanide to lower intracellular ATP and thereby mimic anoxia and ischemia. Treatment with cyanide caused the activation of a potassium current in a sub-population of nigral neurones with distinct pharmacological and electrophysiological properties. The response to cyanide was abolished by the sulphonylurea tolbutamide, a potent blocker of the K_ATP channel. These results suggest that in the substantia nigra, the K_ATP channel plays a pivotal role in normal mechanisms of neuronal homeostasis in response to anoxia and ischaemia. The significance of these findings for our understanding of the cellular mechanisms in Parkinsonian degeneration is discussed.     

All or none block of single Na+ channels by tetrodotoxin

The effects of tetrodotoxin on single Na+-channel currents recorded from excised patches of neuroblastoma cells were examined. Tetrodotoxin was found to cause a dose-dependent reduction in the frequency at which Na+ channels conduct during a series of depolarizations. Surviving conducting states had normal open times and current amplitudes. These effects could be explained by a model which includes initial binding of tetrodotoxin to a closed state of the channel with stable, complete block during the time the channel would normally be gated open.     

Characterization of potassium channels in respiratory cells

In a previous study [26] we described the properties of potassium channels in cultured respiratory cells derived from cystic fibrosis patients (CF) and normal individuals (N). In the present study we examine the regulatory mechanisms of these channels by the patch clamp technique. Since there were no apparent differences in the properties of CF and N K⁺ channels the results were pooled. In the excised inside/out configuration the channel was blocked by different K⁺ channel blockers. Barium (5 · 10^–3 mol/l), retraethylammoniumchloride (5 · 10^–3 mol/l), quinidine 10^–3 mol/l) and lidocaine (5 · 10^–3 mol/l), when added to the cytosolic side, inhibited K⁺ channels reversibly. An increase in the calcium concentration from 10^–7 mol/l to 10^–6 mol/l led to a marked increase in the open channel probability (P _o). Further increases in Ca²⁺ concentration increasedP _o only slightly. No pH effects on the cytosolic side of the channel were observed. The channel open probability was reduced when ATP was present on the cytosolic side at a concentration of 10^–4 mol/l to 10^–3 mol/l. Non hydrolysable adenosine 5-[,-methylene] triphosphate had the same inhibitory effect as ATP. The inhibition by ATP was blunted by the simultaneous addition of 1 mmol/l ADP. The inhibition of K⁺ channels by cytosolic ATP may represent a channel regulatory mechanism in the intact cell. This would allow for coupling between the activity of the (Na⁺+K⁺)-pump and the basolateral K⁺ conductance.     

Polymyxin B has multiple blocking actions on the ATP-sensitive potassium channel in insulin-secreting cells

The action of polymyxin B (0.1 M) on ATP-sensitive K⁺ (K⁺ _ATP) channels in RINm5F insulin-secreting cells was investigated by patch-clamp techniques. Using inside-out patches, open-cells and outside-out patches, polymyxin B was found to block K⁺ _ATP channels by, on average, approximately 90–95% of the initial control level of channel activity. The effects were rapid in onset, sustained and readily reversible. Similar effects were found in patches excised from cells pretreated overnight with 1 M of the phorbol ester phorbol myristate acetate (PMA). External block of channels was associated with a marked decrease in single-channel current amplitude, whereas these effects were not seen when polymyxin B was added to the inside face of the membrane. In patches bathed with internally applied ATP (0.5 mM) and ADP (0.5 mM), polymyxin B inhibited channels but its actions were not reversible upon removal of the compound. However, when the same protocol was undertaken upon cells pre-treated with PMA, the effects of polymyxin B were readily reversed. Our data suggests that polymyxin B is a novel modulator of K⁺ _ATP channels, exhibiting multiple blocking actions that may possibly involve a direct effect upon the channel and indirect effects mediated through the inhibition of endogenous protein kinase(s).To be considered as equal first author.     

Regional expression of the anesthetic-activated potassium channel TRESK in the rat nervous system

The two-pore-domain potassium (K_2P) channels contribute to background (leak) potassium currents maintaining the resting membrane potential to play an important role in regulating neuronal excitability. As such they may contribute to nociception and the mechanism of action of volatile anesthetics. In the present study, we examined the protein expression pattern of the K_2P channel TRESK in the rat central nervous system (CNS) and peripheral nervous system (PNS) by immunohistochemistry. The regional distribution expression pattern of TRESK has both similarities and significant differences from that of other K_2P channels expressed in the CNS. TRESK expression is broadly found in the brain, spinal cord and dorsal root ganglia (DRG). TRESK expression is highest in important CNS structures, such as specific cortical layers, periaqueductal gray (PAG), granule cell layer of the cerebellum, and dorsal horn of the spinal cord. TRESK expression is also high in small and medium sized DRG neurons. These results provide an anatomic basis for identifying functional roles of TRESK in the rat nervous system.     

Iptakalim protects against hypoxic brain injury through multiple pathways associated with ATP-sensitive potassium channels

The rapid and irreversible brain injury produced by anoxia when stroke occurs is well known. Cumulative evidence suggests that the activation of neuronal ATP-sensitive potassium (KATP) channels may have inherent protective effects during cerebral hypoxia, yet little information regarding the therapeutic effects of KATP channel openers is available. We hypothesized that pretreatment with a KATP channel opener might protect against brain injury induced by cerebral hypoxia. In this study, adult Wistar rats were treated with iptakalim, a new KATP channel opener, which is selective for SUR2 type KATP channels, by intragastric administration at doses of 2, 4, or 8 mg/kg/day for 7 days before being exposed to simulated high altitude equivalent to 8000 m in a decompression chamber for 8 h leading to hypoxic brain injury. By light and electron microscopic images, we observed that hypobaric hypoxia-induced brain injury could be prevented by pretreatment with iptakalim. It was also observed that the permeability of the blood-brain barrier, water content, Na+ and Ca2+ concentration, and activities of Na+,K+-ATPase, Ca2+-ATPase and Mg2+-ATPase in rat cerebral cortex were increased and the gene expression of the occludin or aquaporin-4 was down- or upregulated respectively, which could also be prevented by the pretreatment with iptakalim at doses of 2, 4, or 8 mg/kg in a dose-dependent manner. Furthermore, we found that in an oxygen-and-glucose-deprived model in ECV304 cells and rat cortical astrocytes, pretreatment with iptakalim significantly increased survived cell rates and decreased lactate dehydrogenate release, which were significantly antagonized by glibenclamide, a K(ATP) channel blocker. We conclude that iptakalim is a promising drug that may protect against brain injury induced by acute hypobaric hypoxia through multiple pathways associated with SUR2-type K(ATP) channels, suggesting a new therapeutic strategy for stroke treatment.     

The extracellular signal-regulated kinase signaling pathway is involved in the modulation of morphine-induced reward by mPer1

Although there are clear interactions between circadian rhythms and drug addiction, mechanisms for such interactions remain unknown. Studies have shown that the circadian clock gene Period in Drosophila melanogaster could influence behavioral responses to cocaine, and the mouse homologues, mPer1 and mPer2, modulate cocaine sensitization and reward. In the present study, we applied DNAzyme targeting mPer1 to interfere the expression of mPer1 in CNS in mice, and studied its effects on morphine-induced reward and its molecular mechanism. The results demonstrated that the DNAzyme could attenuate the expression of mPer1 in CNS in mice and downregulate the increased extracellular signal-regulated kinase (ERK) activity induced by morphine in whole brain and the nucleus accumbens, the key region of drug addiction. Mice treated with morphine and injected intracerebroventricularly with DNAzyme did not show preference to the morphine-trained side. These results indicate that drug dependence seems to be influenced at least partially by mPer1 and its mechanism may involve the ERK signal pathway.     

Cysteine-modifying reagents alter the gating of the rat cloned potassium channel Kv1.4

The effects of cysteine-modifying reagents on the gating of rat cloned Kv1.4 channels expressed in HEK-293 cells were examined using the whole-cell patch-clamp technique. Cells transfected with Kv1.4 expressed a rapidly inactivating K⁺ current with a midpoint of activation of –31 mV and a slope factor of 5 mV measured with tail current protocols in 35 mM Rb⁺ external solutions. The cysteine-specific oxidizing agents 2,2-dithiobis-5-nitropyridine (DTBNP, 50 M) and chloramine-T (CL-T, 500 M) removed inactivation of Kv1.4. These effects were reversed by the reducing agent dithiothreitol (DTT, 10 mM). In addition, DTBNP and CL-T also slowed Kv1.4 deactivation and increased the voltage sensitivity of deactivation. The action of cysteine-modifying reagents on Kv1.4 suggests that redox state affects channel gating, with oxidation tending to stabilize the open state of the channel, both by removing inactivation and slowing deactivation.     

The sodium-activated potassium channel Slack is modulated by hypercapnia and acidosis

Slack (Slo 2.2), a member of the Slo potassium channel family, is activated by both voltage and cytosolic factors, such as Na(+) ([Na(+)](i)) and Cl(-) ([Cl(-)](i)). Since the Slo family is known to play a role in hypoxia, and since hypoxia/ischemia is associated with an increase in H(+) and CO(2) intracellularly, we hypothesized that the Slack channel may be affected by changes in intracellular concentrations of CO(2) and H(+). To examine this, we expressed the Slack channel in Xenopus oocytes and the Slo 2.2 protein was allowed to be inserted into the plasma membrane. Inside-out patch recordings were performed to examine the response of Slack to different CO(2) concentrations (0.038%, 5%, 12%) and to different pH levels (6.3, 6.8, 7.3, 7.8, 8.3). In the presence of low [Na(+)](i) (5 mM), the Slack channel open probability decreased when exposed to decreased pH or increased CO(2) in a dose-dependent fashion (from 0.28+/-0.03, n=3, at pH 7.3 to 0.006+/-0.005, n=3, P=0.0004, at pH 6.8; and from 0.65+/-0.17, n=3, at 0.038% CO(2) to 0.22+/-0.07, n=3, P=0.04 at 12% CO(2)). In the presence of high [Na(+)](i) (45 mM), Slack open probability increased (from 0.03+/-0.01 at 5 mM [Na(+)](i), n=3, to 0.11+/-0.01, n=3, P=0.01) even in the presence of decreased pH (6.3). Since Slack activity increases significantly when exposed to increased [Na(+)](i), even in presence of increased H(+), we propose that Slack may play an important role in pathological conditions during which there is an increase in the intracellular concentrations of both acid and Na(+), such as in ischemia/hypoxia.     

Single calcium-activated potassium channel in cultured mammary epithelial cells

The properties of Ca²⁺-activated K⁺ channels in mouse mammary epithelial cells in primary culture were studied by the patch-clamp technique. In cell-attached patches, spontaneous channel openings were sometimes observed; the slope conductance of the currents was about served; the slope conductance of the currents was about 12 pS at negative membrane potentials with a physiological solution (152 mM Na⁺, 5.4 mM K⁺) in the pipette. External application of A23187, a calcium ionophore, activated this channel. In excised inside-out patches, the channel was activated by increasing the internal Ca²⁺ concentration (10^–7 to 10^–6 M). No voltage dependence of the channel activity was observed. Internal Na⁺ blocked the outward K⁺ current in a voltage dependent manner and this block led to the non-linear I–V relationship at positive membrane potentials. The channel was blocked by internal Ba²⁺ (0.1 mM) and tetracthylammonium (TEA⁺, 20–50 mM). Ba²⁺ reduced the open probability but not the single channel conductance, whereas TEA⁺ reduced the single channel conductance. The single channel conductance of this channel, measured from the inward current with a high-K⁺ solution (150 mM K⁺) in the pipette, was large (about 40 pS), and showed inward rectification. These results suggest that this channel is different from the usual small conductance Ca²⁺-activated K⁺ channels observed in many other cells.     

Cation channel blocked by extracellular Ca2+ in the apical membrane of the chick embryonic ectoderm

In the chick embryo (20 h incubation, gastrula stage), the apical membrane of the ectodermal cells shows a high density of a non-selective cation channel which is blocked by very low extracellular Ca²⁺ concentrations. Properties of this channel were studied at the single-channel level using the patch-clamp technique in the cell-attached mode.With 1 mmol/l Ca²⁺ in the pipette, only outward current was present and the channel conductance measured at +120 mV was 25.5 pS. In the absence of Ca²⁺, also inward current through the channel was observed. The conductances measured at –50 mV were 49.5 pS with Na⁺ as the charge carrier, 72.5 pS with K⁺, 49.1 pS with Cs⁺, and 18.5 pS with Li⁺. The conductance measured at +80 mV was around 23 pS in all four cases. The reversal potential was similar (around 25 mV) for all four ions, which indicates a poor selectivity of the channel. In the absence of Ca²⁺ and the presence of 1 mmol/l ethylene-bis(oxonitrilo) tetraacetate (EGTA), the kinetics of the channel were characterized by bursts of the order of seconds. During a burst, the channel flickered between one open and one closed level. The open time was constant between –30 mV and –80 mV, while the closed time decreased with hyperpolarization. The open time varied according to the permeant ion (K⁺+=Cs⁺+).Extracellular Ca²⁺ blocked the inward current in a voltage-dependent manner. The K _d values, 1 mol/l at –30 mV and 3.2 mol/l at –80 mV, indicate that Ca²⁺ ions exit the channel toward the intracellular side. A weak voltage dependency of the association rate constant suggests that the Ca²⁺-binding site is close to the outside mouth. Extracellular Ca²⁺ was much less efficient at blocking the outward current (K _d about 1 mmol/l at 80 mV). Tetracaine, but not uraniumdioxide, decreased the opening probability of the channel. The embryonic channel shows similarities with the Ca²⁺-blockable, poorly selective channel described in the epithelium of toad urinary bladder.     

Modulation of L-type Ca channel activity by P2-purinergic agonist in cardiac cells

The mechanism of enhancement of the L-type Ca current by a P₂-purinergic agonist adenosine-5-O-(3-thiotriphosphate) (ATPS) was studied by recording single channel activity from cell-attached patches on rat isolated ventricular cells using patch pipettes containing 110 mM Ba²⁺. The application of ATPS to the patch membrane through the pipette solution did not affect single channel activity. The addition of ATPS to the bath containing a depolarizing solution was ineffective due to the voltage dependence of the purinergic stimulation. Bath application of ATPS (100 M) to control 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES) solution increased the amplitude of ensemble average currents both by decreasing the probability of a blank sweep occurring and by increasing the number of openings per non-blank sweep. The single channel conductance (17 pS) was not changed by ATPS. Both activation and inactivation curves were shifted towards hyperpolarized potentials by about 10 mV under P₂-purinergic stimulation. Since ATPS increased channel activity when applied via the bath, it must be supposed that a diffusible messenger is involved.     

Characterization of potassium channels in respiratory cells

Potassium channels present in the basolateral membrane of respiratory epithelial cells play an important role in the process of chloride secretion. Utilizing the patch clamp technique, we examined human cultured respiratory epithelial cells derived from patients with cystic fibrosis (CF) and normals individual (N) for the existence of and for the properties of K+ channels. We obtained qualitatively and quantitatively identical results for both preparations (CF and N). K+ channels were spontaneously present in cell attached patches. The channels showed burst appearance with rapid flickering within the bursts. When the pipette was filled with 145 mmol/l KCl, a mean conductance of 131 +/- 25 pS (n = 15) was read from the I/V-curve at a clamp voltage (Vc) of 0 mV. After excision, the conductance read from the I/V-curve at Vc = 0 mV was 212 +/- 11 pS (Pipette: 145 mmol/l KCl, bath: 145 mmol/l NaCl) (n = 61). With NaCl in the pipette and KCl in the bath, a similar conductance was obtained (g = 210 pS; n = 2). When both, pipette and bath contained KCl, the conductance was increased to 302 +/- 19 (n = 7). The channel was highly selective for potassium over sodium: PK + /PNa + greater than 40. The channel open probability was only slightly voltage dependent i.e. the open probability increased slightly with depolarisation. For most of the channels one open time constant (to = 6.3 +/- 1.6 ms; n = 22) and one closed time constant (tc = 1.8 +/- 0.3 ms; n = 21) was obtained.(ABSTRACT TRUNCATED AT 250 WORDS)