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Query: UMLS:C0848283 (rundown)
 502 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Agonists that elevate calcium in T84 cells stimulate chloride secretion by activating KBIC, an inwardly rectifying K channel in the basolateral membrane. We have studied the regulation of this channel by calcium, nucleotides and phosphorylation using patch clamp and short-circuit current (ISC) techniques. Open probability (Po) was independent of voltage but declined spontaneously with time after excision. Rundown was slower if patches were excised into a bath solution containing ATP (10 microM-5 mM), ATP (0.1 mM)+protein kinase A (PKA; 180 nM), or isobutylmethylxanthine (IBMX; 1 mM). Analysis of event durations suggested that the channel has at least two open and two closed states, and that rundown under control conditions is mainly due to prolongation of the long closed time. Channel activity was restimulated after rundown by exposure to ATP, the poorly hydrolyzable ATP analogue AMP-PNP, or ADP. Activity was further enhanced when PKA was added in the presence of MgATP, but only if free calcium concentration was elevated (400 nM). Nucleotide stimulation and inward rectification were both observed in nominally Mg-free solutions. cAMP modulation of basolateral potassium conductance in situ was confirmed by measuring currents generated by a transepithelial K gradient after permeabilization of the apical membrane using alpha-toxin. Finally, protein kinase C (PKC) inhibited single KBIC channels when it was added directly to excised patches. These results suggest that nonhydrolytic binding of nucleotides and phosphorylation by PKA and PKC modulate the responsiveness of the inwardly rectifying K channel to Ca-mediated secretagogues.
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PMID:Regulation of an inwardly rectifying K channel in the T84 epithelial cell line by calcium, nucleotides and kinases. 753 42

Dissociated single fibers from the mouse flexor digitorum brevis (FDB) muscle were used in patch clamp experiments to investigate the mechanisms of activation and inactivation of KATP in mammalian skeletal muscle. Spontaneous rundown of channel activity, in many excised patches, occurred gradually over a period of 10-20 min. Application of 1.0 mM free-Ca2+ to the cytoplasmic side of the patch caused irreversible inactivation of KATP within 15 sec. Ca(2+)-induced rundown was not prevented by the presence of 1.0 microM okadaic acid or 2.0 mg ml-1 of an inhibitor of calcium-activated neutral proteases, a result consistent with the conclusion that phosphatases or calcium-activated neutral proteases were not involved in the rundown process. Application of 1.0 mM Mg.ATP to Ca(2+)-inactivated KATP caused inhibition of residual activity but little or no reactivation of the channels upon washout of ATP, even in the presence of the catalytic subunit of cyclic AMP-dependent protein kinase (10 U ml-1). Mg.ATP also failed to reactivate KATP, even after only partial spontaneous rundown, despite the presence of channels that could be activated by the potassium channel opener BRL 38227. Nucleotide diphosphates (500 microM; CDP, UDP, GDP and IDP) caused immediate and reversible opening of Ca(2+)-inactivated KATP. Reactivation of KATP by ADP (100 microM) increased further upon removal of the nucleotide. In contrast to KATP from cardiac and pancreatic cells, there was no evidence for phosphorylation of KATP from the surface sarcolemma of dissociated single fibers from mouse skeletal muscle.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Rundown and reactivation of ATP-sensitive potassium channels (KATP) in mouse skeletal muscle. 780 25

1. A mechanism underlying reactivation of the adenosine 5'-triphosphate-sensitive K+ (K+ATP) channels by MgATP complexes after run-down was examined in guinea-pig ventricular myocytes using the patch-clamp technique with inside-out patch configuration. 2. After run-down was induced by exposure of the intracellular side of the membrane patch to Ca2+ (1 mM), channel activity was reactivated by exposure and subsequent wash-out of MgATP (2 mM). Addition of inhibitors of various serine/threonine protein kinases to the MgATP solution did not suppress reactivation of the run-down channels. 3. Non- or poorly hydrolysable ATP analogues were unable to reactivate run-down channels. 4. The degree of channel recovery was dependent upon the duration of MgATP exposure. The apparent half-activation value (K1/2) of MgATP for reactivation was decreased with increasing exposure time. 5. Various products of ATP hydrolysis were unable to reactivate run-down channels except a relatively low concentration (100 microM) of ADP exposure. 6. Other nucleotide triphosphates, in the presence of Mg2+, were unable to reactivate rundown channels. 7. Fluorescein 5-isothiocyanate (50 microM), which interacts with lysine residues of the nucleotide-binding site on various ATPases, inhibited K+ATP channel activity. After wash-out, channel activity recovered only slightly. 8. These data suggest that the hydrolysis of ATP is important for reactivation of run-down K+ATP channels but that protein phosphorylation by serine/threonine protein kinases may not be involved. Since no products of ATP hydrolysis could reproduce MgATP-induced channel reactivation and since the degree of channel recovery was dependent upon the duration of MgATP application, the hydrolysis energy appears to be utilized for channel reactivation.
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PMID:Mechanism for reactivation of the ATP-sensitive K+ channel by MgATP complexes in guinea-pig ventricular myocytes. 799 37

Treatment with trypsin of the cytoplasmic surface of excised inside-out membrane patches from guinea pig ventricular myocytes altered multiple regulatory properties of ATP-sensitive K+ (KATP) channels including their sensitivity to intracellular ATP (ATPi), intracellular ADP (ADPi), glibenclamide, and cromakalim. The single-channel conductance, reversal potential, and inward rectification (in the presence of intracellular Mg2+) were unaltered after trypsin treatment. KATP channels also remained sensitive to intracellular Ca(2+)-induced rundown after trypsin treatment (n = 6). The effects of trypsin were not prevented by including either 15 mM ATPi (n = 7), 1 mM ADPi (n = 4), or 10 microM glibenclamide (n = 4) during exposure to trypsin, suggesting that occupancy of these binding sites did not prevent access of trypsin to the proteolytic sites responsible for its effects. Treatment of excised membrane patches with 1 mM phenylglyoxal (n = 4) or 5 mM glyoxal (n = 4), which cleave polypeptides at arginine residues, did not increase the dissociation constant for suppression of KATP channels by ATPi. Because trypsin cleaves peptides at both arginine and lysine residues, these results suggest that modification of the regulatory properties of KATP channels by trypsin may result from proteolytic digestion of lysine residues located in cytosolic regions of the channel protein.
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PMID:Effects of trypsin on cardiac ATP-sensitive K+ channels. 814 63

Activation and reactivation of the ATP-sensitive K+ channel (IK.ATP) were studied with the patch-clamp technique in guinea-pig ventricular myocytes. The K+ channel openers, nicorandil and pinacidil, activated IK.ATP in an internal ATP-dependent manner. Both drugs increased the open probability of IK.ATP without changing the channel conductance. They prolonged lifetimes of bursts and shortened interburst intervals without influencing the fast gating within bursts. These effects were the opposite of those of internal ATP. However, the interaction between ATP and either nicorandil or pinacidil appeared not to be simple competition. We found that three carbonyl compounds--3,4-dihydroxybenzaldehyde, 2,3-dihydroxybenzaldehyde, and 2,4-dihydroxyacetophenone--could activate IK.ATP through an intracellular mechanism that was dependent upon the presence of ADP and Mg2+. It has been suggested that these three carbonyl compounds bind covalently to proteins to form a Schiff base, which may be responsible for their effects upon IK.ATP. Internal application of the proteolytic enzyme trypsin prevented both the spontaneous and Ca(2+)-induced rundown of the KK.ATP channel. Tryptic digestion did not change either the channel's sensitivity to inhibition by ATP nor the fast gating kinetics of IK.ATP. Internal application of an exopeptidase, carboxypeptidase A, but not leu-aminopeptidase, prevented the spontaneous and Ca(2+)-induced rundown of the IK/ATP channel, effects similar to those of trypsin treatment. These results suggest that the target site of trypsin digestion may be located on the carboxy (C)-terminal of the channel proteins or associated regulatory units.
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PMID:Activation and reactivation of the ATP-sensitive K+ channel of the heart can be modified by drugs. 825 28

The inside-out configuration of the patch-clamp method was used to study the effects of trypsin on the activity of ATP-sensitive potassium (K-ATP) channels from isolated mouse pancreatic beta-cells. Trypsin (20 micrograms/ml) irreversibly enhanced channel activity around twofold by reducing the interburst intervals without altering the burst kinetics. No effect on the single channel conductance or the inward rectification produced by internal Mg2+ was observed: however, the protease did reduce the inhibitory effect of Mg2+ on channel activity. Trypsin both prevented rundown of K-ATP channel activity and reactivated the channels after complete rundown. These effects of trypsin were absent in the presence of trypsin inhibitor. The protease also reduced the inhibitory effect of ATP on channel activity, increasing the dissociation constant from 7 to 49 microM. Trypsin removed the activating effect of ADP (0.1 mmol/l) on channel activity and reduced the inhibitory effect of tolbutamide (0.5 mmol/l). Carboxypeptidase A did not activate K-ATP channels in excised patches, although it was able to slightly reactivate channels after complete rundown, whereas chymotrypsin increased K-ATP channel activity but it did not produce reactivation. The effects of papain were similar to those of trypsin.
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PMID:Modification of K-ATP channels in pancreatic beta-cells by trypsin. 835 Dec 6

The molecular mechanisms underlying the actions of K channel openers (KCOs) on KATP channels were studied with the patch clamp technique in excised inside-out patches from frog skeletal muscle fibers. Benzopyran KCOs (levcromakalim and SR 47063) opened channels partially blocked by ATP, ADP, or ATP gamma s, with and without Mg2+, but they had no effects in the absence of internal nucleotides, even after channel activity had significantly declined because of rundown. The effects of KCOs could therefore be attributed solely to a competitive interaction between KCOs and nucleotides, as confirmed by observations that ATP decreased the apparent affinity for KCOs and that, conversely, KCOs decreased ATP or ADP sensitivity. Protons antagonized the action of the non-benzopyran KCOs, pinacidil and aprikalim, by enhancing their dissociation rate. This effect resembled the effect of acidification on benzopyran KCOs (Forestier, C., Y. Depresle, and M. Vivaudou. FEBS Lett. 325:276-280, 1993), suggesting that, in spite of their structural diversity, KCOs could act through the same binding sites. Detailed analysis of the inhibitory effects of protons on channel activity induced by levcromakalim or SR 47063 revealed that, in the presence of 100 microM ATP, this effect developed steeply between pH 7 and 6 and was half maximal at pH 6.6. These results are in quantitative agreement with an allosteric model of the KATP channel possessing four protonation sites, two nucleotidic sites accessible preferentially to Mg(2+)-free nucleotides, and one benzopyran KCO site. The structural implications of this model are discussed.
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PMID:Mechanism of action of K channel openers on skeletal muscle KATP channels. Interactions with nucleotides and protons. 872 62

Levosimendan, a new Ca++-sensitizing and positive inotropic agent, was reported to act as a coronary vasodilator and protect ischemic myocardium. To elucidate the mechanisms of these actions, the possible electrophysiological effects of levosimendan on isolated rat ventricular cells were examined by the patch-clamp technique with whole-cell and single-channel recordings. Levosimendan (3 and 10 microM) markedly shortened action potential duration and activated an outward current at potentials positive to -70 mV. The increased current was abolished by glibenclamide, a blocker of the ATP-sensitive K+ (K[ATP]) current. Stimulation of K[ATP] current was dose dependent, with an EC50 value of 4.7 microM; a maximal effect occurred at 30 microM. The L-type Ca++ current was not affected by levosimendan (0.2-10 microM). In single-channel current recording in open cell-attached patches, K[ATP] channels, which had been inhibited by 0.3 mM ATP, were activated by levosimendan. However, levosimendan did not stimulate the K[ATP] channels that exhibited high spontaneous activity in ATP-free solution. Levosimendan also could not stimulate K[ATP] channels that had rundown in ATP-free solution. However, levosimendan could stimulate rundown K[ATP] channels that were reactivated by nucleotide diphosphates. K[ATP] channels inhibited by 0.5 mM AMP-PNP, a nonhydrolyzable ATP analog, were not stimulated by levosimendan; however, the channels were stimulated by levosimendan in the presence of 30 to 50 microM ADP. Levosimendan stimulates cardiac K[ATP] channels that are suppressed by intracellular ATP. It appears that levosimendan acts synergistically with nucleotide diphosphates. These properties of levosimendan may help protect ischemic myocardium because activation of K[ATP] channels by levosimendan would likely occur in ischemic regions in which intracellular ADP concentration is increased and intracellular ATP concentration is decreased.
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PMID:The novel calcium sensitizer levosimendan activates the ATP-sensitive K+ channel in rat ventricular cells. 933 46

The effects of intracellular application of various concentrations of adenine nucleoside phosphates and nucleotide analogs on the M-type K current (IM) of single neurons isolated from sympathetic ganglia were studied. With 1 mM MgATP intracellularly IM decreased to 25% of its initial level 39 min after the start of whole-cell recording. In the absence of ATP the current decreased more rapidly. Addition of glucose and pyruvate extracellularly was equivalent to adding 1 mM MgATP intracellularly. AMP-PNP, a nonhydrolyzable ATP analog, at a concentration of 1 or 3 mM was unable to maintain IM in the absence of ATP. When ATP and AMP-PNP were combined in the pipette, however, the maintenance of IM was prolonged. A series of nucleotides and analogs have been combined with ATP to test for their ability to maintain IM and to alter calcineurin phosphatase activity. There was a positive correlation between the ability of a nucleotide to prevent the rundown of IM and its ability to inhibit calcineurin phosphatase activity. These findings show that the amplitude of IM is dually regulated by cellular levels of adenine nucleotide diphosphates and triphosphates. A hydrolyzable form of ATP is necessary to maintain the M current. The maintenance of IM is further enhanced by the simultaneous presence of ADP or other adenine nucleotides that alter calcineurin activity, but not by higher concentrations of ATP alone. These results are consistent with regulation of IM by phosphorylation events that maintain IM and dephosphorylation events that lead to current rundown.
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PMID:Regulation of M-type potassium current by intracellular nucleotide phosphates. 969 18

Inwardly rectifying K+ current (IKir) in freshly isolated bovine retinal pigment epithelial (RPE) cells was studied in the whole cell recording configuration of the patch-clamp technique. When cells were dialyzed with pipette solution containing no ATP, IKir ran down completely in <10 min [half time (t1/2) = 1.9 min]. In contrast, dialysis with 2 mM ATP sustained IKir for 10 min or more. Rundown was also prevented with 4 mM GTP or ADP. When 0.5 mM ATP was used, IKir ran down by approximately 71%. Mg2+ was a critical cofactor because rundown occurred when the pipette solution contained 4 mM ATP but no Mg2+ (t1/2 = 1.8 min). IKir also ran down when the pipette solution contained 4 mM Mg2+ + 4 mM 5'-adenylylimidodiphosphate (t1/2 = 2.7 min) or 4 mM adenosine 5'-O-(3-thiotriphosphate) (t1/2 = 1.9 min), nonhydrolyzable and poorly hydrolyzable ATP analogs, respectively. We conclude that the sustained activity of IKir in bovine RPE requires intracellular MgATP and that the underlying mechanism may involve ATP hydrolysis.
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PMID:ATP-dependent regulation of inwardly rectifying K+ current in bovine retinal pigment epithelial cells. 981 87


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