Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

ATP-sensitive K+ (KATP) channels are thought only to open during conditions of metabolic impairment (e.g., myocardial ischemia). However, the regulation of KATP channel opening during ischemia remains poorly understood. We tested whether thiol (SH) group oxidation, which is known to occur during ischemia, may be involved in KATP channel regulation. Inside-out membrane patches were voltage clamped at a constant potential (O mV) in asymmetrical K+ solutions. The effects of compounds that specifically modify SH groups [p-chloromercuri-phenylsulfonic acid (pCMPS), 5-5'-dithio-bis(2-nitrobenzoic acid) [DTNB], and thimerosal] were tested. The membrane-impermeable compound, pCMPS (> or = 5 microM), caused a quick and irreversible inhibition of KATP channel activity. The reducing agent, dl-dithiothreitol (DTT) (3 mM) was able to reverse this inhibition. DTNB (500 microM) caused a rapid, but spontaneously reversible, block of KATP channel activity. After DTNB, no change was observed in single channel conductance. Oxidized glutathione (GSSG, 3 mM) did not block KATP channel activity. Thimerosal (100-500 microM) induced a DTT-reversible block of partially rundown KATP channels, or channels that underwent complete rundown; these channels were reactivated with trypsin (1 mg/ml). Thimerosal did not block KATP channels that had a high degree of activity. However, the ATP sensitivity was decreased; the concentration of ATP needed to half-maximally inhibit the channel (Ki) was increased from 47 +/- 12 to 221 +/- 35 microM (n = 6, P < 0.05). This was not due to a spontaneous change with time.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effects of thiol-modifying agents on KATP channels in guinea pig ventricular cells. 750 58

The effects of the proteolytic enzyme trypsin upon ATP-sensitive potassium (KATP) channel activity were examined in the CRI-G1 insulin-secreting cell line. Trypsin activated channels only when applied to the intracellular surface of the cell membrane. The activation could be prevented by the concomitant application of trypsin inhibitor or by heat inactivation of the enzyme. The trypsin-induced change in channel activity was accompanied by a reduction in the rate of channel rundown. However, trypsin did not affect the mean single channel conductance (55.2 pS), the ionic selectivity, or rectification of the KATP channel. Concentration response curves for various KATP channel inhibitors were constructed in the presence and absence of intracellular trypsin. The EC50 for tolbutamide was shifted from 30.0 +/- 4.5 microM, with 100 micrograms/ml heat-inactivated trypsin present to 9.7 +/- 1.0 mM with active trypsin in the intracellular solution. Treatment of the cells' external surface with 1 mg/ml trypsin did not alter the potency of tolbutamide. Intracellular trypsin also produced a significant fall in the potency of glibenclamide, meglitinide, and phentolamine but did not alter the effectiveness of thiopentone. Radioligand binding studies demonstrated a total loss of 3H-labeled glibenclamide binding when the intracellular surface of the cells was exposed to trypsin. In contrast, 3H-labeled glibenclamide binding was not affected when the enzyme was applied to the external surface. Trypsin treatment, therefore, alters a number of characteristics of KATP channel pharmacology, and we suggest that this is due to action at possibly more than one site but includes the functional cleavage of the sulfonylurea receptor from the KATP channel.
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PMID:The effects of trypsin on ATP-sensitive potassium channel properties and sulfonylurea receptors in the CRI-G1 insulin-secreting cell line. 805 51

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

1. The adenosine 5'-triphosphate (ATP)-sensitive K+ channel current was recorded in guinea-pig ventricular myocytes using the patch clamp technique with inside-out patch configuration. Modification of the channel activity by intracellular application of an endoprotease trypsin was studied, and was related to a possible model of regulation of this channel. 2. Maximal ATP-sensitive K+ channel activity was observed immediately upon formation of inside-out patches in the ATP-free internal solution, thereafter activity declined both spontaneously and gradually with time; a phenomenon known as rundown. When trypsin (1 mg/ml) was applied to the intracellular side of the membrane upon formation of inside-out patches, spontaneous run-down did not occur, and this trypsin action was irreversible. Neither trypsin (1 mg/ml) applied with trypsin inhibitor (0.25 mg/ml) nor heat-denatured trypsin (1 mg/ml) could mimic this effect. When trypsin was applied to the patches after run-down, channels were reactivated at approximately 13 min. 3. Treatment with trypsin did not affect unitary current amplitude, channel gating kinetics, or sensitivity to intracellular ATP. 4. Intracellularly applied Ca2+ induced run-down of channel activity in a dose-dependent manner. In membrane patches that were treated with trypsin (1 mg/ml) for 20 min, intracellularly applied Ca2+ up to 1 mM did not induce run-down of channel activity. 5. Intracellular application of an exopeptidase, carboxypeptidase A (1 mg/ml), but not Leu-aminopeptidase (0.5 mg/ml), prevented spontaneous or Ca(2+)-induced run-down of channel activity. 6. As postulated for several other channels, such as Na+ and Ca2+ channels, there may be a possible 'chemical gate' that is responsible for run-down of this channel activity. Application of trypsin might somehow modify this 'chemical gate', resulting in prevention of spontaneous or Ca(2+)-induced run-down. This target site for trypsin may be situated on the carboxy-terminus of the channel proteins, or of associated regulatory units. Because ATP sensitivity remained intact after trypsin treatment, the trypsin-selective site for channel inhibition is not related physically to the ATP binding site.
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PMID:Modification of the adenosine 5'-triphosphate-sensitive K+ channel by trypsin in guinea-pig ventricular myocytes. 841 Jul 13

1. We have used patch-clamp methods to study the effects of the detergents, Cremophor, Tween 80 and Triton X100 on the K(ATP) channel in the pancreatic beta-cell from mouse. 2. All three detergents blocked K(ATP) channel activity with the following order of potency: Tween 80 (Ki< approximately 83 nM)>Triton X100 (Ki=350 nM)>Cremophor. In all cases the block was poorly reversible. 3. Single-channel studies suggested that at low doses, the detergents act as slow blockers of the K(ATP) channel. 4. Unlike the block produced by tolbutamide, that produced by detergent was not affected by intracellular Mg2+-nucleotide, diazoxide or trypsin treatment, nor did it involve an acceleration of rundown or increase in ATP sensitivity of the chanel. 5. The detergents could block the pore-forming subunit, Kir6.2deltaC26, which can be expressed independently of SUR1 (the regulatory subunit of the K(ATP) channel). These data suggest that the detergents act on Kir6.2 and not SUR1. 6. The detergents had no effect on another member of the inward rectifier family: Kir1.1a (ROMK1). 7. Voltage-dependent K-currents in the beta-cell were reversibly blocked by the detergents with a far lower potency than that found for the K(ATP) channel. 8. Like other insulin secretagogues that act by blocking the K(ATP) channel, Cremophor elevated intracellular Ca2+ in single beta-cells to levels that would be expected to elicit insulin secretion. 9. Given the role of the K(ATP) channel in many physiological processes, we conclude that plasma borne detergent may have pharmacological actions mediated through blockage of the K(ATP) channel.
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PMID:Inhibition of the ATP-sensitive potassium channel from mouse pancreatic beta-cells by surfactants. 964 78