Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0848283 (rundown)
 502 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The role of membrane-bound protein serine/threonine phosphatases (PP) in modulating the renal ATP-sensitive K+ (KATP) channel was examined using the patch-clamp technique in principal cells of rat cortical collecting duct. In the absence of ATP, channel activity rapidly (11.2 s) declines (channel "rundown") upon excision of the membrane patches into control bath solutions (1 mM Mg2+, Ca2+ free). Both orthovanadate (5 mM), a broad-spectrum inhibitor of phosphatases except for Ca(2+)-dependent PP (PP-2B), and okadaic acid (OA, 1 microM), a potent inhibitor of PP types 1 and 2A (PP-1 and PP-2A), significantly slowed channel rundown. Removal of Mg2+ from the bath also slowed the rundown process. Incubation of cells with OA in the absence of Mg2+ or with orthovanadate in ATP-free solution maintained channel activity at levels of approximately 70% of control values for 3 min after membrane excision. In contrast, Ca2+ (0.1 mM) and calmodulin (1 microM) in the presence of 1 mM Mg2+, a condition in which PP-2B is stimulated, had no significant effect on the channel activity that persisted in the presence of OA and orthovanadate. Application of exogenous PP-2A (1 U/ml) to the cytosolic side of membrane in inside-out patches significantly inhibited channel activity to 35.0% of control, but the inhibitory-effects of PP-1 (1 U/ml) and PP-2B (20 micrograms/ml) were minor. These results suggest that rundown of the renal KATP channel after membrane excision results mainly from dephosphorylation of the channel or an associated protein by membrane-bound phosphatases.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Regulation of ATP-sensitive K+ channel by membrane-bound protein phosphatases in rat principal tubule cell. 757 84

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

The beta cell KATP channel is an octameric complex of four pore-forming subunits (Kir6.2) and four regulatory subunits (SUR1). A truncated isoform of Kir6.2 (Kir6.2DeltaC26), which expresses independently of SUR1, shows intrinsic ATP sensitivity, suggesting that this subunit is primarily responsible for mediating ATP inhibition. We show here that mutation of C166, which lies at the cytosolic end of the second transmembrane domain, to serine (C166S) increases the open probability of Kir6.2DeltaC26 approximately sevenfold by reducing the time the channel spends in a long closed state. Rundown of channel activity is also decreased. Kir6.2DeltaC26 containing the C166S mutation shows a markedly reduced ATP sensitivity: the Ki is reduced from 175 microM to 2.8 mM. Substitution of threonine, alanine, methionine, or phenylalanine at position C166 also reduced the channel sensitivity to ATP and simultaneously increased the open probability. Thus, ATP does not act as an open channel blocker. The inhibitory effects of tolbutamide are reduced in channels composed of SUR1 and Kir6.2 carrying the C166S mutation. Our results are consistent with the idea that C166 plays a role in the intrinsic gating of the channel, possibly by influencing a gate located at the intracellular end of the pore. Kinetic analysis suggests that the apparent decrease in ATP sensitivity, and the changes in other properties, observed when C166 is mutated is largely a consequence of the impaired transition from the open to the long closed state.
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PMID:Molecular analysis of ATP-sensitive K channel gating and implications for channel inhibition by ATP. 972 93

1. The degree of cell-to-cell coupling between ventricular myocytes of neonatal rats appeared well preserved when studied in the perforated version of the patch clamp technique or, in double whole-cell conditions, when ATP was present in the patch pipette solution. In contrast, when ATP was omitted, the amplitude of junctional current rapidly declined (rundown). 2. To examine the mechanism(s) of ATP action, an 'internal perfusion technique' was adapted to dual patch clamp conditions, and reintroduction of ATP partially reversed the rundown of junctional channels. 3. Cell-to-cell communication was not preserved by a non-hydrolysable ATP analogue (5'-adenylimidodiphosphate, AMP-PNP), indicating that the effect most probably did not involve direct interaction of ATP with the channel-forming proteins. 4. An ATP analogue supporting protein phosphorylation but not active transport processes (adenosine 5'-O-(3-thiotriphosphate), ATPgammaS) maintained normal intercellular communication, suggesting that the effect was due to kinase activity rather than to altered intracellular Ca2+. 5. A broad spectrum inhibitor of endogenous serine/threonine protein kinases (H7) reversibly reduced the intercellular coupling. A non-specific exogenous protein phosphatase (alkaline phosphatase) mimicked the effects of ATP deprivation. The non-specific inhibition of endogenous protein phosphatases resulted in the preservation of substantial cell-to-cell communication in ATP-free conditions. 6. The activity of gap junctional channels appears to require both the presence of ATP and protein kinase activity to counteract the tonic activity of endogenous phosphatase(s).
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PMID:ATP counteracts the rundown of gap junctional channels of rat ventricular myocytes by promoting protein phosphorylation. 1008 44

Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are regulated tightly by protein kinases and phosphatases. The regulatory domain of CFTR has about 20 potential sites for phosphorylation by protein kinases A (PKA) and C (PKC). The reason for this large number of sites is not known, however their conservation from fish to humans implies that they play important roles in vivo. PKA is an important activator, and its stimulation of CFTR is enhanced by PKC via mechanisms which are not fully understood. The physiological stimuli of CFTR are not known for some epithelia, and it appears likely that other serine/threonine and even tyrosine kinases also regulate CFTR in particular tissues. Phosphatases that deactivate CFTR have yet to be identified definitively at the molecular level, however CFTR is regulated by a membrane-bound form of protein phosphatase-2C (PP2C) in several cell types. Patch-clamp studies of channel rundown, co-immunoprecipitation, chemical cross-linking studies, and pull-down assays all indicate that CFTR and PP2C are closely associated within a stable regulatory complex. Understanding the regulation of CFTR by PP2C is a priority due to its potential as a target for pharmacotherapies in the treatment of cystic fibrosis.
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PMID:Regulation of the CFTR channel by phosphorylation. 1184 11

Dihydropyridine-sensitive, voltage-activated calcium channels respond to membrane depolarization with two distinct modes of activity: short bursts of very short openings (mode 1) or repetitive openings of much longer duration (mode 2). Here we show that both the dihydropyridine, BayK8644 (BayK), and the inhibitor of SerThr protein phosphatases, okadaic acid, have identical effects on the gating of the recombinant cardiac calcium channel, Ca(V)1.2 (alpha(1)C). Each produced identical mode 2 gating in cell-attached patches, and each prevented rundown of channel activity when the membrane patch was excised into ATP-free solutions. These effects required Ser or Thr at position 1142 in the domain III pore loop between transmembrane segments S5 and S6, where dihydropyridines bind to the channel. Mutation of Ser-1142 to Ala or Cys produced channels with very low activity that could not be modulated by either BayK or okadaic acid. A molecular model of Ca(V)1.2 indicates that Ser-1142 is unlikely to be phosphorylated, and thus we conclude that BayK binding stabilizes mode 2 gating allosterically by either protecting a phospho Ser/Thr on the alpha(1)C subunit or mimicking phosphorylation at that site.
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PMID:Modulation of cardiac Ca(V)1.2 channels by dihydropyridine and phosphatase inhibitor requires Ser-1142 in the domain III pore loop. 1260 Nov 59

ATP-sensitive K+ (K(ATP)) channels are known to be gated by several intracellular molecules, but the gating mechanisms remain unclear. To understand the relationship of channel gating to ligand binding, we studied Kir6.2 channel gating by ATP and protons, which inhibit and activate the channel, respectively. We have previously shown that a threonine residue (Thr71) is critical for the pH sensitivity of Kir6.2 channel. If this site is involved in channel gating rather than ligand binding, it should affect channel gating by both ATP and proton. To test this hypothesis we performed a mutation analysis. Site-specific mutations of Thr71 to a bulky residue reduced the ATP sensitivity by >100-fold and eliminated the pH sensitivity. Single-channel activity of these mutants was stabilized at the open state with no detectable rundown. Mutations to a small amino acid had little effect on the ATP and pH sensitivities. Mutations to intermediate amino acids reduced but did not abolish the ATP and pH sensitivities. Hydrophobicity is not critical, as both polar and nonpolar amino acids are found in each group. Mutation to a positively charged lysine markedly exacerbated the pH- but not ATP-sensitivity, whereas mutation to glutamate moderately reduced ATP and pH sensitivities. These results indicate that the residue mass is critical for Kir6.2 channel gating, a mass that should be below 120 daltons with no charge. The existence of such a site as Thr71 involved in channel gating by both ATP and proton suggests that channel gating in the K(ATP) channel likely is separate from ligand binding.
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PMID:A threonine residue (Thr71) at the intracellular end of the M1 helix plays a critical role in the gating of Kir6.2 channels by intracellular ATP and protons. 1268 99

Protein phosphorylation is crucial for regulating synaptic transmission. We describe a novel mechanism for the phosphorylation of the GABA(A) receptor, which mediates fast inhibition in the brain. A protein copurified and coimmunoprecipitated with the phosphorylated receptor alpha1 subunit; this receptor-associated protein was identified by purification and microsequencing as the key glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Molecular constructs demonstrated that GAPDH directly phosphorylates the long intracellular loop of GABA(A) receptor alpha1 subunit at identified serine and threonine residues. GAPDH and the alpha1 subunit were found to be colocalized at the neuronal plasma membrane. In keeping with the GAPDH/GABA(A) receptor molecular association, glycolytic ATP produced locally at plasma membranes was consumed for this alpha1 subunit phosphorylation, possibly within a single macrocomplex. The membrane-attached GAPDH is thus a dual-purpose enzyme, a glycolytic dehydrogenase, and a receptor-associated kinase. In acutely dissociated cortical neurons, the rundown of the GABA(A) responses was essentially attributable to a Mg(2+)-dependent phosphatase activity, which was sensitive to vanadate but insensitive to okadaic acid or fluoride. Rundown was significantly reduced by the addition of GAPDH or its reduced cofactor NADH and nearly abolished by the addition of its substrate glyceraldehyde-3-phosphate (G3P). The prevention of rundown by G3P was abolished by iodoacetamide, an inhibitor of the dehydrogenase activity of GAPDH, indicating that the GABA(A) responses are maintained by a glycolysis-dependent phosphorylation. Our results provide a molecular mechanism for the direct involvement of glycolysis in neurotransmission.
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PMID:Glyceraldehyde-3-phosphate dehydrogenase is a GABAA receptor kinase linking glycolysis to neuronal inhibition. 1534 27

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel expressed at the apical surface of epithelia. Although the regulation of CFTR by protein kinases is well documented, channel deactivation by phosphatases is not well understood. We find that the serine/threonine phosphatase PP2A can physically associate with the CFTR COOH terminus. PP2A is a heterotrimeric phosphatase composed of a catalytic subunit and two divergent regulatory subunits (A and B). The cellular localization and substrate specificity of PP2A is determined by the unique combination of A and B regulatory subunits, which can give rise to at least 75 different enzymes. By mass spectrometry, we identified the exact PP2A regulatory subunits associated with CFTR as Aalpha and B'epsilon and find that the B'epsilon subunit binds CFTR directly. PP2A subunits localize to the apical surface of airway epithelia and PP2A phosphatase activity co-purifies with CFTR in Calu-3 cells. In functional assays, inhibitors of PP2A block rundown of basal CFTR currents and increase channel activity in excised patches of airway epithelia and in intact mouse jejunum. Moreover, PP2A inhibition in well differentiated human bronchial epithelial cells results in a CFTR-dependent increase in the airway surface liquid. Our data demonstrate that PP2A is a relevant CFTR phosphatase in epithelial tissues. Our results may help reconcile differences in phosphatase-mediated channel regulation observed for different tissues and cells. Furthermore, PP2A may be a clinically relevant drug target for CF, which should be considered in future studies.
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PMID:The cystic fibrosis transmembrane conductance regulator is regulated by a direct interaction with the protein phosphatase 2A. 1623 22