Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.16 (calcineurin)
17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. Rundown of L-type calcium channels was studied in inside-out patches made from single isolated rabbit ventricular myocytes, using barium as the charge carrier. 2. In the cell-attached patches single-channel activity was stable for more than 15 min after the patch pipette sealed. beta-Receptor stimulation by isoprenaline caused a characteristic increase in opening probability and the appearance of prolonged openings. When the patch was excised to the inside-out configuration and exposed to a simple ionic solution, channel activity disappeared within 1-2 min and never reappeared spontaneously. 3. After rundown of L-type channel activity in the excised patch, exposure of the inside face of the patch to MgATP and the catalytic subunit of the cyclic AMP-dependent protein kinase (PKAc) resulted in recovery of Ca2+ channel activity. Under these conditions channel activity could be even greater than under control cell-attached conditions, resembling channel activity after exposure to isoprenaline. This recovery of activity persisted many minutes, usually until the patch was lost. Addition of MgATP alone caused a small transient increase in channel activity in some patches. 4. Recovery of activity by MgATP and PKAc could be prevented by prior exposure of the excised patch to protein kinase inhibitor (PKI), or it could be abruptly terminated by exposure to PKI after recovery of activity. Addition to the pipette solution of okadaic acid, a protein phosphatase inhibitor, greatly slowed rundown. These findings support the proposal that dephosphorylation is an important component of rundown, and that phosphorylation is needed for channel opening activity. 5. Single-channel conductance was not altered by patch excision, but it was reduced after exposure of the excised patch to MgATP and PKAc. Mg2+ was responsible for this effect, probably by direct channel block from the inside, and Mg2+ also caused a negative shift in the channel activation, as expected from shielding of inside fixed negative charges.
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PMID:Phosphorylation restores activity of L-type calcium channels after rundown in inside-out patches from rabbit cardiac cells. 133 10

A calcium-activated potassium channel in posterior pituitary nerve terminals was modulated by phosphorylation and dephosphorylation. Nearly every patch of membrane containing this channel also contained both membrane bound protein phosphatase and membrane-bound protein kinase. By examining the statistical and kinetic nature of phosphorylation and dephosphorylation in excised patches, it was possible to evaluate two contrasting models for these enzymatic reactions. One of these models treated catalysis as an intermolecular process in which the enzyme and substrate are separate molecular species that diffuse and encounter one another during collisions. The second model treated catalysis as an intramolecular process in which the enzyme and substrate reside within a stable macromolecular complex. The study began with a Poisson analysis of the distribution of channel number in patches, and of the number of protein phosphatase-free and protein kinase-free patches. Subsequent kinetic analysis of dephosphorylation yielded an estimate of the mean number of protein phosphatase molecules per patch that was similar to the value obtained from Poisson analysis. Because these two estimates were independent predictions based on the intermolecular model, their agreement supported this model. Analysis of channel number in protein phosphatase-free patches and of the rarity of patches showing partial but incomplete rundown provided additional support for the intermolecular model over the intramolecular model. Furthermore, dephosphorylation exhibited monotonic kinetics with a rate well below the diffusion limit. Thus, several different lines of analysis support the intermolecular model for dephosphorylation, in which the protein phosphatase must encounter its substrate to effect catalysis. In contrast to the monotonic kinetics of dephosphorylation, the phosphorylation reaction exhibited sigmoidal kinetics, with a rate that depended on membrane potential. Voltage dependence is an unlikely property for a kinetic step involving encounters resulting from diffusion. Furthermore, the velocity of the phosphorylation reaction exceeded the diffusion limit, and this observation is inconsistent with the intermolecular model. Thus, both intermolecular and intramolecular enzymatic mechanisms operate in the modulation of the calcium-activated potassium channel of the posterior pituitary. These studies provide a functional characterization of the interactions between enzyme and substrate in intact patches of cell membrane.
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PMID:Intramolecular and intermolecular enzymatic modulation of ion channels in excised membrane patches. 752 Dec 26

1. Acute homologous desensitization of mu-opioid receptor-induced currents was pharmacologically characterized in locus coeruleus (LC) neurones by use of intracellular and whole cell recording in superfused brain slices. 2. Following desensitization of opioid receptors by perfusion with a high concentration of [Met5] enkephalin (ME) for 5 min, there was a reduction in the maximum response and a rightward shift of the concentration-response curves for ME, [D-Ala2, N-MePhe4, Gly-ol]enkephalin (DAMGO) and normorphine. 3. By simultaneously fitting the operational model to the paired pre- and post-desensitization concentration-response data for each agonist, estimates of the level of desensitization were obtained. The values obtained for the three agonists (between 88% and 96%) were similar and did not vary according to the efficacy of the agonist used. 4. Use of whole cell patch recording techniques caused a slow rundown in the amplitude of ME currents (approx. 40% reduction over 60 min) but did not greatly affect the expression of acute desensitization of opioid currents. 5. When included in the patch recording solution, the phosphatase inhibitors, microcystin (50 nM-4 microM) and okadaic acid (1 microM) had no effect on the induction of desensitization or the normal ability of opioid or alpha 2-adrenoceptors to produce currents. Microcystin decreased the rate of recovery of the ME (300 nM) currents following desensitization; however, okadaic acid had little effect on the rate of recovery from desensitization. 6. Strong calcium buffering with BAPTA (10-20 mM) had no effect on desensitization or the recovery from desensitization. 6. Strong calcium buffering with BAPTA (10-20 mM) had no effect on desensitization or the recovery from desensitization.7 These results suggest that acute homologous desensitization of micro-opioid receptors in LC neurones entails a rapid loss of responsiveness that involves a majority of the receptor population. The mechanism by which desensitization is reversed may involve a non-calcium-dependent protein phosphatase but the processess that cause desensitization remain unclear.
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PMID:Characterization of acute homologous desensitization of mu-opioid receptor-induced currents in locus coeruleus neurones. 758 22

1. N-methyl-D-aspartate (NMDA) channel activity was studied on cultured rat hippocampal neurons in whole-cell voltage-clamp mode. NMDA responses were evoked by rapid application of NMDA and the cytosol was modified using pipette dialysis and intracellular perfusion. 2. In the presence of 2 mM [Ca2+]o with 2.4 mM BAPTA (1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) and 0.4 mM Ca2+ in the whole-cell pipette, the response evoked by regular applications of 10 microM NMDA gradually decreased during prolonged whole-cell recording. After 25 min the peak current was reduced to 56 +/- 1.6% of control. Channel 'rundown' could be prevented by inclusion of an ATP regenerating solution in the pipette. 3. Rundown did not occur in Ca(2+)-free medium even in the absence of added ATP regenerating solution. Rundown was also prevented by increasing [BAPTA]i to 10 mM whereas raising [Ca2+]i by inhibiting the Na(+)-Ca2+ exchanger or by perfusing the patch pipette with high [Ca2+]i (15-1000 microM) reversibly inhibited the NMDA current. By contrast, the rundown of kainate responses was Ca(2+)-independent. 4. The rate and reversibility of rundown was use-dependent. Rundown did not occur with infrequent NMDA applications (0.2/min). Following channel rundown in Ca(2+)-containing medium, a 5 min pause in agonist applications or adding ATP regenerating solution by intracellular perfusion resulted in complete recovery. However, rundown did not recover following large currents evoked by 300 microM NMDA or when 10 mM EGTA was used as the intracellular buffer. Protease inhibitors did not prevent irreversible rundown. 5. ATP-gamma-S (4 mM) was less effective than the ATP regenerating solution in preventing rundown. Likewise, intracellular dialysis with alkaline phosphatase, phosphatase 1 or calcineurin did not induce rundown and addition of phosphatase inhibitors also did not block rundown. Thus receptor dephosphorylation did not appear to be primarily responsible for channel rundown. 6. The mean open time and unitary conductance of the NMDA channel were unaffected by rundown as estimated by fluctuation analysis. The conductance was 42.8 +/- 2.9 nS before and 43.7 +/- 2.8 nS after rundown. The mean open times were 17.3 and 4.0 ms before and 15.9 and 4.0 ms after rundown. However the open probability was reduced following rundown as determined by the onset of MK-801 block of steady-state NMDA currents. 7. Our results suggest that an increase in intracellular calcium leads to channel rundown during whole-cell recording by reducing the open probability of the NMDA channel.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Rundown of N-methyl-D-aspartate channels during whole-cell recording in rat hippocampal neurons: role of Ca2+ and ATP. 830 51

Vasodilating agents induce relaxation of mesangial cells, in part through cGMP-mediated activation of large calcium-activated potassium channels (BKCa). Normally quiescent in cell-attached patches, the response of BKCa to nitric oxide, atrial natriuretic peptide, and dibutyryl cGMP (Bt2cGMP) is characterized by a biphasic increase and then decrease ("rundown") in open probability. Using the patch-clamp method in conjunction with phosphatase inhibitors, we investigated whether the run-down phase was the result of dephosphorylation by an endogenous protein phosphatase. In cell-attached patches, cantharidic acid (500 nM), okadaic acid (100 nM), and calyculin A (100 nM), nondiscriminant inhibitors of protein phosphatases 1 (PP1) and 2A (PP2A) at these concentrations, caused a significantly greater and sustained response of BKCa to Bt2cGMP. Within 2 min, the response of BKCa to the combination of cantharidic acid and Bt2cGMP was greater than the response to these agents added separately. Incubation of mesangial cells with okadaic acid for 20 min at a concentration (5 nM) specific for PP2A increased the basal open probability of BKCa and completely inhibited rundown after activation by Bt2cGMP. Incubation with calyculin A (10 nM), a more potent inhibitor of PP1, did not affect BKCa activity. In inside-out patches, Bt2cGMP plus MgATP caused a sustained activation of BKCa that was inhibited by exogenous PP2A but not PP1. It is concluded that either BKCa or a tightly associated regulator of BKCa is a common substrate for endogenous cGMP-activated protein kinase, which activates BKCa, and PP2A, which inactivates BKCa, in human mesangial cells.
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PMID:Regulation of large calcium-activated potassium channels by protein phosphatase 2A. 909 28

1. We have used non-stationary variance analysis to examine the single channel conductance and the probability of channel opening at the peak of the homomeric GluR6 response (Po,peak) to 100-200 ms application (10-90% exchange time, 0.3 ms) of glutamate onto excised membrane patches from transiently transfected human embryonic kidney cells (HEK 293). 2. Our determinations of both Po,peak and single channel conductance of simulated current responses are insensitive to system filtering, response rise time, desensitization rate and measured variation in our drug perfusion speed. Isolation of stochastic current fluctuations using the local mean response waveform minimizes problems associated with modest rundown of response amplitude during the experiment. 3. The slope conductance calculated from the weighted mean unitary currents for the channels activated in response to glutamate application is 16 pS. Chord conductance between-40 and -80 mV is independent of agonist concentration. Conversion of the codon for glutamine621 to arginine (Q621R) by RNA editing reduces conductance by more than 35-fold to less than 0.4 pS without changing response time course, desensitization, or Po,peak. 4. Po,peak is high at saturating glutamate concentrations (0.65 +/- 0.23; mean +/- S.D.) and varies with agonist concentrations. The half-maximally effective glutamate concentration (EC50) determined for Po,peak (0.2 mM; Hill slope = 0.6) is similar to that determined for the macroscopic peak current amplitude (0.5 mM; Hill slope = 1.0) in response to rapid agonist application. 5. Inclusion of the purified catalytic subunit of cAMP-dependent protein kinase A (PKA) in the patch pipette increases Po,peak to 0.85 +/- 0.12 and co-transfection of cells with a cDNA encoding the catalytic subunit of PKA (C alpha-PKA) increases Po,peak to 0.94 +/- 0.09. 6. Inclusion of purified calcineurin plus its coactivators 200 nM Ca2+ and calmodulin in the patch pipette decreases Po,peak to 0.48 +/- 0.10. The calcineurin-stimulated decrease of Po,peak in cells co-transfected with C alpha-PKA is blocked by 800 nM deltamethrin, a calcineurin inhibitor. Calmodulin, 200 nM Ca2+ and deltamethrin have no effect on Po,peak in the absence of calcineurin. As predicted from its effects on Po,peak, inclusion of calcineurin in the patch pipette accelerates the run-down of whole cell GluR6 responses in cells co-transfected with C alpha-PKA. 7. The effects of both calcineurin and PKA on Po,peak for GluR6 receptors in excised patches occur without any detectable changes to response time course, desensitization, or chord conductance. 8. We conclude that the binding of glutamate to homomeric GluR6 receptors is associated with a high probability of channel opening, which is under the control of two signalling systems that are known to be co-localized at the neuronal membrane: PKA (Po,peak near 1.0) and calcineurin (Po,peak near 0.5).
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PMID:Control of rat GluR6 glutamate receptor open probability by protein kinase A and calcineurin. 937 2

In the present study, rundown of gamma-aminobutyric acid (GABA)-activated Cl- channels was studied in recombinant GABAA receptors stably expressed in human embryonic kidney cells (HEK 293), with conventional whole-cell and amphotericin B-perforated patch recording. When [ATP]i was lowered to 1 mM and resting [Ca++]i was buffered to a relatively high level, the response of alpha 3 beta 2 gamma 2 GABAA receptors to relatively low [GABA] (up to 50 microM) did not show rundown in the whole-cell configuration. However, high [GABA] (greater than 200 microM) induced significant rundown, which was observed by decreases in both the maximum GABA-induced current and GABA EC50. Rundown was prevented completely with a solution containing 4 mM Mg(++)-ATP and low resting [Ca++]i, or during perforated patch recording. The magnitude of rundown was comparable in alpha 1 beta 2 gamma 2 and beta 2 gamma 2 receptors. Neither stimulation nor inhibition of protein kinase A or protein kinase C had a significant effect on rundown. However, sodium metavanadate, an inhibitor of protein tyrosine phosphatase, significantly reduced rundown. In addition, inhibition of protein tyrosine kinase activity by either genistein or lavendustin A induced rundown of the GABA response. Inhibition of the Ca++/calmodulin-dependent phosphatase calcineurin with fenvalerate also prevented rundown of the response to GABA. Our results demonstrate that rundown of GABAA receptor function is concentration-dependent, due to depletion of ATP and/or unbuffered [Ca++]i, and does not depend on the presence or subtype of the alpha subunit. We propose that protein phosphorylation at a tyrosine kinase-dependent site, and a distinct unidentified site, which is dephosphorylated by calcineurin, maintains the function of GABAA receptors.
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PMID:Maintenance of recombinant type A gamma-aminobutyric acid receptor function: role of protein tyrosine phosphorylation and calcineurin. 965 66

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

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

Locus coeruleus (LC) is the significant nucleus for consciousness and it is sensitive to metabolic inhibition. We investigated the effects of a metabolic inhibitor sodium cyanide (NaCN) on the rat dissociated LC neurons using nystatin-perforated patch recordings. Under voltage-clamp (VH=-40 mV), application of NaCN evoked outward currents composed of ATP-sensitive and Ca2+-dependent K+ channel currents (IKATP and IKCa2+). Onset of IKATP was faster than that of IKCa2+. Prolonged application of NaCN brought IKATP rundown but not IKCa2+ rundown. Okadaic acid prevented IKATP rundown, indicating that KATP channels are deactivated by dephosphorylation with protein phosphatase.
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PMID:ATP-sensitive and Ca2+-activated K+ channel activities in the rat locus coeruleus neurons during metabolic inhibition. 1032 Jul 42


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