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

---

Query: EC:2.7.11.11 (AMPK)
12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Voltage-dependent sodium channels from a variety of tissues are known to be phosphorylated by the cAMP-dependent protein kinase, protein kinase A. However, the functional significance of sodium channel phosphorylation is not clearly understood. Using whole-cell voltage-clamp techniques, we show that sodium currents (INas) in rabbit cardiac myocytes are enhanced by isoproterenol (ISO). This enhancement of INa by ISO 1) is holding potential dependent, 2) can be mimicked by forskolin and dibutyryl cAMP, and 3) is accompanied by an increase in the rate of Na+ channel inactivation. In single-channel, inside-out patch experiments, the catalytic subunit of protein kinase A also enhances INa and increases the rate of inactivation, suggesting that cardiac Na+ channel phosphorylation may be physiologically important. Addition of the protein kinase A inhibitor to the pipette solution in whole-cell experiments blocks the stimulatory effect of forskolin without blocking the effect of ISO, suggesting that ISO also enhances INa through a cAMP-independent pathway. To determine if ISO may stimulate INa through a direct G protein pathway, single channels were recorded in the presence of the Gs-activating GTP analogue, GTP gamma S, and the stimulatory G protein subunit, Gs alpha. Both of these agents enhanced INa without affecting the rate of Na+ channel inactivation. These results suggest that ISO enhances rabbit cardiac INa through a dual (direct and indirect) G protein regulatory pathway.
...
PMID:Enhancement of rabbit cardiac sodium channels by beta-adrenergic stimulation. 130 15

Antibodies were raised against three peptides corresponding to the potential protein phosphorylation sites of rat-brain sodium channels by the cAMP-dependent protein kinase (PKA). One of the antibody against sequence (C561-575) reacted to the channel molecule. This immunoreaction occurred in a sequence-specific manner, as it was inhibited by the antigen peptide itself but not inhibited by two other peptides. Although PKA phosphorylates two synthetic peptides, C561-575 and C681-689, of the three, anti-(C561-575) antibody can only inhibit the phosphorylation of peptide (C561-575). PKA catalyzed the incorporation of 3.1-3.5 mol of phosphates into the alpha subunit of the purified sodium channel. The anti-(C561-575) antibody inhibited the channel phosphorylation by 40%. Digestion of the phosphorylated sodium channel with lysyl endoproteinase yielded four major phosphorylated fragments of 3.5, 5.0, 7.0, and 10 kDa. However, similar digestion of the channel that was phosphorylated in the presence of anti-(C561-575) antibody did not yield the phosphorylated fragment of 3.5 kDa and gave the 7.0 kDa fragment in reducing yield. Inspection of these phosphorylated fragments by the predicted sizes of the peptide fragments containing the five potential phosphorylation sites gives a conclusion that anti-(C561-575) antibody inhibits the phosphorylation on Ser-573 completely, and on either Ser-610 or Ser-623 partially, probably due to their proximity orientation in the tertiary structure.
...
PMID:A site-directed antibody that inhibits phosphorylation of the rat-brain sodium channel by cyclic-AMP-dependent protein kinase. 133 1

Immunoprecipitation, radiophosphorylation and SDS-PAGE autoradiography enable the characterization of sodium channel polypeptides in the central nervous system of insects belonging to four phylogenetically distinct orders: grasshoppers, cockroaches, flies and moth larvae. It has been shown that the insect sodium channels: (1) Are recognized by the previously described (Gordon et al. (1988) Biochemistry 27, 7032-7038) site directed antibodies corresponding to a highly conserved segment linking the homologous domains III and IV in the vertebrate sodium channel alpha subunits. (2) Serve as substrates for phosphorylation by cAMP-dependent protein kinase. (3) Are devoid of disulfide linkage to smaller subunits unlike sodium channels in vertebrate brain. (4) Are glycoproteins as shown in the grasshopper by the decrease of apparent molecular weight following endoglycosidase F treatment and specific binding to the lectins concanavalin A and wheat germ agglutinin. (5) Reveal a diversity with regard to their (a) apparent molecular masses which range from 240 to 280 kDa and (b) V8 proteinase digestion phosphopeptides indicating either differences in the positioning of the enzymatic cleavage and/or phosphorylation sites. These results provide the first evidence for structural diversity of sodium channel subtypes among various insect orders and are compared to their mammalian counterparts.
...
PMID:Sodium channel polypeptides in central nervous systems of various insects identified with site directed antibodies. 216 10

We have studied cAMP-dependent phosphorylation of sodium channels in rat brain neurons maintained in primary culture. In back phosphorylation studies, cells were treated with drugs to increase intracellular cAMP and sodium channels were solubilized and isolated by immunoprecipitation. Surface and intracellular pools of sodium channels were isolated separately. Purified channels were then phosphorylated with [gamma-32P]ATP by the catalytic subunit of cAMP-dependent protein kinase to incorporate 32P into available cAMP-dependent phosphorylation sites. The amount of 32P incorporated in vitro is inversely proportional to the extent of endogenous phosphorylation. Incubation of cells with forskolin (0.1-100 microM), 8-Br-cAMP (0.1-10 mM), or isobutylmethylxanthine (0.01-1.0 mM) inhibited subsequent incorporation of 32P into isolated sodium channels by 70-80%, indicating that treatment of cells with these drugs had increased endogenous phosphorylation to nearly maximum levels. The phosphopeptides phosphorylated in vivo and in vitro were identical. To examine the magnitude of basal phosphorylation and the extent of stimulated phosphorylation, the amount of 32P incorporated into sodium channels from control and stimulated cells was compared to that from matched samples which had been dephosphorylated with calcineurin. Sodium channels from control cells incorporated approximately 2-fold more 32P after dephosphorylation, indicating that cAMP-dependent sites on the channel are at least 47% phosphorylated in the basal state. Sodium channels from forskolin-treated cells incorporated 7-8-fold more 32P after dephosphorylation, indicating that cAMP-dependent phosphorylation sites are 80-90% phosphorylated after stimulation. Cell surface and intracellular pools of sodium channels were phosphorylated similarly. In cells metabolically labeled with 32P, cell surface sodium channels incorporated 2.7 mol of phosphate/mol of channel. Forskolin stimulated 32P incorporation into sodium channels 1.3-fold, consistent with the results obtained by back phosphorylation. We conclude that the rat brain sodium channel is substantially phosphorylated in both the cell surface and intracellular pools in vivo in unstimulated rat brain neurons, and the extent of phosphorylation is increased to 80-90% of maximum phosphorylation by agents that elevate intracellular cAMP.
...
PMID:Cyclic-AMP-dependent phosphorylation of voltage-sensitive sodium channels in primary cultures of rat brain neurons. 244 66

Cyclic AMP-dependent protein kinase catalyzes the incorporation of 3-4 mol of phosphate into the alpha subunit of rat brain sodium channels in vitro or in situ. Digestion of phosphorylated sodium channels with CNBr yielded three major phosphorylated fragments of 25, 31, and 33 kDa. These fragments were specifically immunoprecipitated with site-directed antisera establishing their location within an intracellular loop between the first and second homologous domains containing residues 448 to 630 of sodium channel RI or residues 450-639 of sodium channel RII. Five of the seven major tryptic phosphopeptides generated from intact sodium channel alpha subunits were contained in each of the 25-, 31-, and 33-kDa CNBr fragments, indicating that most cAMP-dependent phosphorylation sites are in this domain. Since CNBr digestion of sodium channels which had been metabolically labeled with 32P in intact neurons yielded the same phosphorylated fragments, the phosphorylated region we have identified is the major location of phosphorylation in situ. Only serine residues were phosphorylated by cAMP-dependent protein kinase in vitro, while approximately 16% of the phosphorylation in intact neurons was on threonine residues that must lie outside the domain we have identified. Since this domain is phosphorylated in intact neurons, our results show that it is located on the intracellular side of the plasma membrane. These results are considered with respect to models for the transmembrane orientation of the alpha subunit.
...
PMID:Identification of an intracellular domain of the sodium channel having multiple cAMP-dependent phosphorylation sites. 244 73

The effects of pyrethroids and DDT on the alpha-subunit protein of the rat brain sodium channel were studied by using both native and exogenously added cAMP-dependent protein kinases. For this purpose, the sodium channel was partially purified, using the method of Hartshorne and Catterall [J Biol Chem 259: 1667-1675, 1984], and 32P-phosphorylated using [gamma-32P]ATP and exogenously added catalytic subunit of cAMP-dependent protein kinase. By comparing the phosphorylation patterns of the isolated sodium channel to those of the partially purified or unpurified (i.e. intact synaptosomes) preparations, it was concluded that the alpha-subunit of the voltage-sensitive sodium channel protein is the only phosphorylatable protein present at the 260 kD molecular weight range on the sodium dodecyl sulfate-polyacrylamide gel electrophoretogram. Phosphorylation of the alpha-subunit was induced by depolarization, and this process was inhibited by 10(-6) to 10(-10) M 1R-deltamethrin, but not by 1S-deltamethrin, the latter being an inactive enantiomer of the former. DDT produced a similar effect, but only at a higher concentration range. By using lysed synaptosomal membranes, it was possible to study the direct effects of these compounds on the alpha-subunit, which were similar to those produced by depolarization of intact synaptosomes.
...
PMID:Modification by pyrethroids and DDT of phosphorylation activities of rat brain sodium channel. 254 81

The alpha subunit of the rat brain sodium channel is phosphorylated by cAMP-dependent protein kinase in vitro and in situ at multiple sites which yield seven tryptic phosphopeptides. Phosphopeptides 1-4 and 7 are derived from phosphorylation sites between residues 554 and 623 in a single large CNBr fragment from the cytoplasmic segment connecting homologous domains I and II of the alpha subunit (Rossie, S., Gordon, D., and Catterall, W. A. (1987) J. Biol. Chem. 262, 17530-17535). In the present work, antibodies were prepared against a synthetic peptide corresponding to residues 676-692 (AbSP15), which contain one additional potential phosphorylation site at Ser686-Ser687 in a different predicted CNBr fragment of this same intracellular segment. AbSP15 recognizes native and denatured sodium channels specifically and immunoprecipitates phosphorylated CNBr fragments of low molecular mass that contain a new site phosphorylated by cAMP-dependent protein kinase. Comparison of tryptic phosphopeptides derived from intact alpha subunits with those derived from the phosphorylated CNBr fragments isolated by immunoprecipitation with AbSP15 indicates that the two previously unidentified phosphopeptides 5 and 6 derived from the intact alpha subunit arise from phosphorylation of the site containing Ser686-Ser687. These results identify a new cAMP-dependent phosphorylation site and show that the major cAMP-dependent phosphorylation sites of the rat brain sodium channel, which are phosphorylated both in vitro and in intact neurons, are all located in a cluster between residues 554 and 687 in the intracellular segment between domains I and II.
...
PMID:Phosphorylation of the alpha subunit of rat brain sodium channels by cAMP-dependent protein kinase at a new site containing Ser686 and Ser687. 254 90

Antibodies against a peptide (SP19) corresponding to a highly conserved, predicted intracellular region of the sodium channel alpha subunit bind rat brain sodium channels with a similar affinity as the peptide antigen, indicating that the corresponding segment of the alpha subunit is fully accessible in the intact channel structure. These antibodies recognize sodium channel alpha subunits from rat or eel brain, rat skeletal muscle, rat heart, eel electroplax, and locust nervous system. alpha subunits from all these tissues except rat skeletal muscle are substrates for phosphorylation by cAMP-dependent protein kinase. Disulfide linkage of alpha and beta 2 subunits was observed for both the RI and RII subtypes of rat brain sodium channels and for sodium channels from eel brain but not for sodium channels from rat heart, eel electroplax, or locust nerve cord. Treatment with neuraminidase reduced the apparent molecular weight of sodium channel alpha subunits from rat and eel brain and eel electroplax by 22,000-58,000, those from heart by 8000, and those from locust nerve cord by less than 4000. Our results provide the first identification of sodium channel alpha subunits from rat heart and locust brain and nerve cord and show that sodium channel alpha subunits are expressed with different subunit associations and posttranslational modifications in different excitable tissues.
...
PMID:Biochemical properties of sodium channels in a wide range of excitable tissues studied with site-directed antibodies. 284 76

The alpha subunit of the sodium channel purified from rat brain is rapidly and selectively phosphorylated by the catalytic subunit of cAMP-dependent protein kinase to a level of 3 to 4 mol of 32P/mol of saxitoxin-binding activity. The rate of phosphorylation is comparable to that of the synthetic peptide analog of the phosphorylation site of pyruvate kinase, one of the best substrates for cAMP-dependent protein kinase. An endogenous cAMP-dependent protein kinase that is present in the partially purified sodium channel preparations also selectively phosphorylates the alpha subunit. The specificity and rapidity of the phosphorylation reaction are consistent with the hypothesis that the alpha subunit is phosphorylated by cAMP-dependent protein kinase in vivo.
...
PMID:Selective phosphorylation of the alpha subunit of the sodium channel by cAMP-dependent protein kinase. 628 61

In purified preparations of voltage-sensitive sodium channels, the alpha subunit is selectively phosphorylated by cAMP-dependent protein kinase (Costa, M. R. C., Casnellie, J. E., and Catterall, W. A. (1982) J. Biol. Chem., 7918-7921). We have developed methods to measure sodium channel phosphorylation in both lysed synaptosomal membranes and intact synaptosomes. Incubation of lysed synaptosomal membranes with exogenously added catalytic subunit of cAMP-dependent kinase and [gamma-32P]ATP resulted in rapid phosphorylation of the alpha subunit as detected by specific immuno-precipitation, sodium dodecyl sulfate-gel electrophoresis, and autoradiography. Analysis of tryptic phosphopeptides revealed five major sites of reaction. The level of phosphorylation of these sites on the sodium channel in intact synaptosomes was monitored using a rephosphorylation method in which those sites not phosphorylated in situ were labeled with [gamma-32P]ATP and exogenously added protein kinase after lysis of the synaptosomes. Incubation of synaptosomes with 8-Br-cAMP completely blocked labeling of the alpha subunit in rephosphorylation indicating marked stimulation of phosphorylation of the sites on the sodium channel in situ. Phosphorylation was complete in 15 s and all four of the tryptic phosphopeptides detected under these conditions could be phosphorylated in situ. These results show that the sodium channel can be rapidly phosphorylated by endogenous cAMP-dependent protein kinase in intact synaptosomes. In addition, since ATP and protein kinase are only available inside the synaptosomes, they also show that the alpha subunit is a transmembrane polypeptide exposed on both sides of the synaptosomal membrane. The functional consequences of 8-Br-cAMP-stimulated phosphorylation were examined using ion flux and neurotoxin-binding methods. Binding of saxitoxin and scorpion toxin were unaffected, but neurotoxin-activated 22Na+ influx mediated by the sodium channel was reduced 16 to 26% (P less than 0.01) under various experimental conditions. The potential physiological significance of this action is considered.
...
PMID:Cyclic AMP-dependent phosphorylation of the alpha subunit of the sodium channel in synaptic nerve ending particles. 633 Jan 3


1 2 Next >>

---