Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The existence of dihydropyridine receptor in crayfish striated muscle was proved by Northern blot analysis and 3H PN 200--110 binding. The alpha 1 subunit is encoded by a 8300 nt mRNA population and is expressed as 190 kD protein in crayfish T-tubular system, which binds 3H PN 200--110 (Bmax 1.5 +/- 0.4 pmol/mg protein and KD 6.2 +/- 0.8 nmol/l). The purified protein is phosphorylated by cAMP-dependent protein kinase. The dihydropyridine receptor in crayfish striated muscle also contains alpha 2 subunit, which on Northern blot gives the same signal as the alpha 2 subunit from rabbit skeletal muscle.
 ...
PMID:The dihydropyridine receptor: expression of 190 kD alpha 1 subunit in crayfish muscle. 165 59

The dihydropyridine receptor is associated with the L-type Ca2+ channel in the cell membrane. In this study we have examined the effects of group-specific modification on dihydropyridine binding in heart sarcolemmal membranes isolated from the rabbit. Specifically, dithiothreitol and glutathione were employed to assess the possible role of disulfide (-SS-) bonds in the binding of [3H]dihydropyridines. NEM, PCMS and iodoacetamide were employed to examine the effect of blocking free sulfhydryl groups (-SH) on the binding of [3H]dihydropyridines to their receptor in heart sarcolemma. Glutathione inhibited [3H]PN200-110 binding to sarcolemmal membranes 100%, with an IC50 value of 50 microM, while DTT inhibited maximally by 75% with an IC50 value in the millimolar range. Alkylation of free sulfhydryl groups by NEM or iodoacetamide inhibited binding of [3H]PN200-110 binding in cardiac sarcolemma approx. 40-60%. Blocking of free sulfhydryl groups by PCMS completely inhibited [3H]PN200-110 binding to their receptor in sarcolemmal membranes in a dose-dependent manner with an IC50 value of 20 microM. These results suggest the involvement of disulfide bonds and free sulfhydryl groups in DHP binding to the L-type Ca2+ channel in heart muscle. We also examined the effect of membrane phosphorylation on the specific binding of the dihydropyridine [3H]nitrendipine to its receptor. Phosphorylation was studied in cardiac sarcolemmal as well as skeletal muscle transverse-tubule membranes. Phosphorylation due to endogenous protein kinase and cAMP-dependent protein kinase was without effect on [3H]nitrendipine binding in both cardiac sarcolemmal and skeletal muscle membranes. Addition of exogenous calmodulin under conditions known to promote Ca2+/calmodulin-dependent phosphorylation increased [3H]nitrendipine binding 20% with no alteration in KD in both types of membrane preparation. These results suggest a role for calmodylin in dihydropyridine binding to L-type Ca2+ channels.
 ...
PMID:Dihydropyridine binding to the L-type Ca2+ channel in rabbit heart sarcolemma and skeletal muscle transverse-tubules: role of disulfide, sulfhydryl and phosphate groups. 215 49

Isolated triads from rabbit skeletal muscle were shown to contain an intrinsic protein kinase which was neither Ca2+/calmodulin-dependent nor cAMP-dependent. The protein substrates phosphorylated by this protein kinase exhibited apparent molecular weights of 300,000, 170,000, 90,000, 80,000, 65,000, 56,000, 52,000, 51,000, 40,000, 25,000, 22,000, and 15,000. Purification of the 1,4-dihydropyridine receptor from phosphorylated triads has demonstrated that the 170,000- and 52,000-Da subunits of the 1,4-dihydropyridine receptor are phosphorylated by this intrinsic protein kinase in isolated triads. Monoclonal antibodies to the 170,000-Da subunit of the dihydropyridine receptor immunoprecipitated the 170,000-Da phosphoprotein from detergent extracts of phosphorylated triads. The mobility of the 170,000-Da phosphoprotein in sodium dodecyl sulfate-polyacrylamide gels was not changed with or without reduction, demonstrating that the 170,000-Da phosphoprotein is not the glycoprotein subunit of the receptor. Our results demonstrate that the 170,000- and 52,000-Da subunits of the dihydropyridine receptor are phosphorylated by an intrinsic protein kinase in isolated triads. In addition, our results also demonstrate that the 175,000-Da glycoprotein subunit of the dihydropyridine receptor is not phosphorylated in isolated triads by the intrinsic protein kinase, cAMP-dependent protein kinase, or endogenous Ca2+/calmodulin-dependent protein kinase.
 ...
PMID:Phosphorylation of the 1,4-dihydropyridine receptor of the voltage-dependent Ca2+ channel by an intrinsic protein kinase in isolated triads from rabbit skeletal muscle. 243 99

The purified 1,4-dihydropyridine receptor from skeletal muscle has been incorporated into planar bilayers, and its channel characteristics have been investigated. Conductances showed the characteristics of an L-type Ca2+ channel: divalent cation selectivity (PBa/PNa approximately equal to 30), blockage of Na+ conductance by micromolar Ca2+, and blockage of the Ca2+ channel by D890 and by Cd2+. The alpha 1 subunit of the receptor must be phosphorylated by the cAMP-dependent protein kinase to give channel activity. BAY K 8644 did not activate nonphosphorylated channels, and (+)-PN200-110 caused dramatic prolongation of mean open times when applied after phosphorylation. Channel properties were found to be dependent on association of receptor molecules in the bilayer. Single receptor molecules form channels of 0.9 pS (100 mM Ba2+) and show no voltage-dependent gating. Upon association, both voltage-dependent gating and higher conductance events are recovered; stabilized conductance levels assume values of even multiples of 0.9 pS, predominately 7.5 and 15 pS and multiples of these values up to 60 pS. Thus, individual channels become functionally coupled (synchronous opening and closing) with association, reinstating the characteristics of one larger unitary channel. It is concluded that the L-type Ca2+ channel represents an oligomer of 1,4-dihydropyridine-receptor protein complexes, each of which constitutes a channel, where the array of channels (oligochannel) opens and closes in concerted action.
 ...
PMID:Purified skeletal muscle 1,4-dihydropyridine receptor forms phosphorylation-dependent oligomeric calcium channels in planar bilayers. 245 67

Evidence from electrophysiological and ion flux studies has established that dihydropyridine-sensitive calcium channels are subject to regulation by neurotransmitter-mediated phosphorylation and dephosphorylation reactions. In the present study, we have further characterized the phosphorylation by cAMP-dependent protein kinase and a multifunctional Ca/calmodulin-dependent protein kinase of the membrane-associated form of the 165-kDa polypeptide identified as the skeletal muscle dihydropyridine receptor. The initial rates of phosphorylation of the 165-kDa peptide by both protein kinases were found to be relatively good compared to the rates of phosphorylation of established substrates of the enzymes. Phosphorylation of the 165-kDa peptide by both protein kinases was additive. Prior phosphorylation by either one of the kinases alone did not preclude phosphorylation by the second kinase. The cAMP-dependent protein kinase phosphorylated the 165-kDa peptide preferentially at serine residues, although a small amount of phosphothreonine was also formed. In contrast, after phosphorylation of the 165-kDa peptide by the Ca/calmodulin-dependent protein kinase, slightly more phosphothreonine than phosphoserine was recovered. Phosphopeptide mapping indicated that the two kinases phosphorylated the peptide at distinct as well as similar sites. Notably, one major site phosphorylated by the cAMP-dependent protein kinase was not phosphorylated by the Ca/calmodulin-dependent protein kinase, while other sites were phosphorylated to a high degree by the Ca/calmodulin-dependent protein kinase, but to a much lesser degree by the cAMP-dependent protein kinase. The results show that the 165-kDa dihydropyridine receptor from skeletal muscle can be multiply phosphorylated at distinct sites by the cAMP- and Ca/calmodulin-dependent protein kinases. As the 165-kDa peptide may be the major functional unit of the dihydropyridine-sensitive Ca channel, the results suggest that the phosphorylation-dependent modulation of Ca channel activity by neurotransmitters may involve phosphorylation of the 165-kDa peptide at multiple sites.
 ...
PMID:Multiple phosphorylation sites in the 165-kilodalton peptide associated with dihydropyridine-sensitive calcium channels. 284 83

The dihydropyridine receptor was purified from rabbit skeletal muscle microsomes in the presence of [3H]nitrendipine plus diltiazem or [3H](+)PN 200-110 to an apparent density of 1.5-2 nmol binding sites/mg protein. Sodium dodecyl sulfate gel electrophoresis in the absence of reducing agents yielded three peptide bands of 142, 56 and 30 kDa in a relative ratio of 11:1:1.3, whereas in the presence of 40 mM dithiothreitol bands of 142, 122, 56, 31, 26 and 22 kDa were obtained in a relative ratio of 5.5:2.2:1:0.9:14:0.09. This gel pattern was observed regardless of whether the receptor was purified as a complex with nitrendipine plus diltiazem or with (+)PN 200-110. cAMP-dependent protein kinase phosphorylated preferentially the 142-kDa band up to a stoichiometry of 0.82 +/- 0.07 (15) mol phosphate/mol peptide. The 56-kDa band was phosphorylated only in substoichiometric amounts. [3H]PN 200-110 bound at 4 degrees C to one site with apparent Kd and Bmax values of 9.3 +/- 1.7 nM and 2.2 +/- 0.3 (3) nmol/mg protein, respectively. The binding was stereospecific and was not observed in the presence of 1 mM EGTA. Desmethoxyverapamil interfered with the binding of [3H]PN 200-110 in an apparent allosteric manner. (-)Desmethoxyverapamil inhibited the binding of [3H]PN 200-110 at 37 degrees C and stimulated it at 18 degrees C. In agreement with these results, (-)desmethoxyverapamil increased the dissociation rate of [3H]PN 200-110 from 0.29 min-1 to 0.38 min-1 at 37 degrees C and decreased it threefold from 0.046 min-1 to 0.017 min-1 at 18 degrees C. The (+)isomer of desmethoxyverapamil inhibited PN 200-110 binding at all temperatures tested. d-cis-Diltiazem stimulated the binding of [3H]PN 200-110 at 37 degrees C with an apparent EC50 of 1.4 microM and decreased the dissociation rate from 0.29 min-1 to 0.11 min-1. The stimulatory effect of d-cis-diltiazem was temperature-dependent and was seen only at temperatures above 18 degrees C. These results suggest that the purified dihydropyridine receptor retains the basic properties of the membrane-bound receptor and contains separate sites for at least dihydropyridines and phenylalkylamines.
 ...
PMID:Purification of a functional receptor for calcium-channel blockers from rabbit skeletal-muscle microsomes. 302 84

Cyclic AMP-mediated phosphorylation of calcium channel subunits was studied in vitro and in vivo in preparations from dog heart. Calcium channels in native cardiac membranes were phosphorylated by cAMP-dependent protein kinase (PKA) solubilized with digitonin and subsequently immunoprecipitated using a polyclonal antibody generated against the deduced carboxy-terminal sequence of the cardiac beta subunit. A 62 kDa protein was identified as the major PKA-substrate in the immunoprecipitates. In the intact myocardium, this putative beta subunit was found to be phosphorylated in response to cAMP elevating agents. In contrast, no phosphorylation of a protein with an electrophoretic mobility similar to the alpha 1 subunit was detected, although 1,4-dihydropyridine receptor sites were recovered in the immunoprecipitates. Thus, we suggest that PKA-mediated phosphorylation of the beta subunit is the major mechanism for beta-adrenergic regulation of cardiac L-type calcium channel activity.
 ...
PMID:Phosphorylation of the L-type calcium channel beta subunit is involved in beta-adrenergic signal transduction in canine myocardium. 825

We investigated the modulation of the skeletal muscle L-type Ca2+ channel/dihydropyridine receptor in response to insulin-like growth factor-1 receptor (IGF-1R) activation in single extensor digitorum longus muscle fibers from adult C57BL/6 mice. The L-type Ca2+ channel activity in its dual role as a voltage sensor and a selective Ca2+-conducting pore was recorded in voltage-clamp conditions. Peak Ca2+ current amplitude consistently increased after exposure to 20 ng/ml IGF-1 (EC50 = 5.6 +/- 1.8 nM). Peak IGF-1 effect on current amplitude at -20 mV was 210 +/- 18% of the control. Ca2+ current potentiation resulted from a shift in 13 mV of the Ca2+ current-voltage relationship toward more negative potentials. The IGF-1-induced facilitation of the Ca2+ current was not associated with an effect on charge movement amplitude and/or voltage distribution. These phenomena suggest that the L-type Ca2+ channel structures involved in voltage sensing are not involved in the response to the growth factor. The modulatory effect of IGF-1 on L-type Ca2+ channel was blocked by tyrosine kinase and PKC inhibitors, but not by a cAMP-dependent protein kinase inhibitor. IGF-1-dependent phosphorylation of the L-type Ca2+ channel alpha1 subunit was demonstrated by incorporation of [gamma-32P]ATP to monolayers of adult fast-twitch skeletal muscles. IGF-1 induced phosphorylation of a protein at the 165 kDa band, corresponding to the L-type Ca2+ channel alpha1 subunit. These results show that the activation of the IGF-1R facilitates skeletal muscle L-type Ca2+ channel activity via a PKC-dependent phosphorylation mechanism.
 ...
PMID:Regulation of mouse skeletal muscle L-type Ca2+ channel by activation of the insulin-like growth factor-1 receptor. 927 27