The MinK Potassium Channel Exists in Functional and Nonfunctional Forms When Expressed in the Plasma Membrane of \u3cem\u3eXenopus\u3c/em\u3e Oocytes

The minK protein induces a slowly activating voltage-dependent potassium current when expressed in Xenopus oocytes. In order to measure the levels of minK protein in the plasma membrane, we have modified the minK gene by inserting a 9 amino acid epitope into the N- terminal domain of the protein sequence. When intact live oocytes are injected with the modified minK RNA and subsequently incubated with an antibody to this epitope, specific binding is detected, indicating that the N-terminal domain is extracellular. We found that when oocytes are injected with amounts of minK mRNA up to 50 ng, the levels of protein at the surface are proportional to the amount of injected mRNA. In contrast, the amplitude of the minK current recorded in the oocytes saturates at 1 ng of injected mRNA. Although the amplitude of the currents is not altered by increasing mRNA levels above 1 ng, the kinetics of activation of the current differ in oocytes with high or low levels of minK RNA. In particular, activation is slower with higher levels of minK protein in the plasma membrane. Finally, we find that increasing intracellular cAMP levels, which increases the amplitude of minK currents, does not alter surface expression of the minK protein but produces a small increase in the rate of activation of the current. Our results support a model in which minK protein forms functional potassium channels by association with a factor endogenous to the oocyte

Modulation by cAMP of a Slowly Activating Potassium Channel Expressed in \u3cem\u3eXenopus\u3c/em\u3e Oocytes

When expressed in the Xenopus oocyte, the minK protein induces a slowly activating voltage-dependent potassium current (Isk). We studied the modulation of this current by altering intracellular cAMP levels and found that the amplitude of Isk is dramatically increased by treatments that raise cAMP levels and decreased by agents that lower cAMP levels. Preinjection of a protein inhibitor of the cAMP-dependent protein kinase blocked the effects of increased cAMP levels. There were no changes in the voltage dependence or kinetics of Isk. Mutations that eliminate a potential phosphorylation site on the minK protein did not block the effects of activating the kinase. In addition, the membrane capacitance of the oocyte increased and decreased in parallel with Isk. Our results fit a mechanism in which channel proteins are selectively inserted into and removed from the plasma membrane in response to changes in kinase activity

Arachidonic Acid as a Possible Negative Feedback Inhibitor of Nicotinic Acetylcholine Receptors on Neurons

Neuronal acetylcholine receptors, being highly permeable to calcium, are likely to regulate calcium-dependent events in neurons. Arachidonic acid is a membrane-permeant second messenger that can be released from membrane phospholipids by phospholipases in a calcium-dependent manner. We show here that activation of neuronal acetylcholine receptors triggers release of 3H-arachidonic acid in a calcium-dependent manner from neurons preloaded with the fatty acid. Moreover, low concentrations of arachidonic acid reversibly inhibit the receptors and act most efficiently on receptors likely to have the highest permeability to calcium, namely receptors containing α7 subunits. Low concentrations of arachidonic acid also reversibly inhibit α7- containing receptors expressed in Xenopus oocytes following injection of α7 cRNA. The oocyte results indicate following injection of α7 cRNA. The oocyte results indicate that the inhibition is a feature of the receptors rather than a consequence of neuron-specific machinery. The inhibition is not mediated by specific metabolites of arachidonic acid because the effects can be mimicked by other fatty acids; their effectiveness correlates with their content of double bonds. In contrast to arachidonic effects on calcium currents, inhibition of neuronal nicotinic receptors by the fatty acid cannot be prevented by blocking production of free radicals or by inhibiting protein kinase C. An alternative mechanism is that arachidonic acid binds directly to the receptors or perturbs the local environment in such a manner as to constrain receptor function

Adaptation of the protein kinase filter paper assay to a 96-well microtiter format.

Journal ArticleThe most widely used method for assaying protein kinase activities involves incorporation of radioactive phosphate into a protein or peptide substrate with subsequent binding or precipitaion of the radiolabeled substrate onto filter paper squares. We have adapted this assay for use with readily available 96-well microtiter plate technologies. The sensitivity and reproducibility of the modified 96-well protein kinase assay are comparable to standard filter paper assay methods, but assay throughput is much greater and reagent costs are significantly reduced. This technique has been used to assay several different protein kinase activities using both P81 phosphocellulose and 3MM filter papers. The general methods described here could be readily adapted for use with other kinds of filter materials or used with automated assay systems

Antifreeze Proteins in the Primary Urine of Larvae of the Beetle \u3cem\u3eDendroides canadensis\u3c/em\u3e

To avoid freezing while overwintering beneath the bark of fallen trees, Dendroides canadensis (Coleoptera: Pyrochroidae) larvae produce a family of antifreeze proteins (DAFPs) that are transcribed in specific tissues and have specific compartmental fates. DAFPs and associated thermal hysteresis activity (THA) have been shown previously in hemolymph and midgut fluid, but the presence of DAFPs has not been explored in primary urine, a potentially important site that can contain endogenous ice-nucleating compounds that could induce freezing. A maximum mean THA of 2.65±0.33°C was observed in primary urine of winter-collected D. canadensis larvae. THA in primary urine increased significantly through autumn, peaked in the winter and decreased through spring to levels of 0.2–0.3°C in summer, in a pattern similar to that of hemolymph and midgut fluid. THA was also found in hindgut fluid and excreted rectal fluid, suggesting that these larvae not only concentrate AFPs in the hindgut, but also excrete AFPs from the rectal cavity. Based on dafp transcripts isolated from Malpighian tubule epithelia, cDNAs were cloned and sequenced, identifying the presence of transcripts encoding 24 DAFP isoforms. Six of these Malpighian tubule DAFPs were known previously, but 18 are new. We also provide functional evidence that DAFPs can inhibit ice nucleators present in insect primary urine. This is potentially critical because D. canadensis larvae die if frozen, and therefore ice formation in any body fluid, including the urine, would be lethal

Nuclear Factor-κB-Independent Anti-Inflammatory Action of Salicylate in Human Endothelial Cells

In contrast to aspirin, salicylate, its active metabolite, possesses profound anti-inflammatory properties without blocking cyclooxygenase. Inhibition of the transcription factor nuclear factor-κB (NF-κB) has been discussed to play a role in the anti-inflammatory profile of salicylate. However, NF-κB-independent effects of salicylate have been assumed but have up to now been poorly investigated. Therefore, the aim of the present study was to investigate NF-κB-independent anti-inflammatory mechanisms of salicylate in human umbilical vein endothelial cells using interleukin-4 (IL-4) as NF-κB-independent proinflammatory stimulus and P-selectin as inflammatory read-out parameter. Using quantitative real-time reverse transcriptionpolymerase chain reaction, we found that salicylate decreases IL-4-induced P-selectin expression. As judged by Western blot analysis, salicylate increased endothelial heme oxygenase-1 (HO-1) protein levels. Using both the HO-1 inhibitor tin(II) protoporphyrin IX and HO-1 antisense oligonucleotides, we causally linked the induction of HO-1 to the decrease of P-selectin. Moreover, we were interested in the signaling mechanisms leading to the up-regulation of HO-1 by salicylate. c-Jun NH2-terminal kinase (JNK) was found to be activated by salicylate, and we could causally link this activation to the induction of HO-1 by using the JNK inhibitor 1,9-pyrazoloanthrone. By applying activator protein-1 (AP-1) decoys, it was shown that the transcription factor AP-1 is crucially involved in the up-regulation of HO-1 downstream of JNK. In summary, our study introduces HO-1 as novel NF-κB-independent anti-inflammatory target of salicylate in human endothelial cells. Moreover, we elucidated the JNK/AP-1 pathway as crucial for the induction of HO-1 by salicylate

Characterization of the regulatory domain of the gamma-subunit of phosphorylase kinase. The two noncontiguous calmodulin-binding subdomains are also autoinhibitory.

Journal ArticlePhosphorylase kinase is a multimeric protein kinase (alpha 4 beta 4 gamma 4 delta 4) whose enzymatic activity is conferred by its gamma-subunit. A library of 18 overlapping synthetic peptides spanning residues 277-386 of the gamma-subunit has been prepared to use in identifying important regulatory structures in the protein. In the present study, the library was screened to identify regions that might function as autoinhibitory domains. Peptides from two distinct regions were found to inhibit the Ca2(+)-activated holoenzyme. The same regions were previously found to bind calmodulin (i.e. the delta-subunit; Dasgupta, M. Honeycutt, T., and Blumenthal, D. K. (1989) J. Biol. Chem. 264, 17156-17163). The most potent substrate antagonist peptides were PhK13 (residues 302-326; Ki = 300 nM) and PhK5 (residues 342-366; Ki = 20 microM). Both peptides inhibited the holoenzyme competitively with respect to phosphorylase b and noncompetitively with respect to Mg.ATP. When the pattern of inhibition with both peptides present was analyzed, inhibition was observed to be synergistic and modestly cooperative indicating that the two peptides can simultaneously occupy the protein substrate-binding site(s). These data are consistent with a model in which the regions of the gamma-subunit represented by PhK5 and PhK13 work in concert as regulatory subdomains that transduce Ca2(+)-induced conformational changes in the delta-subunit to the catalytic gamma-subunit through a pseudosubstrate autoinhibitory mechanism

Dephosphorylation of cAMP-dependent protein kinase regulatory subunit (type II) by calmodulin-dependent protein phosphatase. Determinants of substrate specificity.

Journal ArticleCalmodulin-dependent protein phosphatase purified from bovine cardiac muscle catalyzed the rapid dephosphorylation of Ser-95 of bovine cardiac cAMP-dependent protein kinase regulatory subunit (RII). The kinetic constants determined for the reaction (Km = 20 microM; Vmax = 2 mumol min-1 mg-1) are comparable to those determined for other good substrates of this phosphatase. Because little is known about the determinants of substrate specificity for the calmodulin-dependent phosphatase, various phosphopeptides were used to investigate the structural features important for substrate recognition. Limited proteolysis of phospho-RII with trypsin and chymotrypsin yielded fragments (residues 93-400 and 91-400, respectively) that were poor substrates, whereas digestion with Staphylococcal aureus V8 protease produced three phosphopeptides that were all dephosphorylated as rapidly as intact RII. The sequence of the shortest phosphopeptide produced by S. aureus V8 protease was determined by sequence analysis to be Asp-Leu-Asp-Val-Pro-Ile-Pro-Gly-Arg-Phe-Asp-Arg-Arg-Val-Ser-Val-Cys-Ala-Glu, corresponding to residues 81-99 of RII. Synthetic phosphopeptides corresponding to residues 81-99, 85-99, 90-99, and 91-99 were prepared to determine the minimum sequence necessary for substrate recognition. Only the 19-residue peptide (81-99) was dephosphorylated with kinetics comparable to RII (Km = 26 microM, Vmax = 1.7 mumol min-1 mg-1). Structural analysis of this peptide indicates that an amphipathic beta-sheet structure may be an important structural determinant for some substrates of the calmodulin-dependent phosphatase

Characterization of the regulatory domain of the γ-subunit of phosphorylase kinase

Phosphorylase kinase is a multimeric protein kinase (α4β4γ4δ4) whose enzymatic activity is conferred by its γ-subunit. A library of 18 overlapping synthetic peptides spanning residues 277-386 of the γ-subunit has been prepared to use in identifying important regulatory structures in the protein. In the present study, the library was screened to identify regions that might function as autoinhibitory domains. Peptides from two distinct regions were found to inhibit the Ca2+-activated holoenzyme. The same regions were previously found to bind calmodulin (i.e. the δ-subunit; Dasgupta, M. Honeycutt, T. and Blumenthal, D. K.(1989) J. Biol. Chem. 264, 17156-17163). The most potent substrate antagonist peptides were PhK13 (residues 302-326; Ki = 300 nM) and PhK5 (residues 342-366; Ki = 20 μM). Both peptides inhibited the holoenzyme competitively with respect to phosphorylase b and noncompetitively with respect to Mg•ATP. When the pattern of inhibition with both peptides present was analyzed, inhibition was observed to be synergistic and modestly cooperative indicating that the two peptides can simultaneously occupy the protein substrate-binding site(s). These data are consistent with a model in which the regions of the γ-subunit represented by PhK5 and PhK13 work in concert as regulatory subdomains that transduce Ca2+-induced conformational changes in the δ-subunit to the catalytic γ-subunit through a pseudosubstrate autoinhibitory mechanism

Effects of deletions in the central helix of calmodulin on enzyme activation and peptide binding.

Journal ArticleUsing site-directed mutagenesis we have expressed in Escherichia coli three engineered calmodulins (CaM) containing deletions in the solvent-exposed region of the central helix. These are CaM delta 84, Glu-84 removed; CaM delta 83-84, Glu-83 and Glu-84 removed; and CaM delta 81-84, Ser-81 through Glu-84 removed. The abilities of these proteins to activate skeletal muscle myosin light chain kinase, plant NAD kinase, and bovine brain calcineurin activities were determined, as were their abilities to bind a synthetic peptide based on the calmodulin-binding domain of skeletal muscle myosin light chain kinase. Similar results were obtained with all three deletion proteins. Vm values for enzymes activated by the deletion proteins are all within 10-20% of those values obtained with bacterial control calmodulin. Relative to bacterial control values, changes in Kact or Kd values associated with the deletions are all less than an order of magnitude: Kact values for NAD kinase and myosin light chain kinase are increased 5-7-fold, Kd values for binding of the synthetic peptide are increased 4-7-fold, and Kact values for calcineurin are increased only 1-3-fold. In assays of NAD kinase and myosin light chain kinase activation some differences between bovine calmodulin and bacterial control calmodulin were observed. With NAD kinase, Kact values for the bacterial control protein are increased 4-fold relative to values for bovine calmodulin, and Vm values are increased by 50%; with myosin light chain kinase, Kact values are increased 2-fold and Vm values are decreased 10-15% relative to those values obtained with bovine calmodulin. These differences between bacterial control and bovine calmodulins probably can be attributed to known differences in postranslational processing of calmodulin in bacterial and eucaryotic cells. No differences between bovine and control calmodulins were observed in assays of calcineurin activation or peptide binding. Our observations indicate that contacts with the deleted residues, Ser-81 through Glu-84, are not critical in the calmodulin-target complexes we have evaluated. Formation of these calmodulin-target complexes also does not appear to be greatly affected by the global alterations in the structure of calmodulin that are associated with the deletions. In models in which the central helix is maintained in the altered calmodulins, each deleted residue causes the two lobes of calmodulin to be twisted 100 degrees relative to one another and brought 1.5 A closer together.(ABSTRACT TRUNCATED AT 400 WORDS