PKA and phosphatases attached to the CaV1.2 channel regulate channel activity in cell-free patches

2016 ◽  
Vol 310 (2) ◽  
pp. C136-C141 ◽  
Author(s):  
Jianjun Xu ◽  
Lifeng Yu ◽  
Etsuko Minobe ◽  
Liting Lu ◽  
Ming Lei ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Ventricular Myocytes ◽  
Catalytic Subunit ◽  
Channel Activity ◽  
Activity Recovery ◽  
Dependent Protein ◽  
Excised Patches ◽  
Inside Out

Calmodulin (CaM) + ATP can reprime voltage-gated L-type Ca2+ channels (CaV1.2) in inside-out patches for activation, but this effect decreases time dependently. This suggests that the CaV1.2 channel activity is regulated by additional cytoplasmic factors. To test this hypothesis, we examined the role of cAMP-dependent protein kinase A (PKA) and protein phosphatases in the regulation of CaV1.2 channel activity in the inside-out mode in guinea pig ventricular myocytes. CaV1.2 channel activity quickly disappeared after the patch was excised from the cell and recovered to only 9% of that in the cell-attached mode on application of CaM + ATP at 10 min after the inside out. However, immediate exposure of the excised patch to the catalytic subunit of PKA + ATP or the nonspecific phosphatase inhibitor okadaic acid significantly increased the CaV1.2 channel activity recovery by CaM + ATP (114 and 96%, respectively) at 10 min. Interestingly, incubation of the excised patches with cAMP + ATP also increased CaM/ATP-induced CaV1.2 channel activity recovery (108%), and this effect was blocked by the nonspecific protein kinase inhibitor K252a. The channel activity in the inside-out mode was not maintained by either catalytic subunit of PKA or cAMP + ATP in the absence of CaM, but was stably maintained in the presence of CaM for more than 40 min. These results suggest that PKA and phosphatase(s) attached on or near the CaV1.2 channel regulate the basal channel activity, presumably through modulation of the dynamic CaM interaction with the channel.

scholarly journals ATP mediates both activation and inhibition of K(ATP) channel activity via cAMP-dependent protein kinase in insulin-secreting cell lines.

1989 ◽  
Vol 94 (4) ◽  
pp. 693-717 ◽  
Author(s):  
B Ribalet ◽  
S Ciani ◽  
G T Eddlestone
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Single Channel ◽  
K Channel ◽  
Channel Activity ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Insulin Secreting Cells ◽  
Dependent Protein ◽  
Inside Out

The single-channel recording technique was employed to investigate the mechanism conferring ATP sensitivity to a metabolite-sensitive K channel in insulin-secreting cells. ATP stimulated channel activity in the 0-10 microM range, but depressed it at higher concentrations. In inside-out patches, addition of the cAMP-dependent protein kinase inhibitor (PKI) reduced channel activity, suggesting that the stimulatory effect of ATP occurs via cAMP-dependent protein kinase-mediated phosphorylation. Raising ATP between 10 and 500 microM in the presence of exogenous PKI progressively reduced the channel activity; it is proposed that this inactivation results from a reduction in kinase activity owing to an ATP-dependent binding of PKI or a protein with similar inhibitory properties to the kinase. A model describing the effects of ATP was developed, incorporating these two separate roles for the nucleotide. Assuming that the efficacy of ATP in controlling the channel activity depends upon the relative concentrations of inhibitor and catalytic subunit associated with the membrane, our model predicts that the channel sensitivity to ATP will vary when the ratio of these two modulators is altered. Based upon this, it is shown that the apparent discrepancy existing between the sensitivity of the channel to low ATP concentrations in the excised patch and the elevated intracellular level of ATP may be explained by postulating a change in the inhibitor/kinase ratio from 1:1 to 3:2 owing to the loss of protein kinase after patch excision. At a low concentration of ATP (10-20 microM), a nonhydrolyzable ATP analogue, AMP-PNP, enhanced the channel activity when present below 10 microM, whereas the analogue blocked the channel activity at higher concentrations. It is postulated that AMP-PNP inhibits the formation of the kinase-inhibitor complex in the former case, and prevents phosphate transfer in the latter. A similar mechanism would explain the interaction between ATP and ADP which is characterized by enhanced activity at low ADP concentrations and blocking at higher concentrations.

Calmodulin reverses rundown of L-type Ca2+ channels in guinea pig ventricular myocytes

2004 ◽  
Vol 287 (6) ◽  
pp. C1717-C1724 ◽  
Author(s):  
Jian-Jun Xu ◽  
Li-Ying Hao ◽  
Asako Kameyama ◽  
Masaki Kameyama
Keyword(s):  
Protein Kinase ◽  
Guinea Pig ◽  
Kinase Inhibitors ◽  
Ventricular Myocytes ◽  
Protein Kinase Inhibitors ◽  
Channel Activity ◽  
Dependent Protein ◽  
Dose Dependent ◽  
Inside Out ◽  
Ca2 Channels

Calmodulin (CaM) is implicated in regulation of Ca2+ channels as a Ca2+ sensor. The effect of CaM on rundown of L-type Ca2+ channels in inside-out patch form was investigated in guinea pig ventricular myocytes. Ca2+ channel activity disappeared within 1–3 min and did not reappear when the patch was excised and exposed to an artificial intracellular solution. However, application of CaM (0.03, 0.3, 3 μM) + 3 mM ATP to the intracellular solution within 1 min after patch excision resulted in dose-dependent activation of channel activity. Channel activity averaged 11.2%, 94.7%, and 292.9%, respectively, of that in cell-attached mode. Channel activity in inside-out patch mode was induced by CaM + ATP at nanomolar Ca2+ concentrations ([Ca2+]); however, increase to micromolar [Ca2+] rapidly inactivated the channel activity induced, revealing that the effect of CaM on the channel was Ca2+ dependent. At the 2nd, 4th, 6th, 8th, and 10th minutes after patch excision, CaM (0.75 μM) + ATP induced Ca2+ channel activity to 150%, 100%, 96.9%, 29.3%, and 16.6%, respectively, revealing a time-dependent action of CaM on the channel. CaM added with adenosine 5′-(β,γ-imido)triphosphate (AMP-PNP) also induced channel activity, although with much lower potency and shorter duration. Protein kinase inhibitors KN-62, CaM-dependent protein kinase (CaMK)II 281-309, autocamtide-related CaMKII inhibitor peptide, and K252a (each 1–10 μM) did not block the effect of CaM, indicating that the effect of CaM on the Ca2+ channel was phosphorylation independent. Neither CaM nor ATP alone induced Ca2+ channel activity, showing a cooperative effect of CaM and ATP on the Ca2+ channel. These results suggest that CaM is a crucial regulatory factor of Ca2+ channel basal activity.

Nitric oxide and cGMP protein kinase activity in aged ventricular myocytes

2001 ◽  
Vol 281 (6) ◽  
pp. H2304-H2309 ◽  
Author(s):  
Qihang Zhang ◽  
Bruno Molino ◽  
Lin Yan ◽  
Todd Haim ◽  
Yakir Vaks ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Ventricular Myocytes ◽  
Nitric Oxide Donor ◽  
Protein Kinase Activity ◽  
Edge Detector ◽  
Negative Effects ◽  
Dependent Protein

We tested the hypothesis that nitric oxide-induced negative functional effects through cGMP would be reduced in aged cardiac myocytes. Maximum rate of shortening ( R max) and percent shortening of ventricular myocytes from young (6 mo) and old (3 y) rabbits were studied using a video edge detector. cGMP-dependent phosphorylation was examined by electrophoresis and autoradiography. Myocytes received a nitric oxide donor S-nitroso- N-acetyl-penicillamine (SNAP, 10−7, 10−6, and 10−5 M) followed by KT-5823 (10−6 M), a cGMP protein kinase inhibitor. Baseline function was similar in young and old myocytes (89.1 ± 4.5 young vs. 86.4 ± 8.3 μm/s old R max, 5.6 ± 0.3 vs. 5.2 ± 0.7%shortening). SNAP (10−5 M) decreased R max in both young (25%, n = 6) and old myocytes (24%, n = 7). SNAP also reduced percent shortening by 28% in young and 23% in old myocytes. The negative effects of SNAP were partially reversed by KT-5823 only in young myocytes. Multiple proteins were phosphorylated by cGMP, and KT-5823 could reduce this effect. The degree of phosphorylation was significantly less in old myocytes. These results suggest that the functional response of ventricular myocytes to nitric oxide was preserved during aging. However, the importance of cGMP-dependent protein phosphorylation was decreased, indicating a shift to other pathways.

The protein kinase A catalytic subunit Cβ2: molecular characterization and distribution of the splice variant

2000 ◽  
Vol 351 (1) ◽  
pp. 123-132 ◽  
Author(s):  
Sandra THULLNER ◽  
Frank GESELLCHEN ◽  
Stefan WIEMANN ◽  
Walter PYERIN ◽  
Volker KINZEL ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Splice Variant ◽  
Cardiac Tissue ◽  
Kinase Inhibitor ◽  
Morphological Changes ◽  
Affinity Purification ◽  
Catalytic Subunit ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

Cβ2, a 46 kDa splice variant of the Cβ isoform, is the largest isoform so far described for catalytic subunits from cAMP-dependent protein kinase in mammals. It differs from Cβ in the first 15 N-terminal residues which are replaced with a 62-residue domain with no similarity to other known proteins. The Cβ2 protein was identified in cardiac tissue by MS, microsequencing and C-subunit-isoform-selective antibodies. The Cβ2 protein has a very low abundance of about 2% of total affinity-purified C subunits from bovine cardiac tissue. This, and the similarity of its biochemical properties to Cα and Cβ, are probably some of the reasons why the Cβ2 protein has escaped detection so far. The abundance of the Cβ2 protein differs dramatically between tissues, with most protein detected in heart, liver and spleen, and the lowest level in testis. Cβ2 protein shows kinase activity against synthetic substrates, and is inhibited by the protein kinase inhibitor peptide PKI(5-24). The degree of Cβ2 removal from tissue extracts by binding to PKI(5-24) depends on the cAMP level, i.e. on the dissociation state of the holoenzyme. Two sites in the protein are phosphorylated: Thr-244 in the activation segment and Ser-385 close to the C-terminus. By affinity purification and immunodetection Cβ2 was found in cattle, pig, rat, mouse and turkey tissue and in HeLa cells. In the cAMP-insensitive CHO 10260 cell line, which has normal Cβ but is depleted of Cα, stable transfection with Cβ2 restored most of the cAMP-induced morphological changes. Cβ2 is a ubiquitously expressed protein with characteristic properties of a cAMP-dependent protein kinase catalytic subunit.

Intracellular Cs+ activates the PKA pathway, revealing a fast, reversible, Ca2+-dependent inactivation of L-type Ca2+ current

2003 ◽  
Vol 285 (2) ◽  
pp. C310-C318 ◽  
Author(s):  
Fabien Brette ◽  
Alain Lacampagne ◽  
Laurent Sallé ◽  
Ian Findlay ◽  
Jean-Yves Le Guennec
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Ventricular Myocytes ◽  
Cyclopiazonic Acid ◽  
Dependent Protein Kinase ◽  
Pipette Solution ◽  
Camp Dependent Protein Kinase ◽  
Dependent Inactivation ◽  
Dependent Protein ◽  
Pka Pathway

Inactivation of the L-type Ca2+ current ( ICaL) was studied in isolated guinea pig ventricular myocytes with different ionic solutions. Under basal conditions, ICaL of 82% of cells infused with Cs+-based intracellular solutions showed enhanced amplitude with multiphasic decay and diastolic depolarization-induced facilitation. The characteristics of ICaL in this population of cells were not due to contamination by other currents or an artifact. These phenomena were reduced by ryanodine, caffeine, cyclopiazonic acid, the protein kinase A inhibitor H-89, and the cAMP-dependent protein kinase inhibitor. Forskolin and isoproterenol increased ICaL by only ∼60% in these cells. Cells infused with either N-methyl-d-glucamine or K+-based intracellular solutions did not show multiphasic decay or facilitation under basal conditions. Isoproterenol increased ICaL by ∼200% in these cells. In conclusion, we show that multiphasic inactivation of ICaL is due to Ca2+-dependent inactivation that is reversible on a time scale of tens of milliseconds. Cs+ seems to activate the cAMP-dependent protein kinase pathway when used as a substitute for K+ in the pipette solution.

Regulation of inositol 1,4,5-trisphosphate-induced Ca2+ release. II. Effect of cAMP-dependent protein kinase

1990 ◽  
Vol 258 (6) ◽  
pp. C1086-C1091 ◽  
Author(s):  
P. Volpe ◽  
B. H. Alderson-Lang
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Catalytic Subunit ◽  
Scatchard Analysis ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Plot Analysis ◽  
Receptor Sites ◽  
Inositol 1,4,5 Trisphosphate ◽  
Dependent Protein

The effect of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) on Ca2+ loading, inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release, and [3H]IP3 binding of canine cerebellar membrane fractions was investigated. PKA in the presence of cAMP and the catalytic subunit of PKA did not change Ca2+ loading yet increased the extent of IP3-induced Ca2+ release by approximately 35%. Hill plot analysis indicated that the catalytic subunit of PKA increased the apparent Michaelis constant of IP3-induced Ca2+ release twofold, from 0.3 to 0.7 microM IP3. The protein kinase inhibitor reversed these changes. cAMP affected neither Ca2+ loading nor IP3-induced Ca2+ release. The catalytic subunit of PKA did not appreciably affect the maximum binding and dissociation constant of [3H]IP3 binding, as judged by Scatchard analysis. Thus the catalytic subunit of PKA influences the opening of Ca2+ channels by IP3 without interfering with the binding of IP3 to its receptor sites.

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