Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We determined the effect of nucleotides and protein kinase A (PKA) on the Ca(2+)-dependent gating of the cloned intermediate conductance, Ca(2+)-dependent K(+) channel, hIK1. In Xenopus oocytes, during two-electrode voltage-clamp, forskolin plus isobutylmethylxanthine induced a Ca(2+)-dependent increase in hIK1 activity. In excised inside-out patches, addition of ATP induced a Ca(2+)-dependent increase in hIK1 activity (NP(o)). In contrast, neither nonhydrolyzable (AMP-PNP, AMP-PCP) nor hydrolyzable ATP analogs (GTP, CTP, UTP, and ITP) activated hIK1. The ATP-dependent activation of hIK1 required Mg(2+) and was reversed by either exogenous alkaline phosphatase or the PKA inhibitor PKI(5-24). The Ca(2+) dependence of hIK1 activation was best fit with a stimulatory constant (K(s)) of 350 nM and a Hill coefficient (n) of 2.3. ATP increased NP(o) at [Ca(2+)] >100 nM while having no effect on K(s) or n. Mutation of the single PKA consensus phosphorylation site at serine 334 to alanine (S334A) had no effect on the PKA-dependent activation during either two-electrode voltage-clamp or in excised inside-out patches. When expressed in HEK293 cells, ATP activated hIK1 in a Mg(2+)-dependent fashion, being reversed by alkaline phosphatase. Neither PKI(5-24) nor CaMKII(281-309) or PKC(19-31) affected the ATP-dependent activation. Northern blot analysis revealed hIK1 expression in the T84 colonic cell line. Endogenous hIK1 was activated by ATP in a Mg(2+)- and PKI(5-24)-dependent fashion and was reversed by alkaline phosphatase, whereas CaMKII(281-309) and PKC(19-31) had no effect on the ATP-dependent activation. The Ca(2+)-dependent activation (K(s) and n) was unaffected by ATP. In conclusion, hIK1 is activated by a membrane delimited PKA when endogenously expressed. Although the oocyte expression system recapitulates this regulation, expression in HEK293 cells does not. The effect of PKA on hIK1 gating is Ca(2+)-dependent and occurs via an increase in NP(o) without an effect on either Ca(2+) affinity or apparent cooperativity.
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PMID:Kinase-dependent regulation of the intermediate conductance, calcium-dependent potassium channel, hIK1. 1061 55

We describe an in vitro system in which post-Golgi vesicles containing metabolically labeled, sialylated, vesicular stomatitis virus (VSV) G protein molecules (VSV-G) are produced from the trans-Golgi network (TGN) of an isolated Golgi membrane fraction. This fraction is prepared from VSV-infected Madin-Darby canine kidney (MDCK) cells in which the (35)S-labeled viral envelope glycoprotein was allowed to accumulate in the trans-Golgi network during a prolonged incubation at 20 degrees C. The vesicles produced in this system are separated from the remnant Golgi membranes by differential centrifugation or by velocity sedimentation in a sucrose gradient. Vesicle production, quantified as the percentage of labeled VSV-G released from the Golgi membranes, is optimal at 37 degrees C and does not occur below 20 degrees C. It requires GTP and the small GTP-binding protein Arf (ADP-ribosylation factor), as well as coat protein type I (COPI) coat components (coatomer) and vesicle scission factors-one of which corresponds to the phosphatidylinositol transfer protein (PITP). Formation of the vesicles does not require GTP hydrolysis which, however, is necessary for their uncoating. Thus, vesicles generated in the presence of the nonhydrolyzable GTP analogs, GTPgammaS or GMP-PNP, retain a coatomer coat visible in the electron microscope, sediment more rapidly in sucrose density gradients than those generated with ATP or GTP, and can be captured with anticoatomerantibodies. The process of coatomer-coated vesicle formation from the TGN can be dissected into two distinct sequential phases, corresponding to coat assembly/bud formation and vesicle scission. The first phase is completed when Golgi fractions are incubated with cytosolic proteins and nonhydrolyzable GTP analogs at 20 degrees C. The scission phase, which leads to vesicle release, takes place when coated Golgi membranes, recovered after phase I, are incubated at higher temperatures in the presence of cytosolic proteins. The scission phase does not take place if protein kinase C inhibitors are added during the first phase, even though these inhibitors do not prevent membrane coating and bud formation. The phosphorylating activity of a protein kinase C, however, plays no role in vesicle formation, since this process does not require ATP.
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PMID:In vitro generation from the trans-Golgi network of coatomer-coated vesicles containing sialylated vesicular stomatitis virus-G protein. 1072 Apr 65

Using the patch-clamp technique, we have identified an intermediate conductance Ca(2+)-activated K(+) channel from bullfrog (Rana catesbeiana) erythrocytes and have investigated the regulation of channel activity by cytosolic ATP. The channel was highly selective for K(+) over Na(+), gave a linear I-V relationship with symmetrical 117.5 mM K(+) solutions and had a single-channel conductance of 60 pS. Channel activity was dependent on Ca(2+) concentration (K(1/2) = 600 nM) but voltage-independent. These basic characteristics are similar to those of human and frog erythrocyte Ca(2+)-activated K(+) (Gardos) channels previously reported. However, cytoplasmic application of ATP reduced channel activity with block exhibiting a novel bell-shaped concentration dependence. The channel was inhibited most by approximately 10 microM ATP (P(0) reduced to 5% of control) but less blocked by lower and higher concentrations of ATP. Moreover, the novel type of ATP block did not require Mg(2+), was independent of PKA or PKC, and was mimicked by a nonhydrolyzable ATP analog, AMP-PNP. This suggests that ATP exerts its effect by direct binding to sites on the channel or associated regulatory proteins, but not by phosphorylation of either of these components.
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PMID:A novel type of ATP block on a Ca(2+)-activated K(+) channel from bullfrog erythrocytes. 1086 55

The properties of the inward-rectifying Cl- conductance in rat choroid plexus epithelial cells were investigated to allow comparisons to be made with ClC-2. All experiments were performed using the whole-cell configuration of the patch-clamp method. The conductance was transiently activated using an electrode solution which contained 375 nM catalytic subunit of protein kinase A (PKA). PKA failed to activate the conductance, however, when cells were pre-incubated with phorbol esters, which activate protein kinase C [1 microM phorbol 12-myristate 13-acetate (PMA) and 1 microM phorbol 12,13-dibutyrate (PDBu)]. Sustained activation of the conductance by PKA was observed in Ca2+-free conditions (5 mM BAPTA in the electrode solution), or when 100 nM calphostin C, a PKC inhibitor, was added to the electrode solution. The inward-rectifying Cl- conductance in choroid plexus is therefore similar to ClC-2 in that it is inhibited by PKC. The inward-rectifying conductance was blocked when Cd2+ (30 and 300 microM) and Zn2+ (1, 30 and 300 microM) were added to the bath solution. ClC-2 channels are also blocked by Zn2+ and Cd2+. The magnitude of the inward conductance was dependent on the concentration of ATP in the electrode solution. The conductance was not observed when ATP in the electrode was replaced with non-hydrolysable ATP analogues [adenosine 5'-O-(3-thiotriphosphate) (ATP[gamma-S]) and 5'-adenylylimidodiphosphate (AMP-PNP)), but it was supported by UTP and GTP. These data contrast with those of previous studies in which ClC-2 channels were activated in the absence of ATP. In conclusion, the inward-rectifying Cl- channel in rat choroid plexus shares some properties with ClC-2 (inhibition by PKC and block by divalent cations), but differs in that it depends on intracellular ATP.
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PMID:Properties of the inward-rectifying Cl- channel in rat choroid plexus: regulation by intracellular messengers and inhibition by divalent cations. 1104 61

The role of protein kinases on store-operated Ca2+ entry in rat basophilic leukaemia cells (RBL) has been studied using the whole-cell configuration of the patch-clamp technique and Ca2+ imaging with fura-2. Specific inhibitors of tyrosine kinase (lavendustin A), mitogen-activated protein (MAP) kinase (SB 203580, PD 98059), Ca2+/calmodulin-dependent kinase (CaMK, KN-62, KN-93) and protein kinase C (PKC, bisindolylmaleimide I) had no significant effect on peak current amplitude and time constant of activation. Likewise, the broad spectrum kinase blockers H-7 and staurosporine did not alter Ca2+ entry compared to control recordings. Store-mediated Ca2+ entry was unaffected if intracellular ATP was substituted by either adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS) or adenylyl-imidodiphosphate (AMP-PNP). Similarly, buffering intracellular Mg2+, an essential cofactor for protein kinases, had no effect on Ca2+ influx. These results indicate that protein phosphorylation by various kinases is not required for the activation of the store-operated Ca2+ current in RBL cells.
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PMID:Activation of store-operated Ca2+ entry in RBL cells without the contribution of protein kinases. 1141 58

The effect of external ATP on intracellular pH (pH(i)) was investigated using a pH imaging system in a human bronchial epithelial cell line (16HBE14o-) loaded with BCECF-AM. The steady-state pH(i) of 16HBE14o- epithelial monolayers was 7.137 +/- 0.027 (n = 46). Apical addition of ATP (10(-4) M) to epithelial monolayers induced a rapid and sustained pH(i) decrease of 0.164 +/- 0.024 pH units (n = 17; P < 0.001). The intracellular acidification was rapidly reversed upon removal of external ATP. In contrast, the non-hydrolysable ATP analogue AMP-PNP did not produce any significant change in pH(i). Inhibition of purinoreceptors by suramin did not affect the acidification induced by apical ATP. Inhibition of Na+-H+ exchange by apical Na+ removal or addition of amiloride (0.5 mM) reduced the apical ATP-induced pH(i) decrease, suggesting the involvement of a Na+-H+ exchanger or surface pH effects on the ATP-induced pH(i) response. Inhibitors of proton channels such as ZnCl2 (10(-4) M) also partially inhibited the ATP response. The pH(i) response to ATP was dependent on the external pH (pH(o)), with increasing acidification produced at lower pH(o) values. Neither the basal pH(i) nor the ATP-induced intracellular acidification was affected by thapsigargin (a Ca2+-ATPase inhibitor), chelerythrine chloride (a protein kinase C (PKC) inhibitor), RpcAMP (a protein kinase A (PKA) inhibitor) or PMA (a PKC activator). Therefore, the intracellular acidification of human bronchial epithelial cells induced by apical ATP does not involve signalling via Ca2+, PKC or PKA nor binding to a purinoreceptor. We interpret the effect of ATP to produce an intracellular acidification as a three step process: activation of H+ channels, inhibition of Na+-H+ exchange and influx of protonated ATP.
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PMID:Cellular mechanisms for apical ATP effects on intracellular pH in human bronchial epithelium. 1218 Dec 78

In the presence of NMDA receptor open-channel blockers [Mg(2+); (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801); 1-amino-3,5-dimethyladamantane (memantine)] and TTX, high concentrations (30-100 microm) of either 5-hydroxytryptamine (5-HT) or alpha-methyl-5-hydroxytryptamine (alpha-Me-5-HT) significantly potentiated NMDA-induced depolarizations of frog spinal cord motoneurones. Potentiation was blocked by LY-53,857 (10-30 microm), SB 206553 (10 microm), and SB 204741 (30 microm), but not by spiroxatrine (10 microm), WAY 100,635 (1-30 microm), ketanserin (10 microm), RS 102221 (10 microm), or RS 39604 (10-20 microm). Therefore, alpha-Me-5-HT's facilitatory effects appear to involve 5-HT(2B) receptors. These effects were G-protein dependent as they were prevented by prior treatment with guanylyl-5'-imidodiphosphate (GMP-PNP, 100 microm) and H-Arg-Pro-Lys-Pro-Gln-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH(2) (GP antagonist 2A, 3-6 microm), but not by pertussis toxin (PTX, 3-6 ng ml(-1), 48 h preincubation). This potentiation was not reduced by protein kinase C inhibition with staurosporine (2.0 microm), U73122 (10 microm) or N-(2-aminoethyl)-5-isoquinolinesulfonamide HCl (H9) (77 microm) or by intracellular Ca(2+) depletion with thapsigargin (0.1 microm) (which inhibits Ca(2+)/ATPase). Exposure of the spinal cord to the L-type Ca(2+) channel blockers nifedipine (10 microm), KN-62 (5 microm) or gallopamil (100 microm) eliminated alpha-Me-5-HT's effects. The calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide (W7) (100 microm) diminished the potentiation. However, the calcium/calmodulin-dependent protein kinase II (CaM Kinase II) blocker KN-93 (10 microm) did not block the 5-HT enhancement of the NMDA responses. In summary, activation of 5-HT(2B) receptors by alpha-Me-5-HT facilitates NMDA-depolarizations of frog motoneurones via a G-protein, a rise in [Ca(2+)](i) from the entry of extracellular Ca(2+) through L-type Ca(2+) channels, the binding of Ca(2+) to calmodulin and a lessening of the Mg(2+) -produced open-channel block of the NMDA receptor.
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PMID:Mechanisms intrinsic to 5-HT2B receptor-induced potentiation of NMDA receptor responses in frog motoneurones. 1533 59

We investigated, by using the patch clamp technique, Ca2+-mediated regulation of heterologously expressed TRPC6 and TRPC7 proteins in HEK293 cells, two closely related homologues of the transient receptor potential (TRP) family and molecular candidates for native receptor-operated Ca2+ entry channels. With nystatin-perforated recording, the magnitude and time courses of activation and inactivation of carbachol (CCh; 100 microM)-activated TRPC6 currents (I(TRPC6)) were enhanced and accelerated, respectively, by extracellular Ca2+ (Ca2+(o)) whether it was continuously present or applied after receptor stimulation. In contrast, Ca2+(o) solely inhibited TRPC7 currents (I(TRPC7)). Vigorous buffering of intracellular Ca2+ (Ca2+(i)) under conventional whole-cell clamp abolished the slow potentiating (i.e. accelerated activation) and inactivating effects of Ca2+(o), disclosing fast potentiation (EC50: approximately 0.4 mM) and inhibition (IC50: approximately 4 mM) of I(TRPC6) and fast inhibition (IC50: approximately 0.4 mM) of I(TRPC7). This inhibition of I(TRPC6) and I(TRPC7) seems to be associated with voltage-dependent reductions of unitary conductance and open probability at the single channel level, whereas the potentiation of I(TRPC6) showed little voltage dependence and was mimicked by Sr2+ but not Ba2+. The activation process of I(TRPC6) or its acceleration by Ca2+(o) probably involves phosphorylation by calmodulin (CaM)-dependent kinase II (CaMKII), as pretreatment with calmidazolium (3 microM), coexpression of Ca2+-insensitive mutant CaM, and intracellular perfusion of the non-hydrolysable ATP analogue AMP-PNP and a CaMKII-specific inhibitory peptide all effectively prevented channel activation. However, this was not observed for TRPC7. Instead, single CCh-activated TRPC7 channel activity was concentration-dependently suppressed by nanomolar Ca2+(i) via CaM and conversely enhanced by IP3. In addition, the inactivation time course of I(TRPC6) was significantly retarded by pharmacological inhibition of protein kinase C (PKC). These results collectively suggest that TRPC6 and 7 channels are multiply regulated by Ca2+ from both sides of the membrane through differential Ca2+-CaM-dependent and -independent mechanisms.
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PMID:Multiple regulation by calcium of murine homologues of transient receptor potential proteins TRPC6 and TRPC7 expressed in HEK293 cells. 1548 49

The intracellular signaling pathways responsible for extracellualr uridine-5'-triphosphate (UTPo)-induced chloride (Cl-) currents (I(Cl.UTP)) were studied in mouse ventricular myocytes with the whole-cell clamp technique. UTPo (0.1 to 100 microM) activated a whole-cell current that showed a time-independent activation, a linear current-voltage relationship in symmetrical Cl- solutions, an anion selectivity of Cl- > iodide > aspartate, and an inhibition by a thiazolidinone-derived specific inhibitor (CFTR(inh)-172, 10 microM) of cystic fibrosis transmembrane conductance regulator (CFTR), but not by a disulfonic stilbene derivative (DIDS, 100 microM), these properties matching those of CFTR Cl- channels. The potency order of nucleotides for an activation of the Cl- current was UTP = ATP > uridine-5'-diphosphate (UDP) = ADP. Suramin (100 microM), a P2Y receptor antagonist, strongly inhibited the UTPo -activation of the Cl- current, whereas pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 100 microM), another P2Y receptor antagonist, induced little inhibition of I(Cl.UTP). The activation of I(Cl.UTP) was sensitive to protein kinase C (PKC) inhibitor, phospholipase C (PLC) inhibitor, intracellular GDPbetaS (nonhydrolyzable GDP analogue) or anti-Gq/11 antibody. UTPo failed to activate the Cl- current when the cells were dialyzed with nonhydrolyzable ATP analogues (ATPS or AMP-PNP) without ATP, suggesting that ATP hydrolysis is a prerequisite for the current activation. I(Cl.UTP) was persistently activated with a mixture of ATPgammaS + ATP in the pipette, suggesting the involvement of phosphorylation reaction in the current activation process. Our results strongly suggest that I(Cl.UTP) is due to the activation of CFTR Cl- channels through Gq/11-coupled P2Y2 receptor-PLC-PKC signaling and ATP hydrolysis in mouse heart.
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PMID:Regulation of extracellular UTP-activated Cl- current by P2Y-PLC-PKC signaling and ATP hydrolysis in mouse ventricular myocytes. 1729 97

It is well known that angiotensin II (Angio II) mimics most of the muscarinic-mediated excitatory actions of acetylcholine on superior cervical ganglion neurons. For instance, in addition to depolarization and stimulation of norepinephrine release, muscarinic agonists and Angio II modulate the M-type K(+) current and the N-type Ca(2+) current. We recently found that muscarinic receptors modulate the delayed rectifier current I(KV) as well. Therefore a whole cell patch-clamp experiment was carried out in rat cultured sympathetic neurons to assess whether Angio II modulates I(KV). We found that Angio II increased I(KV) by about 30% with a time constant of approximately 30 s. In comparison, inhibition of M-current was faster (tau approximately 8 s) and stronger ( approximately 61%). Modulation of I(KV) was disrupted by the AT(1) receptor-antagonist losartan but not by the AT(2)-antagonist PD123319. I(KV) enhancement was reduced by the G-protein inhibitor GDP-beta-S, whereas current modulation remained unaltered after cell treatment with pertussis toxin. The peptidergic modulation of I(KV) was severely disrupted when internal ATP was replaced by its nonhydrolyzable analogue AMP-PNP. Angio II enhanced I(KV) and further reduced the stimulatory action of a muscarinic agonist on I(KV). Likewise, the muscarinc agonist enhanced I(KV) and occluded the effect of Angio II on I(KV). We have also found that the protein kinase C activator PMA enhanced I(KV), thereby mimicking and further attenuating the action of Angio II on I(KV). These results suggest that AT(1) receptors by coupling to pertussis toxin-insensitive G proteins, stimulate an ATP-dependent and PKC-mediated pathway to modulate I(KV).
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PMID:Modulation of a delayed-rectifier K+ current by angiotensin II in rat sympathetic neurons. 1749 17


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