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

Electrophysiologic and volumetric evidence link the swelling-activated Cl- channels [gCl(Vol)] of nonpigmented ciliary epithelial (NPE) cells with the Cl(-)-channel/Cl(-)-channel regulator protein pICln. However, inhibitors (verapamil and dideoxyforskolin) of another Cl- channel/regulator (MDR1) have been found to inhibit the volume-activated transport response [the regulatory volume decrease (RVD)] of bovine NPE cells. We have addressed the possible molecular basis for the NPE Cl- channels by volumetric measurements of ODM human NPE cells in hypotonic and isotonic test solutions, and by polymerase chain reaction (PCR) cloning and Northern analyses of the same cells. Verapamil and dideoxyforskolin did inhibit the RVD. However, at a concentration (100 microM) which blocks > 90% of the MDR1-associated Cl- currents, forskolin had no effect on the volume-activated Cl- channels or on the inhibition of those channels by protein kinase C. High concentrations of ATP (3.5 and 10 mM) and niflumic acid (IC50 approximately 200 microM) also block [gCl(Vol)]. The RVD is inhibited by 9-phenylanthranilic acid (DPC) and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), unaffected by anthracene-9-carboxylic acid (9-AC), and stimulated by ionomycin. The Cl(-)-channel blockers NPPB, niflumic acid, DPC and 9-AC, and the Ca2(+)-ionophore ionomycin had qualitatively similar effects on the rate of staurosporine-activated isotonic cell shrink-age. These results support the concept that the volume-sensitive protein pICln regulates the Cl- channels, and that the same conduits subserve volume- and staurosporine-activated Cl- release. Of the cloned and sequenced Cl- channels, ClC-3 uniquely conforms to the stationary currents and PKC sensitivity of the NPE Cl- channels. PCR amplifications of human cDNA libraries from ciliary body, NPE cells and retina with primers based on human ClC-3 and ClC-4 cDNA, and Northern analyses using the products generated indicated that ciliary epithelial cells express transcripts for ClC-3 (but not ClC-4). We suggest that ClC-3 provides the same conduit for both volume-activated and isotonically staurosporine-activated Cl- channels of human nonpigmented ciliary epithelial cells.
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PMID:Association of ClC-3 channel with Cl- transport by human nonpigmented ciliary epithelial cells. 866 80

Swelling-activated or volume-sensitive Cl- currents are found in numerous cell types and play a variety of roles in their function; however, molecular characterization of the channels is generally lacking. Recently, the molecular entity responsible for swelling-activated Cl- current in cardiac myocytes has been identified as ClC-3. The goal of our study was to determine whether such a channel exists in smooth muscle cells of the canine colon using both molecular biological and electrophysiological techniques and, if present, to characterize its functional and molecular properties. We hypothesized that ClC-3 is present in colonic smooth muscle and is regulated in a manner similar to the molecular entity cloned from heart. Indeed, the ClC-3 gene was expressed in colonic myocytes, as demonstrated by reverse transcriptase polymerase chain reaction performed on isolated cells. The current activated by decreasing extracellular osmolarity from 300 to 250 mosM was outwardly rectifying and dependent on the Cl- gradient. Current magnitude increased and reversed at more negative potentials when Cl- was replaced by I- or Br-. Tamoxifen ([Z]-1-[p-dimethylaminoethoxy-phenyl]-1,2-diphenyl-1-butene; 10 microM) and DIDS (100 microM) inhibited the current, whereas 25 microM niflumic acid, 10 microM nicardipine, and Ca2+ removal had no effect. Current was inhibited by 1 mM extracellular ATP in a voltage-dependent manner. Cl- current was also regulated by protein kinase C, as phorbol 12,13-dibutyrate (300 nM) decreased Cl- current magnitude, while chelerythrine chloride (30 microM) activated it under isotonic conditions. Our findings indicate that a current activated by hypotonic solution is present in colonic myocytes and is likely mediated by ClC-3. Furthermore, we suggest that the ClC-3 may be an important mechanism controlling depolarization and contraction of colonic smooth muscle under conditions that impose physical stress on the cells.
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PMID:Functional and molecular identification of a novel chloride conductance in canine colonic smooth muscle. 975 47

In many mammalian cells, ClC-3 volume-regulated chloride channels maintain a variety of normal cellular functions during osmotic perturbation. The molecular mechanisms of channel regulation by cell volume, however, are unknown. Since a number of recent studies point to the involvement of protein phosphorylation/dephosphorylation in the control of volume-regulated ionic transport systems, we studied the relationship between channel phosphorylation and volume regulation of ClC-3 channels using site-directed mutagenesis and patch-clamp techniques. In native cardiac cells and when overexpressed in NIH/3T3 cells, ClC-3 channels were opened by cell swelling or inhibition of endogenous PKC, but closed by PKC activation, phosphatase inhibition, or elevation of intracellular Ca2+. Site-specific mutational studies indicate that a serine residue (serine51) within a consensus PKC-phosphorylation site in the intracellular amino terminus of the ClC-3 channel protein represents an important volume sensor of the channel. These results provide direct molecular and pharmacological evidence indicating that channel phosphorylation/dephosphorylation plays a crucial role in the regulation of volume sensitivity of recombinant ClC-3 channels and their native counterpart, ICl.vol.
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PMID:A serine residue in ClC-3 links phosphorylation-dephosphorylation to chloride channel regulation by cell volume. 987 88

The whole-cell patch-clamp technique was used to study the effect of protein kinase C (PKC) stimulation and alpha-adrenergic agonists on the swelling-induced chloride current (ICl,swell) in canine atrial cells. ICl,swell was activated by positive-pressure inflation. 4beta-Phorbol 12, 13-dibutyrate (PDBu) concentration-dependently stimulated ICl,swell. PDBu (500 nM) increased the current density of ICl,swell from 9.1+/-1.3 to 24.2+/-4.8 pA/pF at +20 mV (n=4). This effect developed slowly, reaching a steady-state after more than 5 min of exposure. 4alpha-Phorbol 12, 13-dibutyrate (4alpha-PDBu, 500 nM), an inactive analogue of PDBu, did not affect ICl,swell. The effect of PDBu was inhibited by bisindolylmaleimide I. After down regulation of PKC by phorbol 12-myristate 13-acetate (PMA, 1.6 microM, 24 h), ICl,swell no longer responded to PDBu (n=4). Neither the basal whole-cell current (prior to cell inflation) nor inflation-induced ICl,swell were affected by PKC down regulation. Phenylephrine did not affect ICl,swell. We conclude that PKC activity stimulates and does not prevent the activation of dog atrial ICl,swell. These results contrast with reports of PKC-dependent inhibition of rabbit atrial ICl,swell and currents conducted by ClC-3, a putative clone for ICl,swell. The data suggest species-dependent variations in the modulation of cardiac ICl,swell by PKC.
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PMID:Protein kinase C stimulates swelling-induced chloride current in canine atrial cells. 992 63

1. Volume-activated chloride currents in cultured rat brain endothelial cells were investigated on a functional level using the whole-cell voltage-clamp technique and on a molecular level using the reverse transcriptase-polymerase chain reaction (RT-PCR). 2. Exposure to a hypotonic solution caused the activation of a large, outward rectifying current, which exhibited a slight time-dependent decrease at strong depolarizing potentials. The anion permeability of the induced current was I- (1.7) > Br- (1.2) > Cl- (1.0) > F- (0. 7) > gluconate (0.18). 3. The chloride channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB, 100 microM) rapidly and reversibly inhibited both inward and outward currents. The chloride transport blocker 4,4'-diisothiocyanatostilbene-2, 2'-disulphonic acid (DIDS, 100 microM) also blocked the hypotonicity-induced current in a reversible manner. In this case, the outward current was more effectively suppressed than the inward current. The volume-activated current was also inhibited by the antioestrogen tamoxifen (10 microM). 4. The current was dependent on intracellular ATP and independent of intracellular Ca2+. 5. Activation of protein kinase C by phorbol 12,13-dibutyrate (PDBu, 100 nM) inhibited the increase in current normally observed following hypotonic challenge. 6. Extracellular ATP (10 mM) inhibited the current with a more pronounced effect on the outward than the inward current. 7. Verapamil (100 microM) decreased both the inward and the outward hypotonicity-activated chloride current. 8. RT-PCR analysis was used to determine possible molecular candidates for the volume-sensitive current. Expression of the ClC-2, ClC-3 and ClC-5 chloride channels, as well as pICln, could be shown at the mRNA level. 9. We conclude that rat brain endothelial cells express chloride channels which are activated by osmotic swelling. The biophysical and pharmacological properties of the current show strong similarities to those of ClC-3 channel currents as described in other cell types.
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PMID:Functional and molecular characterization of a volume-sensitive chloride current in rat brain endothelial cells. 1006 24

Anion transport proteins in mammalian cells participate in a wide variety of cell and intracellular organelle functions, including regulation of electrical activity, pH, volume, and the transport of osmolites and metabolites, and may even play a role in the control of immunological responses, cell migration, cell proliferation, and differentiation. Although significant progress over the past decade has been achieved in understanding electrogenic and electroneutral anion transport proteins in sarcolemmal and intracellular membranes, information on the molecular nature and physiological significance of many of these proteins, especially in the heart, is incomplete. Functional and molecular studies presently suggest that four primary types of sarcolemmal anion channels are expressed in cardiac cells: channels regulated by protein kinase A (PKA), protein kinase C, and purinergic receptors (I(Cl.PKA)); channels regulated by changes in cell volume (I(Cl.vol)); channels activated by intracellular Ca(2+) (I(Cl.Ca)); and inwardly rectifying anion channels (I(Cl.ir)). In most animal species, I(Cl.PKA) is due to expression of a cardiac isoform of the epithelial cystic fibrosis transmembrane conductance regulator Cl(-) channel. New molecular candidates responsible for I(Cl.vol), I(Cl.Ca), and I(Cl.ir) (ClC-3, CLCA1, and ClC-2, respectively) have recently been identified and are presently being evaluated. Two isoforms of the band 3 anion exchange protein, originally characterized in erythrocytes, are responsible for Cl(-)/HCO(3)(-) exchange, and at least two members of a large vertebrate family of electroneutral cotransporters (ENCC1 and ENCC3) are responsible for Na(+)-dependent Cl(-) cotransport in heart. A 223-amino acid protein in the outer mitochondrial membrane of most eukaryotic cells comprises a voltage-dependent anion channel. The molecular entities responsible for other types of electroneutral anion exchange or Cl(-) conductances in intracellular membranes of the sarcoplasmic reticulum or nucleus are unknown. Evidence of cardiac expression of up to five additional members of the ClC gene family suggest a rich new variety of molecular candidates that may underlie existing or novel Cl(-) channel subtypes in sarcolemmal and intracellular membranes. The application of modern molecular biological and genetic approaches to the study of anion transport proteins during the next decade holds exciting promise for eventually revealing the actual physiological, pathophysiological, and clinical significance of these unique transport processes in cardiac and other mammalian cells.
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PMID:Anion transport in heart. 1061 65

1. ClC-3 encodes a volume-regulated Cl- channel (ICl,vol) in heart. We studied the regulation of native and recombinant cardiac ICl,vol by intracellular cyclic AMP (cAMPi). 2. Symmetrical high Cl- concentrations were used to effectively separate outwardly rectifying ICl,vol from other non-rectifying Cl- currents, such as the cystic fibrosis transmembrane conductance regulator (CFTR) and Ca2+-activated Cl- currents (ICl,CFTR and ICl,Ca, respectively), which are concomitantly expressed in cardiac myocytes. 3. 8-Bromo-cyclic AMP (8-Br-cAMP) significantly inhibited ICl,vol in most guinea-pig atrial myocytes. In approximately 30 % of the atrial myocytes examined, 8-Br-cAMP increased macroscopic Cl- currents. However, the 8-Br-cAMP-stimulated difference currents exhibited a linear current-voltage (I-V ) relation, consistent with activation of ICl,CFTR, not ICl,vol. 4. In canine atrial myocytes, isoprenaline (1 microM) consistently reduced ICl,vol in Ca2+-free hypotonic bath solutions with strong intracellular Ca2+ (Ca2+i) buffering. In Ca2+-containing hypotonic bath solutions with weak Ca2+i buffering, however, isoprenaline increased net macroscopic Cl- currents. Isoprenaline-stimulated difference currents were not outwardly rectifying, consistent with activation of ICl,Ca, not ICl, vol. 5. In NIH/3T3 cells transfected with gpClC-3 (the gene encoding ICl,vol), 8-Br-cAMP consistently inhibited ICl,ClC-3. These effects were prevented by a protein kinase A (PKA) inhibitor, KT5720, or by mutation of a single consensus protein kinase C (PKC) phosphorylation site (S51A) on the N-terminus of ClC-3, which also mediates PKC inhibition of ICl,ClC-3. 6. We conclude that cAMPi causes inhibition of ICl,vol in mammalian heart due to cross-phosphorylation of the same PKC consensus site on ClC-3 by PKA. Our results suggest that contamination of macroscopic ICl,vol by ICl,CFTR and/or ICl,Ca may account for some of the inconsistent and controversial effects of cAMPi on ICl,vol previously reported in native cardiac myocytes.
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PMID:Intracellular cyclic AMP inhibits native and recombinant volume-regulated chloride channels from mammalian heart. 1071 49

The effect of the novel imidazoline compound 2-[2-(4,5-dihydro-1H-imidazol-2-yl)-1-(5-methyl-2,3-dihydrobenzofuran-7-yl)-ethyl]-pyridine (NNC77-0020) on stimulus-secretion coupling and hormone secretion was investigated in mouse pancreatic islets and isolated alpha- and beta-cells. In the presence of elevated glucose concentrations NNC77-0020 stimulated insulin secretion concentration dependently (EC(50) 64 nM) by 200% without affecting the whole-cell K(+) current or cytoplasmic Ca(2+) levels. Capacitance measurements in single mouse beta-cells showed that intracellular application of NNC77-0020 via the recording pipette enhanced Ca(2+)-dependent exocytosis. This action was dependent on protein kinase C (PKC) and cytoplasmic phospholipase A(2) (cPLA(2)) activity and required functional granular ClC-3 Cl(-) channels. In intact islets NNC77-0020 stimulated glucose-dependent somatostatin secretion, an effect that was also dependent on PKC and cPLA(2) activity. NNC77-0020 also inhibited glucagon secretion. In single mouse alpha-cells this action was not associated with a change in spontaneous electrical activity and resulted from a reduction in the rate of Ca(2+)-dependent exocytosis. Inhibition of exocytosis by NNC77-0020 was pertussis toxin sensitive and mediated by activation of the protein phosphatase calcineurin. In conclusion, our data suggest that the imidazoline compound NNC77-0020 modulates pancreatic hormone secretion in a complex fashion, comprising glucose-dependent stimulation of insulin and somatostatin secretion and inhibition of glucagon release. These mechanisms of action constitute an ideal basis for the development of novel imidazoline-containing anti-diabetic compounds.
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PMID:The imidazoline NNC77-0020 affects glucose-dependent insulin, glucagon and somatostatin secretion in mouse pancreatic islets. 1368 90

ClC-3, a member of the large superfamily of ClC voltage-dependent Cl(-) channels, has been proposed as a molecular candidate responsible for volume-sensitive osmolyte and anion channels (VSOACs) in some cells, including heart and vascular smooth muscle. However, the reported presence of native VSOACs in at least two cell types from transgenic ClC-3 disrupted (Clcn3(-/-)) mice casts considerable doubt on this proposed role for ClC-3. We compared several properties of native VSOACs and examined mRNA transcripts and membrane protein expression profiles in cardiac and pulmonary arterial smooth muscle cells from Clcn3(+/+) and Clcn3(-/-) mice to: (1) test the hypothesis that native VSOACs are unaltered in cells from Clcn3(-/-) mice, and (2) test the possibility that targeted inactivation of the Clcn3 gene using a conventional murine global knock-out approach may result in compensatory changes in expression of other membrane proteins. Our experiments demonstrate that VSOAC currents in myocytes from Clcn3(+/+) and Clcn3(-/-) mice are remarkably similar in terms of activation and inactivation kinetics, steady-state current densities, rectification, anion selectivity (I(-) > Cl(-)>> Asp(-)) and sensitivity to block by glibenclamide, niflumic acid, DIDS and extracellular ATP. However, additional experiments revealed several significant differences in other fundamental properties of native VSOACs recorded from atrial and smooth muscle cells from Clcn3(-/-) mice, including: differences in regulation by endogenous protein kinase C, differential sensitivity to block by anti-ClC-3 antibodies, and differential sensitivities to [ATP](i) and free [Mg(2+)](i). These results suggest that in response to Clcn3 gene deletion, there may be compensatory changes in expression of other proteins that alter VSOAC channel subunit composition or associated regulatory subunits that give rise to VSOACs with different properties. Consistent with this hypothesis, in atria from Clcn3(-/-) mice compared to Clcn3(+/+) mice, quantitative analysis of ClC mRNA expression levels revealed significant increases in transcripts for ClC-1, ClC-2, and ClC-3, and protein expression profiles obtained using two-dimensional polyacrylamide gel electrophoresis revealed complex changes in at least 35 different unidentified membrane proteins in cells from Clcn3(-/-) mice. These findings emphasize that caution needs to be exercised in simple attempts to interpret the phenotypic consequences of conventional global Clcn3 gene inactivation.
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PMID:Altered properties of volume-sensitive osmolyte and anion channels (VSOACs) and membrane protein expression in cardiac and smooth muscle myocytes from Clcn3-/- mice. 1502 Jun 89

The serum- and glucocorticoid-inducible kinase (SGK) is a serine/threonine protein kinase (PK) transcriptionally regulated by corticoids, serum, and cell volume. SGK regulates cell volume of various cells by effects on Na(+) and K(+) transport through membrane channels. We hypothesized a role for SGK in the activation of volume-sensitive osmolyte and anion channels (VSOACs) in cultured canine pulmonary artery smooth muscle cells (PASMCs). Intracellular dialysis through the patch electrode of recombinant active SGK, but not kinase-dead Delta60-SGK-K127M, heat-inactivated SGK, or active Akt1, partially activated VSOACs under isotonic conditions. Dialysis of active SGK before cell exposure to hypotonic medium significantly accelerated the activation kinetics and increased the maximal density of VSOAC current. Exposure of PASMCs to hypotonic medium (230 mosM) activated phosphatidylinositol 3-kinases (PI3Ks) and their downstream targets Akt/PKB and SGK but not PKC-epsilon. Inhibition of PI3Ks with wortmannin reduced the activation rate and maximal amplitude of VSOACs. Immunoprecipitated ClC-3 channels were phosphorylated by PKC-epsilon but not by SGK in vitro, suggesting that SGK may activate VSOACs indirectly. These data indicate that the PI3K-SGK cascade is activated on hypotonic swelling of PASMCs and, in turn, affects downstream signaling molecules linked to activation of VSOACs.
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PMID:Hypotonic activation of volume-sensitive outwardly rectifying chloride channels in cultured PASMCs is modulated by SGK. 1527 97


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