Protein Kinase C-dependent Phosphorylation Research Articles

The Enzymatic Function of Kvβ2 Controls Vascular Potassium Channel Activity

Voltage-gated potassium (Kv) channels expressed in vascular smooth muscle control membrane potential and thereby regulate vascular tone and organ perfusion. Native Kv1 channels in the resistance vasculature consist of an alpha pore domain in complex with intracellular beta (Kvβ) proteins, which are active aldo-keto reductases (AKR6A). Recent work suggests that the AKR function of Kvβ2 is required for vascular Kv1 channel NAD(P)(H) redox sensing and O2 sensitivity of resistance arterial tone. Nonetheless, whether acute changes in Kvβ2 AKR function impact vascular Kv1 activity is unknown. We propose that Kv1 gating is sensitive to changes in the levels of endogenous substrates and post-translational modification of Kvβ2. Here, we tested the hypothesis that the availability of the glycolytic byproduct and purported Kvβ substrate, methylglyoxal, and protein kinase C-dependent phosphorylation differentially regulate vascular Kvβ2 function. Using the inside-out configuration of the patch clamp technique, we measured the open probability (nPo) of Kv channels present in patches excised from human coronary smooth muscle cells. Perfusion of methylglyoxal (5 μM) resulted in significantly increased Kv channel nPo (3.3x10−5 ± 9.5x10−6 to 3.2x10−4 ± 2.1x10−4; n=5) and this effect was enhanced (~8-fold) in the presence of 100 μM NADPH, suggesting that Kv sensitivity to methylglyoxal is cofactor-dependent. Based on this, we next tested whether Kvβ phosphorylation regulates AKR catalysis and influences vascular Kv1 redox sensitivity. Using in situ proximity ligation, we found that serine phosphorylation of Kvβ2 in smooth muscle was increased upon treatment with the PKC activator phorbol 12-myristate 13-acetate (PMA; 100 nM), indicating that smooth muscle Kvβ2 is a phosphorylation target under these conditions. In vitro phosphorylation of purified Kvβ2 with PKCα significantly slowed catalysis (11.3 ± 0.3 vs. 6.5 ± 0.2 μmoles/min/mg protein, control and PKCα-treated rat Kvβ2.1, respectively). Pretreatment of cells with 10 nM PMA before excising inside-out membrane patches eliminated the increase in Kv channel nP(o) evoked by methylglyoxal (±NADPH). Moreover, ex vivo treatment of isolated coronary arteries with PMA (10 nM) abolished vasodilation in response to elevated NADH:NAD+ via application of 2–5 mM external L-lactate. Together, these results suggest that vascular smooth muscle Kv1 channels are functionally regulated by endogenous substrates and phosphorylation of intracellular Kvβ proteins. We propose that targeting vascular Kvβ AKR function may represent a novel strategy to modify K+ channel redox sensitivity and thus strengthen the coupling between metabolic demand and organ perfusion. T32-ES011564 and R01HL163818. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Protein Kinase C-Dependent Phosphorylation of Human Pleckstrin Reduces its Capacity for Self-Association and Impairs its Ability to Bind Phosphoinositide

Pleckstrin is a major substrate of protein kinase C in lymphocytes, macrophages, monocytes, granulocytes and platelets. In these cells, pleckstrin is expressed at high levels and represents approximately 1 % of total cellular protein. Pleckstrin plays an important role in protein kinase C-dependent secretion, and abberant pleckstrin phosphorylation has been associated with disease. The mechanism, however, by which phosphorylation regulates pleckstrin function is unknown. Here, we show that native pleckstrin self-associates to form dimers that reduce its binding capacity to phosphoinositides. Phosphomimetic amino acid substitutions at residues normally phosphorylated by protein kinase C result in significantly reduced pleckstrin dimerization. Although phosphomimetic forms of pleckstrin exhibit subtle conformational changes they retain most of their phosphoinositide binding properties. These findings suggest that phosphorylation regulates pleckstrin interaction with membranes via a dimerization-dependent mechanism.

Epileptogenesis and epileptic maturation in phosphorylation site-specific SNAP-25 mutant mice

Snap25(S187A/S187A) mouse is a knock-in mouse with a single amino acid substitution at a protein kinase C-dependent phosphorylation site of the synaptosomal-associated protein of 25 kDa (SNAP-25), which is a target-soluble NSF attachment protein receptor (t-SNARE) protein essential for neurotransmitter release. Snap25(S187A/S187A) mice exhibit several distinct phenotypes, including reductions in dopamine and serotonin release in the brain, anxiety-like behavior, and cognitive dysfunctions. Homozygous mice show spontaneous epileptic convulsions, and about 15% of the mice die around three weeks after birth. The remaining mice survive for almost two years and exhibit spontaneous recurrent seizures throughout their lifetime. Here, we conducted long-term continuous video electroencephalogram recording of the mice and analyzed the process of epileptogenesis and epileptic maturation in detail. Spikes and slow-wave discharges (SWDs) were observed in the cerebral cortex and thalamus before epileptic convulsions began. SWDs showed several properties similar to those observed in absence seizures including (1) lack of in the hippocampus, (2) movement arrest during SWDs, and (3) inhibition by ethosuximide. Multiple generalized seizures occurred in all homozygous mice around three weeks after birth. However, seizure generation stopped within several days, and a seizure-free latent period began. Following a spike-free quiet period, the number of spikes increased gradually, and epileptic seizures reappeared. Subsequently, spontaneous seizures occurred cyclically throughout the life of the mice, and several progressive changes in seizure frequency, seizure duration, seizure cycle interval, seizure waveform, and the number and waveform of epileptic discharges during slow-wave sleep occurred with different time courses over 10 weeks. Anxiety-related behaviors appeared suddenly within three days after epileptic seizures began and were delayed markedly by oral administration of valproic acid. These results showed that Snap25(S187A/S187A) mice exhibited a variety of epilepsy-related phenomena, and thus, they will be useful for understanding the mechanisms of epileptogenesis, epileptic maturation, and the actions of antiepileptic drugs.

Elucidating the interaction of γ-hydroxymethyl-γ-butyrolactone substituents with model membranes and protein kinase C-C1 domains.

The protein kinase C (PKC) family of proteins is an attractive drug target. Dysregulation of PKC-dependent signalling pathways is related to several human diseases like cancer, immunological and other diseases. We approached the problem of altering PKC activities by developing C1 domain-based PKC ligands. In this report γ-hydroxymethyl-γ-butyrolactone (HGL) substituents were investigated in an effort to develop small molecule-based PKC regulators with higher specificity for C1 domain than the endogenous diacylglycerols (DAGs). Extensive analysis of membrane-ligands interaction measurements revealed that the membrane-active compounds strongly interact with the lipid bilayers and the hydrophilic parts of compounds localize at the bilayer/water interface. The pharmacophores like hydroxymethyl, carbonyl groups and acyl-chain length of the compounds are crucial for their interaction with the C1 domain proteins. The potent compounds showed more than 17-fold stronger binding affinity for the C1 domains than DAG under similar experimental conditions. Nonradioactive kinase assay confirmed that these potent compounds have similar or better PKC dependent phosphorylation capabilities than DAG under similar experimental conditions. Hence, our findings reveal that these HGL analogues represent an attractive group of structurally simple C1 domain ligands that can be further structurally altered to improve their potencies.

Neurosteroids promote phosphorylation and membrane insertion of extrasynaptic GABAA receptors.

Neurosteroids are synthesized within the brain and act as endogenous anxiolytic, anticonvulsant, hypnotic, and sedative agents, actions that are principally mediated via their ability to potentiate phasic and tonic inhibitory neurotransmission mediated by γ-aminobutyric acid type A receptors (GABAARs). Although neurosteroids are accepted allosteric modulators of GABAARs, here we reveal they exert sustained effects on GABAergic inhibition by selectively enhancing the trafficking of GABAARs that mediate tonic inhibition. We demonstrate that neurosteroids potentiate the protein kinase C-dependent phosphorylation of S443 within α4 subunits, a component of GABAAR subtypes that mediate tonic inhibition in many brain regions. This process enhances insertion of α4 subunit-containing GABAAR subtypes into the membrane, resulting in a selective and sustained elevation in the efficacy of tonic inhibition. Therefore, the ability of neurosteroids to modulate the phosphorylation and membrane insertion of α4 subunit-containing GABAARs may underlie the profound effects these endogenous signaling molecules have on neuronal excitability and behavior.

Hematopoietic Protein-1 Regulates the Actin Membrane Skeleton and Membrane Stability in Murine Erythrocytes

Hematopoietic protein-1 (Hem-1) is a hematopoietic cell specific member of the WAVE (Wiskott-Aldrich syndrome verprolin-homologous protein) complex, which regulates filamentous actin (F-actin) polymerization in many cell types including immune cells. However, the roles of Hem-1 and the WAVE complex in erythrocyte biology are not known. In this study, we utilized mice lacking Hem-1 expression due to a non-coding point mutation in the Hem1 gene to show that absence of Hem-1 results in microcytic, hypochromic anemia characterized by abnormally shaped erythrocytes with aberrant F-actin foci and decreased lifespan. We find that Hem-1 and members of the associated WAVE complex are normally expressed in wildtype erythrocyte progenitors and mature erythrocytes. Using mass spectrometry and global proteomics, Coomassie staining, and immunoblotting, we find that the absence of Hem-1 results in decreased representation of essential erythrocyte membrane skeletal proteins including α- and β- spectrin, dematin, p55, adducin, ankyrin, tropomodulin 1, band 3, and band 4.1. Hem1−/− erythrocytes exhibit increased protein kinase C-dependent phosphorylation of adducin at Ser724, which targets adducin family members for dissociation from spectrin and actin, and subsequent proteolysis. Increased adducin Ser724 phosphorylation in Hem1−/− erythrocytes correlates with decreased protein expression of the regulatory subunit of protein phosphatase 2A (PP2A), which is required for PP2A-dependent dephosphorylation of PKC targets. These results reveal a novel, critical role for Hem-1 in the homeostasis of structural proteins required for formation and stability of the actin membrane skeleton in erythrocytes.

Kv3.4 Channel Expression, Modulation and Function in a Spinal Cord Injury Model of Neuropathic Pain

We previously showed that Kv3.4 channels underlie most of the high-voltage activated K+ current (IKHV) in dorsal root ganglion (DRG) nociceptors and elimination of Kv3.4 N-type inactivation by protein kinase C-dependent phosphorylation enhances the channel's ability to repolarize action potentials (APs) (Ritter et al. 2012 J Physiol 590:145-61). This function is important as reduction of Kv3.4 channel expression is found in diverse models of neuropathic pain. We hypothesize that phosphorylated Kv3.4 channels underlie an anti-nociceptive homeostatic mechanism in nociceptors that reduces downstream Ca++ signaling and down-regulation of Kv3.4 channel expression destroys this putative homeostatic mechanism, contributing to chronic pain. To test this hypothesis, we investigated a spinal cord injury (SCI) rat model of neuropathic pain in which a C5 hemi-contusion induces bilateral forepaw thermal hyperalgesia and mechanical allodynia 2 weeks post injury. In the contralateral SCI DRG, the peak IKHV is reduced by 55% and the time constant of inactivation (Taui) increases by 2-fold relative to laminectomy controls. Furthermore, while 85% of laminectomy nocicpetors show a fast inactivating Kv3.4 current, the SCI phenotypes fall into three categories: no current (25%), substantial sustained current (46%) or slow inactivation (29%, Taui >2 S.D. of controls). This suggests that Kv3.4 channels are phosphorylated and down-regulated in SCI. Confirming the impact of these changes on AP firing, AP-clamp experiments in SCI nociceptors show altered currents compared to controls. Interestingly, Kv3.4 mRNA splicing is altered in SCI DRG, which might alter the Kv3.4 channels' response to injury. We propose that Kv3.4 channels, their modulation by PKC and regulation of splicing are therapeutic targets in neuropathic pain.Supported by the Farber Family Foundation (MC), Sigma Xi GIAR (DR), Craig Nielson Foundation (AL), and Paralyzed Veterans of America (AL).

Raf-1 Kinase Inhibitory Protein (RKIP) Mediates Ethanol-induced Sensitization of Secretagogue Signaling in Pancreatic Acinar Cells

Excessive alcohol consumption is associated with most cases of chronic pancreatitis, a progressive necrotizing inflammatory disease that can result in pancreatic insufficiency due to acinar atrophy and fibrosis and an increased risk of pancreatic cancer. At a cellular level acute alcohol exposure can sensitize pancreatic acinar cells to secretagogue stimulation, resulting in dysregulation of intracellular Ca(2+) homeostasis and premature digestive enzyme activation; however, the molecular mechanisms by which ethanol exerts these toxic effects have remained undefined. In this study we identify Raf-1 kinase inhibitory protein as an essential mediator of ethanol-induced sensitization of cholecystokinin- and carbachol-regulated Ca(2+) signaling in pancreatic acinar cells. We show that exposure of rodent acinar cells to ethanol induces protein kinase C-dependent Raf-1 kinase inhibitory protein phosphorylation, sensitization of cholecystokinin-stimulated Ca(2+) signaling, and potentiation of both basal and cholecystokinin-stimulated extracellular signal-regulated kinase activation. Furthermore, we show that either suppression of Raf-1 kinase inhibitory protein expression using short hairpin RNA or gene ablation prevented the sensitizing effects of ethanol on cholecystokinin- and carbachol-stimulated Ca(2+) signaling and intracellular chymotrypsin activation in pancreatic acinar cells, suggesting that the modulation of Raf-1 inhibitory protein expression may have future therapeutic utility in the prevention or treatment of alcohol-associated pancreatitis.

Regulatory Domain Determinants That Control PKD1 Activity

The canonical pathway for protein kinase D1 (PKD1) activation by growth factor receptors involves diacylglycerol binding to the C1 domain and protein kinase C-dependent phosphorylation at the activation loop. PKD1 then autophosphorylates at Ser(916), a modification frequently used as a surrogate marker of PKD1 activity. PKD1 also is cleaved by caspase-3 at a site in the C1-PH interdomain during apoptosis; the functional consequences of this cleavage event remain uncertain. This study shows that PKD1-Δ1-321 (an N-terminal deletion mutant lacking the C1 domain and flanking sequence that models the catalytic fragment that accumulates during apoptosis) and PKD1-CD (the isolated catalytic domain) display high basal Ser(916) autocatalytic activity and robust activity toward CREBtide (a peptide substrate) but little to no activation loop autophosphorylation and no associated activity toward protein substrates, such as cAMP-response element binding protein and cardiac troponin I. In contrast, PKD1-ΔPH (a PH domain deletion mutant) is recovered as a constitutively active enzyme, with high basal autocatalytic activity and high basal activity toward peptide and protein substrates. These results indicate that individual regions in the regulatory domain act in a distinct manner to control PKD1 activity. Finally, cell-based studies show that PKD1-Δ1-321 does not substitute for WT-PKD1 as an in vivo activator of cAMP-response element binding protein and ERK phosphorylation. Proteolytic events that remove the C1 domain (but not the autoinhibitory PH domain) limit maximal PKD1 activity toward physiologically relevant protein substrates and lead to a defect in PKD1-dependent cellular responses.

Endogenously Expressed Muscarinic Receptors in HEK293 Cells Augment Up-regulation of Stably Expressed α4β2 Nicotinic Receptors

Nicotine-induced up-regulation of neuronal nicotinic receptors (nAChRs) has been known and studied for more than 25 years. Other nAChR ligands can also up-regulate nAChRs, but it is not known if these ligands induce up-regulation by mechanisms similar to that of nicotine. In this study, we compared up-regulation by three different nicotinic agonists and a competitive antagonist of several different nAChR subtypes expressed in HEK293 cells. Nicotine markedly increased α4β2 nAChR binding site density and β2 subunit protein. Carbachol, a known nAChR and muscarinic receptor agonist, up-regulated both α4β2 nAChR binding sites and subunit protein 2-fold more than did nicotine. This increased up-regulation was shown pharmacologically to involve endogenously expressed muscarinic receptors, and stimulation of these muscarinic receptors also correlated with a 2-fold increase in α4 and β2 mRNA. Muscarinic receptor activation in these cells appears to affect CMV promoter activity only minimally (∼1.2 fold), suggesting that the increase in α4 and β2 nAChR mRNA may not be dependent on enhanced transcription. Instead, other mechanisms may contribute to the increase in mRNA and a consequent increase in receptor subunits and binding site density. These studies demonstrate the possibility of augmenting nAChR expression in a cell model through mechanisms and targets other than the nAChR receptor itself.

Nicotine-induced up regulation of α4β2 neuronal nicotinic receptors is mediated by the protein kinase c-dependent phosphorylation of α4 subunits

Sustained exposure to nicotine is well known to increase the cell surface density of α4β2* neuronal nicotinic receptors both in vivo and in vitro, but the cellular mechanisms mediating this effect are equivocal. Using a pharmacological approach to investigate the effects of nicotine on receptor subunit expression and phosphorylation in SH-EP1 cells expressing human α4 and β2 nicotinic receptor subunits, we have demonstrated that incubation with nicotine for 24 h increased the expression of immature and mature forms of both α4 and β2 subunits in a concentration-dependent manner, and that inhibition of protein kinase C (PKC), but not cAMP-dependent protein kinase (PKA) inhibited the nicotine-induced increased expression of subunits. Incubation of cells with nicotine for 24 h also increased the phosphorylation of immature forms of α4 subunits similar to that induced by activation of either PKC or PKA. When cells were preincubated with nicotine, the PKC-mediated increased phosphorylation was inhibited; the PKA-mediated phosphorylation was unaltered. The phosphopeptide maps for immature α4 subunits following nicotine exposure or PKC activation were identical, and phosphoamino acid analyses indicated phosphorylation on serine residues only. Results indicate that nicotine-induced up regulation of α4β2 neuronal nicotinic receptors involves a PKC-dependent mechanism and likely reflects the ability of nicotine to activate PKC, leading to the phosphorylation of immature α4 subunits, promoting subunit assembly and receptor maturation. Because up regulation of these receptors has been implicated to mediate tolerance, locomotor sensitization and addiction to nicotine, results identify a potential new target for modulating the effects of nicotine on the brain.

Protein kinase C-dependent phosphorylation of Borna disease virus P protein is required for efficient viral spread

Mutational analysis of the phosphate acceptor sites of the Borna disease virus (BDV) phosphoprotein (P) has suggested a role of phosphorylation for viral spread. However, the studied mutant viruses also had two amino acid exchanges in the X protein, because the reading frames of P and X overlap. To determine the relative contribution of P and X to viral attenuation, we studied a P variant with serine-to-leucine substitutions (P(S26L,S28L)) in which the wild-type X sequence was conserved. Viral spread of rBDV-P(S26L,S28L) was impaired in human oligodendroglioma cells and in adult rats. Thus, BDV-P phosphorylation contributes to efficient viral dissemination.

Activation of Dual Oxidases Duox1 and Duox2: differential regulation mediated by camp-dependent protein kinase and protein kinase C-dependent phosphorylation

Activation of Dual Oxidases Duox1 and Duox2: differential regulation mediated by camp-dependent protein kinase and protein kinase C-dependent phosphorylation

Identification of a Protein Kinase C-dependent phosphorylation site involved in sensitization of TRPV4 channel

Transient Receptor Potential (TRP) proteins are non-selective cation channels performing diverse cellular functions. TRPV1 and TRPV4, two calcium-permeable channels of the vanilloid subfamily of TRP proteins, are activated by various physical and chemical stimuli, including noxious heat and mechanical stress, respectively. These channels are also required for exaggerated sensation of painful stimuli, condition referred to as hyperalgesia, which is frequently associated with inflammation. Phosphorylation of TRPV1, involving Protein Kinase C (PKC) and Protein Kinase A (PKA), appears to be the predominant mechanism for channel sensitization and development of heat hyperalgesia. PKC and PKA pathways have also been implicated in the sensitization of TRPV4, but the respective phosphorylation sites remain unknown. Using mass spectrometry, we report now that TRPV4 is phosphorylated on serine 824 by the PKC-activating phorbol 12-myristate 13-acetate. This phosphorylation is prevented by a PKC inhibitor, confirming the involvement of PKC. Ser824, located in the carboxy-terminal cytosolic tail of TRPV4, is also phosphorylated after activation of the PKA pathway by forskolin, albeit less potently. Substitution of Ser824 with aspartic acid, mimicking phosphorylation at this site, increased TRPV4-mediated calcium influx in resting and in stimulated cells, underlining the importance of this residue in TRPV4 regulation. Thus PKC, and possibly PKA, phosphorylate TRPV4 at Ser824 leading to the enhancement of TRPV4 channel function. Our findings suggest an important role of this phosphorylation in TRPV4 sensitization and the development of hyperalgesia.

Differential effects of phosphorylation of regions of troponin I in modifying cooperative activation of cardiac thin filaments

Ischemia and heart failure are associated with protein kinase C (PKC) dependent phosphorylation of cardiac troponin I (cTnI). We investigated the effect of phosphorylation of cTnI PKC sites S43, S45 and T144 under normal (pH 7.0) and acidic (pH 6.5) conditions on tension in skinned fiber bundles from a mouse heart. To mimic the PKC phosphorylation, we exchanged troponin (cTn) in these fiber bundles with cTn complex containing either cTnI-(S43E/S45E) or cTnI-(T144E). We determined how pseudo-phosphorylation and acidic pH affect activation of thin filaments by strongly bound crossbridges by use of n-ethyl maleimide (NEM-S1) to mimic rigor. We hypothesized that PKC phosphorylation of cTnI amplifies the effect of ischemic/hypoxic conditions to depress myofilament force and Ca2+-responsiveness by reducing the ability of rigor crossbridge to activate force. Pseudo-phosphorylation of cTnI at S43/S45 exacerbated the effect of acidic pH to induce a rightward shift in the Ca2+-tension relation. Under acidic conditions, fibers regulated by cTnI-(S43E/S45E) demonstrated a significant reduction in the ability of NEM-S1 to recruit cycling crossbridges, when compared to controls regulated by cTnI. Similar effects of pseudo-phosphorylation of cTnI-(T144) occurred, but to a lesser extent that those of pseudo-phosphorylation of S43/S45. We conclude that under acidic conditions PKC phosphorylation of cTnI residues at S43/S45 and at T144 is likely to have differential, but significant effects in depressing the ability of both Ca2+ and rigor crossbridges to activate force generation. Although these effects of PKC dependent phosphorylation may be maladaptive in heart failure, they may also spare ATP consumption and be cardio-protective in ischemia.

Protein Kinase C-Dependent Phosphorylation Regulates the Cell Cycle-Inhibitory Function of the p73 Carboxy Terminus Transactivation Domain

The transcription factor p73, a member of the p53 family of proteins, is involved in the regulation of cell cycle progression and apoptosis. However, the regulatory mechanisms controlling the distinct roles for p73 in these two processes have remained unclear. Here, we report that p73 is able to induce cell cycle arrest independently of its amino-terminal transactivation domain, whereas this domain is crucial for p73 proapoptotic functions. We also characterized a second transactivation domain in the carboxy terminus of p73 within amino acid residues 381 to 399. This carboxy terminus transactivation domain was found to preferentially regulate genes involved in cell cycle progression. Moreover, its activity is regulated throughout the cell cycle and modified by protein kinase C-dependent phosphorylation at serine residue 388. Our results suggest that this novel posttranslational modification within the p73 carboxy terminus transactivation domain is involved in the context-specific guidance of p73 toward the selective induction of cell cycle arrest.

Signaling at the membrane interface by the DGK/SK enzyme family

The sphingosine (SK) and diacylglycerol (DGK) kinases have become the subject of considerable focus recently due to their involvement as signaling enzymes in a variety of important biological processes. These lipid signaling kinases are closely related by sequence as well as functional properties. These enzymes are soluble, yet their substrates are hydrophobic. Therefore, they must act at the membrane interface. Second, for both of these enzyme families, their substrates (diacylglycerol for DGKs, sphingosine for SKs) as well as their products (phosphatidic acid for DGK, sphingosine-1-phosphate for SK) have signaling function. To understand how the signaling processes emanating from these kinases are regulated it is critical to understand the fundamental mechanisms that control their enzymatic activity. This is particularly true for the rational design of small molecules that would be useful as therapeutic compounds. Here we summarize enzymological properties of the diacylglycerol and SKs. Further, because the three-dimensional structure of the eukaryotic members of this family has yet to be determined, we discuss what can be gleaned from the recently reported structures of related prokaryotic members of this enzyme family.

CPI-17 Silencing–Reduced Responsiveness in Control and TNF-α–Treated Human Bronchi

Under pathophysiologic conditions, the modulation of Ca2+ sensitivity and reactivity of bronchial smooth muscle is controlled by protein kinase C-dependent phosphorylation of the newly described protein, CPI-17. The goal of the present study was to assess the key role of this regulatory protein in airway hyperresponsiveness (AHR) using control and TNF-alpha-treated human bronchi as well as a specific siRNA duplex against human CPI-17 transcripts. Validity of a mixed transfection strategy was assessed using the reversible permeabilization method to introduce X-TremeGene (X-TG)-siRNA complexes in an overreactive model of human bronchi treated with TNF. Data demonstrate that X-TG-siRNA complexes targeted against CPI-17 transcripts resulted in a reduction in mRNA and specific protein expression in human bronchial tissues. This approach revealed that overall reactivity of bronchial smooth muscle to methacholine was reduced, while their relaxing responses to beta2-agonist were increased, when compared with responses triggered in control TNF-alpha-treated bronchi. Quantification analysis showed that Ca2+ -sensitivity in both untreated and TNF-alpha-treated bronchi were largely reduced upon transfection with human CPI-17 siRNA-X-TremeGene complexes, while Western blot analysis corroborated the decrease in CPI-17 and MLC phosphorylation levels in pretreated human bronchi. Identical results were obtained upon treatment with an antiinflammatory eicosanoid, 14,15-EET, known to inhibit CPI-17 phosphorylation. Together these results are consistent with a key molecular role for CPI-17 in AHR, in the absence of bronchial wall remodeling.

Voltage is a partial activator of rat thermosensitive TRP channels

TRPV1 and TRPM8 are sensory nerve ion channels activated by heating and cooling, respectively. A variety of physical and chemical stimuli activate these receptors in a synergistic manner but the underlying mechanisms are unclear. Both channels are voltage sensitive, and temperature and ligands modulate this voltage dependence. Thus, a voltage-sensing mechanism has become an attractive model to explain the generalized gating of these and other thermo-sensitive TRP channels. We show here using whole-cell and single channel measurements that voltage produces only a partial activation of TRPV1 and TRPM8. At room temperature (20-25 degrees C) membrane depolarization evokes responses that saturate at approximately 50-60% of the maximum open probability. Furthermore, high concentrations of capsaicin (10 microm), resiniferatoxin (5 microm) and menthol (6 mm) reveal voltage-independent gating. Similarly, other modes of TRPV1 regulation including heat, protein kinase C-dependent phosphorylation, and protons enhance both the efficacy and sensitivity of voltage activation. In contrast, the TRPV1 antagonist capsazepine produces the opposite effects. These data can be explained by an allosteric model in which voltage, temperature, agonists and inverse agonists are independently coupled, either positively or negatively, to channel gating. Thus, voltage acts separately but in concert with other stimuli to regulate channel activation, and, therefore, a voltage-sensitive mechanism is unlikely to represent a final, gating mechanism for these channels.

Direct Protein Kinase C-dependent Phosphorylation Regulates the Cell Surface Stability and Activity of the Potassium Chloride Cotransporter KCC2

The potassium chloride cotransporter KCC2 plays a major role in the maintenance of transmembrane chloride potential in mature neurons; thus KCC2 activity is critical for hyperpolarizing membrane currents generated upon the activation of gamma-aminobutyric acid type A and glycine (Gly) receptors that underlie fast synaptic inhibition in the adult central nervous system. However, to date an understanding of the cellular mechanism that neurons use to modulate the functional expression of KCC2 remains rudimentary. Using Escherichia coli expression coupled with in vitro kinase assays, we first established that protein kinase C (PKC) can directly phosphorylate serine 940 (Ser(940)) within the C-terminal cytoplasmic domain of KCC2. We further demonstrated that Ser(940) is the major site for PKC-dependent phosphorylation for full-length KCC2 molecules when expressed in HEK-293 cells. Phosphorylation of Ser(940) increased the cell surface stability of KCC2 in this system by decreasing its rate of internalization from the plasma membrane. Coincident phosphorylation of Ser(940) increased the rate of ion transport by KCC2. It was further evident that phosphorylation of endogenous KCC2 in cultured hippocampal neurons is regulated by PKC-dependent activity. Moreover, in keeping with our recombinant studies, enhancing PKC-dependent phosphorylation increased the targeting of KCC2 to the neuronal cell surface. Our studies thus suggest that PKC-dependent phosphorylation of KCC2 may play a central role in modulating both the functional expression of this critical transporter in the brain and the strength of synaptic inhibition.