Intracellular Cysteine Residues Research Articles

A2B adenosine receptor signaling and regulation.

The A2B adenosine receptor (A2BR) is one of the four adenosine-activated G protein-coupled receptors. In addition to adenosine, protein kinase C (PKC) was recently found to activate the A2BR. The A2BR is coupled to both Gs and Gi, as well as Gq proteins in some cell types. Many primary cells and cell lines, such as bladder and breast cancer, bronchial smooth muscle, skeletal muscle, and fat cells, express the A2BR endogenously at high levels, suggesting its potentially important role in asthma, cancer, diabetes, and other conditions. The A2BR has been characterized as both pro- and anti-inflammatory, inducing cell type-dependent secretion of IL-6, IL-8, and IL-10. Theophylline and enprofylline have long been used for asthma treatment, although it is still not entirely clear if their A2BR antagonism contributes to their therapeutic effects or side effects. The A2BR is required in ischemic cardiac preconditioning by adenosine. Both A2BR and protein kinase C (PKC) contribute to cardioprotection, and both modes of A2BR signaling can be blocked byA2BR antagonists. Inhibitors of PKC and A2BR are in clinical cancer trials. Sulforaphane and other isothiocyanates from cruciferous vegetables such asbroccoli and cauliflower have been reported to inhibit A2BR signaling via reaction with an intracellular A2BR cysteine residue (C210). A full, A2BR-selective agonist, critical to elucidate many controversial roles of the A2BR, is still not available, although agonist-bound A2BR structures have recently been reported.

Limb-Clasping Response in NMDA Receptor Palmitoylation-Deficient Mice.

Proper regulation of N-methyl-D-aspartate-type glutamate receptor (NMDA receptor) expression is responsible for excitatory synaptic functions in the mammalian brain. NMDA receptor dysfunction can cause various neuropsychiatric disorders and neurodegenerative diseases. Posttranslational protein S-palmitoylation, the covalent attachment of palmitic acid to intracellular cysteine residues via thioester bonds, occurs in the carboxyl terminus of GluN2B, which is the major regulatory NMDA receptor subunit. Mutations of three palmitoylatable cysteine residues in the membrane-proximal cluster of GluN2B to non-palmitoylatable serine (3CS) lead to the dephosphorylation of GluN2B Tyr1472 in the hippocampus and cerebral cortex, inducing a reduction in the surface expression of GluN2B-containig NMDA receptors. Furthermore, adult GluN2B 3CS homozygous mice demonstrated a definite clasping response without abnormalities in the gross brain structure, other neurological reflexes, or expression levels of synaptic proteins in the cerebrum. This behavioral disorder, observed in the GluN2B 3CS knock-in mice, indicated that complex higher brain functions are coordinated through the palmitoylation-dependent regulation of NMDA receptors in excitatory synapses.

Gating of the capsaicin receptor TRPV1 by UVA-light and oxidants are mediated by distinct mechanisms

Redox-sensitivity is a common property of several transient receptor potential (TRP) ion channels. Oxidants and UVA-light activate TRPV2 by oxidizing methionine pore residues which are conserved in the capsaicin-receptor TRPV1. However, the redox-sensitivity of TRPV1 is regarded to depend on intracellular cysteine residues. In this study we examined if TRPV1 is gated by UVA-light, and if the conserved methionine residues are relevant for redox-sensitivity of TRPV1. Patch clamp recordings were performed to explore wildtype (WT) and mutants of human TRPV1 (hTRPV1).UVA-light induced hTRPV1-mediated membrane currents and potentiated both proton- and heat-evoked currents. The reducing agent dithiothreitol (DTT) prevented and partially reversed UVA-light induced sensitization of hTRPV1. UVA-light induced sensitization was reduced in the mutant hTRPV1-C158A/C387S/C767S (hTRPV1-3C). The remaining sensitivity to UVA-light of hTRRPV1-3C was not further reduced upon exchange of the methionine residues M568 and M645. While UVA-induced sensitization was reduced in the protein kinase C-insensitive mutant hTRPV1-S502A/S801A, the PKC-inhibitors chelerythrine chloride, staurosporine and Gö6976 did not reduce UVA-induced effects on hTRPV1-WT. While hTRPV1-3C was insensitive to the cysteine-selective oxidant diamide, it displayed a residual sensitivity to H2O2 and chloramine-T. However, the exchange of M568 and M645 in hTRPV1-3C did not further reduce these effects.Our data demonstrate that oxidants and UVA-light gate hTRPV1 by cysteine-dependent as well as cysteine-independent mechanisms. In contrast to TRPV2, the methionine residues 568 and 645 seem to be of limited relevance for redox-sensitivity of hTRPV1. Finally, UVA-light induced gating of hTRPV1 does not seem to require activation of protein kinase C.

A Non-covalent Ligand Reveals Biased Agonism of the TRPA1 Ion Channel

The TRPA1 ion channel is activated by electrophilic compounds through the covalent modification of intracellular cysteine residues. How non-covalent agonists activate the channel and whether covalent and non-covalent agonists elicit the same physiological responses are not understood. Here, we report the discovery of a non-covalent agonist, GNE551, and determine a cryo-EM structure of the TRPA1-GNE551 complex, revealing a distinct binding pocket and ligand-interaction mechanism. Unlike the covalent agonist allyl isothiocyanate, which elicits channel desensitization, tachyphylaxis, and transient pain, GNE551 activates TRPA1 into a distinct conducting state without desensitization and induces persistent pain. Furthermore, GNE551-evoked pain is relatively insensitive to antagonist treatment. Thus, we demonstrate the biased agonism of TRPA1, a finding that has important implications for the discovery of effective drugs tailored to different disease etiologies.

Regulation of NCX1 by palmitoylation

Palmitoylation (S-acylation) is the reversible conjugation of a fatty acid (usually C16 palmitate) to intracellular cysteine residues of proteins via a thioester linkage. Palmitoylation anchors intracellular regions of proteins to membranes because the palmitoylated cysteine is recruited to the lipid bilayer. NCX1 is palmitoylated at a single cysteine in its large regulatory intracellular loop. The presence of an amphipathic α-helix immediately adjacent to the NCX1 palmitoylation site is required for NCX1 palmitoylation. The NCX1 palmitoylation site is conserved through most metazoan phlya. Although palmitoylation does not regulate the normal forward or reverse ion transport modes of NCX1, NCX1 palmitoylation is required for its inactivation: sodium-dependent inactivation and inactivation by PIP2 depletion are significantly impaired for unpalmitoylatable NCX1. Here we review the role of palmitoylation in regulating NCX1 activity, and highlight future questions that must be addressed to fully understand the importance of this regulatory mechanism for sodium and calcium transport in cardiac muscle.

The local environment of cysteine 621 determines the rapid electrophilic adduction and activation of hTRPA1

Transient Receptor Potential Ankyrin 1 (TRPA1) is a non‐selective cation channel expressed largely on nociceptive sensory nerves. TRPA1 is rapidly activated by electrophiles, such as iodoacetamide, through covalent modification of intracellular cysteine (Cys) residues. We have previously shown that, at physiologically relevant exposures, iodoacetamide rapidly labels four Cys residues at positions 273, 621, 655, 1085. Of these, Cys residue at position 621 (C621) appears to be crucial for binding and activation of the human channel (hTRPA1). Interestingly, when comparing hTRPA1 with the electrophile‐insensitive rattlesnake homolog, while the amino acids lysine K620 and C621 are conserved the proline at P622 is not. We hypothesized that this rapid binding of C621 residue in human TRPA1 is mediated by neighboring amino acids K620 and P622 rendering it more sensitive to electrophilic adduction. We expressed V5‐His tagged hTRPA1 constructs in HEK293 cells including the wild‐type and the point mutants C621A, K620A and P622A. We used pulse‐chase experiments to label TRPA1 protein with iodoacetamide‐alkyne (IA‐alk), then used click chemistry to label with a fluorescent tag. Binding was measured using immunoprecipitated TRPA1 separated by SDS‐polyacrylamide gel electrophoresis and western blotting. TRPA1 channel activation was measured using ratiometric calcium imaging. The K620A mutant had the effect of greatly reducing electrophilic binding and activation of the channel. Modeling of deprotonation energies suggests that K620 contributes to C621 reactivity by reducing its pKa. The K620 mutant therefore likely reduces the probability that C621 will be thiolate and therefore able to rapidly react with electrophiles. The P622A mutant was also largely insensitive to electrophilic adduction and activation. IA‐alk binding was ~10% of that seen with the wild‐type channel. We hypothesize this is because, in the wild‐type hTRPA1, P622 induces a kink in the protein resulting in the positively charged K620 being closer to C621 compared with the nearby negatively charged glutamates, E625 and E681. The work described here highlights the importance of the amino acids K620 and P622 flanking C621. Our data demonstrate how the integrity of the local environment is essential to TRPA1 function as an electrophilic sensor.Support or Funding InformationThis work was supported by the National Heart Lung and Blood Institute in Bethesda, USA(R01‐HL119802‐S1)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Comparative analysis of palmitoylation sites of serotonin (5-HT) receptors in vertebrates.

BackgroundIn the vertebrate central nervous system as well as in the periphery, serotonin, also known as 5‐hydroxytriptamine (5‐HT), function as a neurotransmitter, a hormone or a mitogen. 5‐HT receptors are composed of 7 family 5‐HT 1‐7 receptors, comprising of 14 structurally and pharmacologically distinct 5‐HT receptor subtypes. Previous experimental studies showed that mouse 5‐HT 1A, 5‐HT 4 and 5‐HT 7 receptors are regulated by post‐translational protein palmitoylation, the reversible attachment of the lipid palmitate to intracellular cysteine residues. Here, we further focused on conservation of these putative palmitoylation sites found in vertebrate 5‐HT receptor orthologs.Methods and ResultsAnalysis of sequence databases provides evidence to suggest that palmitoylation sites of these 5‐HT receptors have been extremely conserved in the vertebrate lineages from jawless fishes to human, in spite of the divergence of 5‐HT 1A, 5‐HT 4 or 5‐HT 7 receptors full‐length amino acid sequences during molecular evolution.ConclusionOur findings mean that dynamic regulation of 5‐HT receptors made possible by reversible post‐translational protein palmitoylation may be critical for refined functions of the vertebrate serotonergic systems.

Subunit Dependent Regulation of LRRC8 Mediated VRAC Currents by Oxidation

Volume-regulated anion channels (VRAC) are heteromers composed of the essential LRRC8A subunit and at least one among the LRRC8B-E subunits [1]. Reactive oxygen species (ROS) play physiological and pathophysiological roles and VRAC channels are known to be highly ROS sensitive. Using fluorescently tagged LRRC8 proteins that give rise to constitutive currents in Xenopus oocytes [2] we tested direct effects of oxidation on different subunit combinations of LRRC8 channels. 8A/8E heteromers were dramatically potentiated (more than 10-fold) by oxidation of intracellular cysteine residues by chloramine-T or tert-butyl hydroperoxide. Oxidation was not necessary for hypotonicity induced activation. In contrast, 8A/8C and 8A/8D heteromers were strongly inhibited by oxidation. In agreement with the non-inactivating current characteristic of VRAC in T lymphocytes that is a typical feature of the 8C subunit, we found that VRAC currents in Jurkat cells are inhibited by oxidation and that the 8E subunit is the least expressed in Jurkat cells. A reduction of VRAC during acute T cell activation associated with ROS production might be of physiological relevance in the immune response.

Activation of the capsaicin-receptor TRPV1 by the acetaminophen metabolite N-arachidonoylaminophenol results in cytotoxicity

AimsThe anandamide reuptake inhibitor N-arachidonoylaminophenol (AM404) and the reactive substance N-acetyl-p-benzoquinone imine (NAPQI) are both metabolites of acetaminophen and may contribute to acetaminophen-induced analgesia by acting at TRPV1 expressed in the peripheral or central nervous system. While NAPQI slowly sensitizes and activates TRPV1 by interacting with distinct intracellular cysteine residues, detailed properties of AM404 as an agonist of TRPV1 have not yet been reported on. We explored the effects of AM404 on recombinant human TRPV1 and in rodent dorsal root ganglion (DRG) neurons. Materials and methodsHEK 293 cells expressing different isoforms of recombinant TRPV1 and rodent DRG neurons were employed for patch clamp and calcium imaging experiments. Cytotoxicity was assessed by propidium iodide and Annexin V staining on TRPV1-HEK 293 cells and with trypan blue staining on DRG neurons. Key findingsAM404 activates hTRPV1 at concentrations >1μM and in a concentration-dependent manner. AM404 also potentiates TRPV1-mediated currents evoked by heat and anandamide. Moreover, AM404-evoked currents are potentiated by NAPQI. While the partly capsaicin-insensitive rabbit (o) TRPV1 fails to respond to AM404, AM404-sensitivity is restored by insertion of the capsaicin binding-domain of rat TRPV1 into oTRPV1. In DRG neurons, AM404-evoked calcium influx as well as cell death is mediated by TRPV1. SignificanceAM404 gates TRPV1 by interacting with the vanilloid-binding site, and TRPV1 is the main receptor for AM404 in DRG neurons. While direct activation of TRPV1 requires high concentrations of AM404, it is possible that synergistic effects of AM404 with further TRPV1-agonists may occur at clinically relevant concentrations.

Subunit-dependent oxidative stress sensitivity of LRRC8 volume-regulated anion channels.

Swelling-activated anion currents are modulated by oxidative conditions, but it is unknown if oxidation acts directly on the LRRC8 channel-forming proteins or on regulatory factors. We found that LRRC8A-LRRC8E heteromeric channels are dramatically activated by oxidation of intracellular cysteines, whereas LRRC8A-LRRC8C and LRRC8A-LRRC8D heteromers are inhibited by oxidation. Volume-regulated anion currents in Jurkat T lymphocytes were inhibited by oxidation, in agreement with a low expression of the LRRC8E subunit in these cells. Our results show that LRRC8 channel proteins are directly modulated by oxidation in a subunit-specific manner. The volume-regulated anion channel (VRAC) is formed by heteromers of LRRC8 proteins containing the essential LRRC8A subunit and at least one among the LRRC8B-E subunits. Reactive oxygen species (ROS) play physiological and pathophysiological roles and VRAC channels are highly ROS sensitive. However, it is unclear if ROS act directly on the channels or on molecules involved in the activation pathway. We used fluorescently tagged LRRC8 proteins that yield large constitutive currents to test direct effects of oxidation. We found that 8A/8E heteromers are dramatically potentiated (more than 10-fold) by oxidation of intracellular cysteine residues by chloramine-T or tert-butyl hydroperoxide. Oxidation was, however, not necessary for hypotonicity-induced activation. In contrast, 8A/8C and 8A/8D heteromers were strongly inhibited by oxidation. Endogenous VRAC currents in Jurkat T lymphocytes were similarly inhibited by oxidation, in agreement with the finding that LRRC8C and LRRC8D subunits were more abundantly expressed than LRRC8E in Jurkat cells. Our results show that LRRC8 channels are directly modulated by oxidation in a subunit-dependent manner.

The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels contain multiple S-palmitoylation sites.

Expression of hyperpolarization-activated cyclic nucleotide-gated channels (HCN1-4) on distal dendrites of neurons is suggested to modify synaptic integration in the central nervous system. However, the mechanisms of dendritic localization are not fully understood. Recent studies have revealed that S-palmitoylation plays an important role in the enrichment of various molecules at the postsynaptic membrane. Thus, we performed an acyl-biotinyl exchange assay, and found that HCN1, HCN2, and HCN4, but not HCN3, were S-palmitoylated in HEK293 cells. Mutation of multiple intracellular cysteine residues at the N-terminus of HCN2 was required for complete inhibition of S-palmitoylation. However, this mutagenesis had a minimal effect on surface expression of HCN2 proteins or electrophysiological properties of HCN2 current when expressed in HEK293 cells or in Xenopus oocytes. These findings provide insight into the physiological roles of S-palmitoylation of HCN channels in native neurons.

Regulation of Pro-Apoptotic Phosphorylation of Kv2.1 K+ Channels.

Caspase activity during apoptosis is inhibited by physiological concentrations of intracellular K+. To enable apoptosis in injured cortical and hippocampal neurons, cellular loss of this cation is facilitated by the insertion of Kv2.1 K+ channels into the plasma membrane via a Zn2+/CaMKII/SNARE-dependent process. Pro-apoptotic membrane insertion of Kv2.1 requires the dual phosphorylation of the channel by Src and p38 at cytoplasmic N- and C-terminal residues Y124 and S800, respectively. In this study, we investigate if these phosphorylation sites are mutually co-regulated, and whether putative N- and C-terminal interactions, possibly enabled by Kv2.1 intracellular cysteine residues C73 and C710, influence the phosphorylation process itself. Studies were performed with recombinant wild type and mutant Kv2.1 expressed in Chinese hamster ovary (CHO) cells. Using immunoprecipitated Kv2.1 protein and phospho-specific antibodies, we found that an intact Y124 is required for p38 phosphorylation of S800, and, importantly, that Src phosphorylation of Y124 facilitates the action of the p38 at the S800 residue. Moreover, the actions of Src on Kv2.1 are substantially decreased in the non-phosphorylatable S800A channel mutant. We also observed that mutations of either C73 or C710 residues decreased the p38 phosphorylation at S800 without influencing the actions of Src on tyrosine phosphorylation of Kv2.1. Surprisingly, however, apoptotic K+ currents were suppressed only in cells expressing the Kv2.1(C73A) mutant but not in those transfected with Kv2.1(C710A), suggesting a possible structural alteration in the C-terminal mutant that facilitates membrane insertion. These results show that intracellular N-terminal domains critically regulate phosphorylation of the C-terminal of Kv2.1, and vice versa, suggesting possible new avenues for modifying the apoptotic insertion of these channels during neurodegenerative processes.

The physiology of protein S-acylation.

Protein S-acylation, the only fully reversible posttranslational lipid modification of proteins, is emerging as a ubiquitous mechanism to control the properties and function of a diverse array of proteins and consequently physiological processes. S-acylation results from the enzymatic addition of long-chain lipids, most typically palmitate, onto intracellular cysteine residues of soluble and transmembrane proteins via a labile thioester linkage. Addition of lipid results in increases in protein hydrophobicity that can impact on protein structure, assembly, maturation, trafficking, and function. The recent explosion in global S-acylation (palmitoyl) proteomic profiling as a result of improved biochemical tools to assay S-acylation, in conjunction with the recent identification of enzymes that control protein S-acylation and de-acylation, has opened a new vista into the physiological function of S-acylation. This review introduces key features of S-acylation and tools to interrogate this process, and highlights the eclectic array of proteins regulated including membrane receptors, ion channels and transporters, enzymes and kinases, signaling adapters and chaperones, cell adhesion, and structural proteins. We highlight recent findings correlating disruption of S-acylation to pathophysiology and disease and discuss some of the major challenges and opportunities in this rapidly expanding field.

Tmem27 dimerization, deglycosylation, plasma membrane depletion, and the extracellular Phe-Phe motif are negative regulators of cleavage by Bace2

The pancreatic β-cell surface protein Tmem27 is promotes the preservation of functional β-cell mass. It is a selective substrate of the protease Bace2, yet the intramolecular features of Tmem27 that regulate its processing by this sheddase have not been characterized. In particular, the importance of homodimerization, glycosylation, trafficking to the plasma membrane (PM), the existence of multiple cleavage sites, and the amino acid residues that govern these features are currently unknown. Using Tmem27 mutational analysis and multiple biochemical approaches, we here show that Tmem27 dimerization is a dynamic process mediated by its intracellular cysteine residue and that prevents Tmem27 cleavage, that extracellular asparagine glycosylation is essential for Tmem27 trafficking to the PM and its processing by Bace2, that the amount of Tmem27 at the PM is proportional to its total cell levels upon glucose stimulation and Bace2 inhibition, and that the double phenylalanine motif in the Tmem27 cleavage site is an intramolecular Bace2 inhibitor. These findings define structural properties of Tmem27 that affect the susceptibility to its protease Bace2 and have implications for the efficiency with which Tmem27 and other Bace2 substrates are cleaved in normal and disease states.

DPP6A Confers Redox Sensitivity to Kv4 Channel Inactivation

Reactive oxygen species (ROS) are important modulators of excitability and may play a critical role in the etiology of many neurodegenerative disorders. Previous studies have shown that the fast N-type inactivation of Kv1.4 channels is suppressed by ROS-mediated cysteine oxidation whereas Kv4 channel N-type inactivation is not. However, in native neurons Kv4 channel subunits form large macromolecular complexes with KChIP and DPL proteins, and fast N-type inactivation is conferred to Kv4 channel complexes by a specific isoform of DPL proteins, either DPP10a or DPP6a. To investigate whether the DPP6a-mediated fast inactivation is regulated by ROS, tert- butyl hydroperoxide (tBHP) was applied to oocytes expressing Kv4.2+KChIP3a+DPP6a channels. tBHP (1 mM) application dramatically increases the peak current amplitude by ∼ 44% and slowed inactivation kinetics. The effects of tBHP are reversed by the reducing agent dithiothreitol (DTT, 10 mM). In contrast, ternary complex channels containing another DPP6 isoform (DPP6K) are not affected by tBHP, indicating the importance of the DPP6a variable N-terminal domain for the tBHP effect. Alignment of N-terminal sequences from DPP6a and DPP10a with Kv1.4 reveals a common cysteine residue in position 13 (Cys-13), which in Kv1.4 is critical for the redox-regulation of N-type inactivation. Substituting Cys-13 of DPP6a with serine (DPP6a/C13S) results in a loss of regulation by tBHP, consistent with a similar critical role for DPP6a Cys-13. To test if other cysteines in the channel are also required for this regulation, we switched to mutant Kv4.1 (C11xA) and KChIP3a (KChIP3a/del2-59) constructs that remove most intracellular cysteine residues. Channels composed of C11xA, KChIP3a/del2-59, and normal DPP6a show disrupted regulation by tBHP, suggesting that ROS likely induces an intersubunit disulfide linkage to regulate DPP6a-mediated N-type inactivation. This work was supported by a grant from the National Institute of Health (R01 GM090029).

Differential Roles of Cysteine Residues in the Cellular Trafficking, Dimerization, and Function of the High-Density Lipoprotein Receptor, SR-BI

The scavenger receptor, class B, type I (SR-BI) binds high-density lipoprotein (HDL) and mediates selective delivery of cholesteryl esters (CEs) to the liver and steroidogenic cells of the adrenal glands and gonads. Although it is clear that the large extracellular domain (ECD) of SR-BI binds HDL, the role of ECD in the selective HDL-CE transport remains poorly understood. In this study, we used a combination of mutational and chemical approaches to systematically evaluate the contribution of cysteine residues, especially six cysteine residues of ECD, in SR-BI-mediated selective HDL-CE uptake, intracellular trafficking, and SR-BI dimerization. Pretreatment of SR-BI-overexpressing COS-7 cells with a disulfide (S-S) bond reducing agent, β-mercaptoethanol (100 mM) or dithiothreitol (DTT) (10 mM), modestly but significantly impaired SR-BI-mediated selective HDL-CE uptake. Treatment of SR-BI-overexpressing COS-7 cells with the optimal doses of membrane permeant alkyl methanethiosulfonate (MTS) reagents, positively charged MTSEA or neutral MMTS, that specifically react with the free sulfhydryl group of cysteine reduced the rate of SR-BI-mediated selective HDL-CE uptake, indicating that certain intracellular free cysteine residues may also be critically involved in the selective cholesterol transport process. In contrast, use of membrane impermeant MTS reagent, positively charged MTSET and negatively charged MTSES, showed no such effect. Next, the importance of eight cysteine residues in SR-BI expression, cell surface expression, dimer formation, and selective HDL-derived CE transport was evaluated. These cysteine residues were replaced either singly or in pairs with serine, and the mutant SR-BIs were expressed in either COS-7 or CHO cells. Four mutations, C280S, C321S, C323S, and C334S, of the ECD, either singly or in various pair combinations, resulted in significant decreases in SR-BI (HDL) binding activity, selective CE uptake, and trafficking to the cell surface. Surprisingly, we found that mutation of the two remaining cysteine residues, C251 and C384 of the ECD, had no effect on either SR-BI expression or function. Other cysteine mutations and substitutions were also without effect. Western blot data indicated that single and double mutations at C280, C321, C323, and C334 residues strongly favor dimer formation. However, they are rendered nonfunctional presumably because of mutation-induced formation of aberrant disulfide linkages resulting in inhibition of optimal HDL binding and, thus, selective HDL-CE uptake. These results provide novel insights into the functional role of four cysteine residues, C280, C321, C323, and C334, of the SR-BI ECD in SR-BI expression and trafficking to the cell surface, its dimerization, and associated selective CE transport function.

Palmitoylation of human proteinase-activated receptor-2 differentially regulates receptor-triggered ERK1/2 activation, calcium signalling and endocytosis

hPAR(2) (human proteinase-activated receptor-2) is a member of the novel family of proteolytically activated GPCRs (G-protein-coupled receptors) termed PARs (proteinase-activated receptors). Previous pharmacological studies have found that activation of hPAR(2) by mast cell tryptase can be regulated by receptor N-terminal glycosylation. In order to elucidate other post-translational modifications of hPAR(2) that can regulate function, we have explored the functional role of the intracellular cysteine residue Cys(361). We have demonstrated, using autoradiography, that Cys(361) is the primary palmitoylation site of hPAR(2). The hPAR(2)C361A mutant cell line displayed greater cell-surface expression compared with the wt (wild-type)-hPAR(2)-expressing cell line. hPAR(2)C361A also showed a decreased sensitivity and efficacy (intracellular calcium signalling) towards both trypsin and SLIGKV. In stark contrast, hPAR(2)C361A triggered greater and more prolonged ERK (extracellular-signal-regulated kinase) phosphorylation compared with that of wt-hPAR(2) possibly through Gi, since pertussis toxin inhibited the ability of this receptor to activate ERK. Finally, flow cytometry was utilized to assess the rate and extent of receptor internalization following agonist challenge. hPAR(2)C361A displayed faster internalization kinetics following trypsin activation compared with wt-hPAR(2), whereas SLIGKV had a negligible effect on internalization for either receptor. In conclusion, palmitoylation plays an important role in the regulation of PAR(2) expression, agonist sensitivity, desensitization and internalization.

Activation of TRPA1 by Membrane Permeable Local Anesthetics

BackgroundLow concentrations of local anesthetics (LAs) suppress cellular excitability by inhibiting voltage-gated Na+ channels. In contrast, LAs at high concentrations can be excitatory and neurotoxic. We recently demonstrated that LA-evoked activation of sensory neurons is mediated by the capsaicin receptor TRPV1, and, to a lesser extent by the irritant receptor TRPA1. LA-induced activation and sensitization of TRPV1 involves a domain that is similar, but not identical to the vanilloid-binding domain. Additionally, activation of TRPV1 by LAs involves PLC and PI(4,5)P2-signalling. In the present study we aimed to characterize essential structural determinants for LA-evoked activation of TRPA1.ResultsRecombinant rodent and human TRPA1 were expressed in HEK293t cells and investigated by means of whole-cell patch clamp recordings. The LA lidocaine activates TRPA1 in a concentration-dependent manner. The membrane impermeable lidocaine-derivative QX-314 is inactive when applied extracellularly. Lidocaine-activated TRPA1-currents are blocked by the TRPA1-antagonist HC-030031. Lidocaine is also an inhibitor of TRPA1, an effect that is more obvious in rodent than in human TRPA1. This species-specific difference is linked to the pore region (transmembrane domain 5 and 6) as described for activation of TRPA1 by menthol. Unlike menthol-sensitivity however, lidocaine-sensitivity is not similarly determined by serine- and threonine-residues within TM5. Instead, intracellular cysteine residues known to be covalently bound by reactive TRPA1-agonists seem to mediate activation of TRPA1 by LAs.ConclusionsThe structural determinants involved in activation of TRPA1 by LAs are disparate from those involved in activation by menthol or those involved in activation of TRPV1 by LAs.

Intracellular Cysteine 346 Is Essentially Involved in Regulating Panx1 Channel Activity

Pannexins constitute a family of proteins exhibiting predominantly hemichannel activity. Pannexin channels have been suggested to participate in a wide spectrum of biological functions such as propagation of calcium waves, release of IL-1β, and responses to ischemic conditions. At present, the molecular mechanisms regulating pannexin hemichannel activity are essentially unknown. Because cysteines have been shown to constitute key elements in regulating hemichannel properties of the connexin-type we performed site-directed mutagenesis of intracellular cysteine residues of Panx1. Cysteine to serine exchange (Cys → Ser) at the C-terminal position amino acid 346 led to a constitutively leaky hemichannel and subsequently to cell death. Increased channel activity was demonstrated by dye uptake and electrophysiological profiling in injected Xenopus laevis oocytes and transfected N2A cells. Mutations of the remaining intracellular cysteines did not result in major changes of Panx1 channel properties. From these data we conclude that the Cys-346 residue is important for proper functioning of the Panx1 channel.

Diabetes Depresses Synaptic Transmission in Sympathetic Ganglia by Inactivating nAChRs through a Conserved Intracellular Cysteine Residue

Most people with diabetes develop severe complications of the autonomic nervous system; yet, the underlying causes of many diabetic-induced dysautonomias are poorly understood. Here we explore the idea that these dysautonomias results, in part, from a defect in synaptic transmission. To test this idea, we investigated cultured sympathetic neurons and show that hyperglycemia inactivates nAChRs through a mechanism involving an elevation in reactive oxygen species and an interaction with highly conserved cysteine residues located near the intracellular mouth of the nAChR channel. Consistent with this, we show that diabetic mice have depressed ganglionic transmission and reduced sympathetic reflexes, whereas diabetic mice expressing mutant postsynaptic nAChRs that lack the conserved cysteine residues on the alpha3 subunit have normal synaptic transmission in sympathetic ganglia and normal sympathetic reflexes. Our work suggests a new model for diabetic-induced dysautonomias and identifies ganglionic nAChRs as targets of hyperglycemia-induced downstream signals.