A single structurally conserved SUMOylation site in CRMP2 controls NaV1.7 function

The neuronal collapsin response mediator protein 2 (CRMP2) undergoes several posttranslational modifications that codify its functions. Most recently, CRMP2 SUMOylation (addition of small ubiquitin like modifier (SUMO)) was identified as a key regulatory step within a modification program that codes for CRMP2 interaction with, and trafficking of, voltage-gated sodium channel NaV1.7. In this paper, we illustrate the utility of combining sequence alignment within protein families with structural analysis to identify, from several putative SUMOylation sites, those that are most likely to be biologically relevant. Co-opting this principle to CRMP2, we demonstrate that, of 3 sites predicted to be SUMOylated in CRMP2, only the lysine 374 site is a SUMOylation client. A reduction in NaV1.7 currents was the corollary of the loss of CRMP2 SUMOylation at this site. A 1.78-Å-resolution crystal structure of mouse CRMP2 was solved using X-ray crystallography, revealing lysine 374 as buried within the CRMP2 tetramer interface but exposed in the monomer. Since CRMP2 SUMOylation is dependent on phosphorylation, we postulate that this state forces CRMP2 toward a monomer, exposing the SUMO site and consequently, resulting in constitutive regulation of NaV1.7.     

Chemical shift perturbation mapping of the Ubc9-CRMP2 interface identifies a pocket in CRMP2 amenable for allosteric modulation of Nav1.7 channels

Drug discovery campaigns directly targeting the voltage-gated sodium channel NaV1.7, a highly prized target in chronic pain, have not yet been clinically successful. In a differentiated approach, we demonstrated allosteric control of trafficking and activity of NaV1.7 by prevention of SUMOylation of collapsin response mediator protein 2 (CRMP2). Spinal administration of a SUMOylation incompetent CRMP2 (CRMP2 K374A) significantly attenuated pain behavior in the spared nerve injury (SNI) model of neuropathic pain, underscoring the importance of SUMOylation of CRMP2 as a pathologic event in chronic pain. Using a rational design strategy, we identified a heptamer peptide harboring CRMP2’s SUMO motif that disrupted the CRMP2-Ubc9 interaction, inhibited CRMP2 SUMOylation, inhibited NaV1.7 membrane trafficking, and specifically inhibited NaV1.7 sodium influx in sensory neurons. Importantly, this peptide reversed nerve injury-induced thermal and mechanical hypersensitivity in the SNI model, supporting the practicality of discovering pain drugs by indirectly targeting NaV1.7 via prevention of CRMP2 SUMOylation. Here, our goal was to map the unique interface between CRMP2 and Ubc9, the E2 SUMO conjugating enzyme. Using computational and biophysical approaches, we demonstrate the enzyme/substrate nature of Ubc9/CRMP2 binding and identify hot spots on CRMP2 that may form the basis of future drug discovery campaigns disrupting the CRMP2-Ubc9 interaction to recapitulate allosteric regulation of NaV1.7 for pain relief.     

SUMOylation alters CRMP2 regulation of calcium influx in sensory neurons

The axon/dendrite specification collapsin response mediator protein 2 (CRMP2) bidirectionally modulates N-type voltage-gated Ca²⁺ channels (CaV2.2). Here we demonstrate that small ubiquitin-like modifier (SUMO) protein modifies CRMP2 via the SUMO E2-conjugating enzyme Ubc9 in vivo. Removal of a SUMO conjugation site KMD in CRMP2 (K374A/M375A/D376A; CRMP2_AAA) resulted in loss of SUMOylated CRMP2 without compromising neurite branching, a canonical hallmark of CRMP2 function. Increasing SUMOylation levels correlated inversely with calcium influx in sensory neurons. CRMP2 deSUMOylation by SUMO proteases SENP1 and SENP2 normalized calcium influx to those in the CRMP2_AAA mutant. Thus, our results identify a novel role for SUMO modification in CRMP2/CaV2.2 signaling pathway.     

Modulation of GABA(A) receptor phosphorylation and membrane trafficking by phospholipase C-related inactive protein/protein phosphatase 1 and 2A signaling complex underlying brain-derived neurotrophic factor-dependent regulation of GABAergic inhibition

Brain-derived neurotrophic factor (BDNF) modulates several distinct aspects of synaptic transmission, including GABAergic transmission. Exposure to BDNF alters properties of GABA(A) receptors and induces changes in the expression level at the cell surface. Although phospholipase C-related inactive protein-1 (PRIP-1) plays an important role in GABA(A) receptor trafficking and function, its role in BDNF-dependent modulation of these receptors, together with the role of PRIP-2, was investigated using neurons cultured from PRIP double knock-out mice. The BDNF-dependent inhibition of whole cell GABA-evoked currents observed in wild type neurons was not detected in neurons cultured from knock-out mice. Instead, a gradual increase in GABA-evoked currents in these neurons correlated with a gradual increase in phosphorylation of GABA(A) receptor beta3 subunit in response to BDNF. To characterize the specific role(s) that PRIP plays as components of underlying molecular machinery, we examined the recruitment of protein phosphatase(s) to GABA(A) receptors. We demonstrate that PRIP associates with phosphatases as well as with beta subunits. PRIP was found to colocalize with GABA(A) receptor clusters in cultured neurons and with recombinant GABA(A) receptors when co-expressed in HEK293 cells. Importantly, a peptide mimicking a domain of PRIP involved in binding to beta subunits disrupted the co-localization of these proteins in HEK293 cells and potently inhibited the BDNF-mediated attenuation of GABA(A) receptor currents in wild type neurons. Together, the results suggest that PRIP plays an important role in BDNF-dependent regulation of GABA(A) receptors by mediating the specific association between beta subunits of these receptors with protein phosphatases.     

Calcium-activated K+ channels increase cell proliferation independent of K+ conductance

The intermediate-conductance calcium-activated potassium channel (IK1) promotes cell proliferation of numerous cell types including endothelial cells, T lymphocytes, and several cancer cell lines. The mechanism underlying IK1-mediated cell proliferation was examined in human embryonic kidney 293 (HEK293) cells expressing recombinant human IK1 (hIK1) channels. Inhibition of hIK1 with TRAM-34 reduced cell proliferation, while expression of hIK1 in HEK293 cells increased proliferation. When HEK293 cells were transfected with a mutant (GYG/AAA) hIK1 channel, which neither conducts K(+) ions nor promotes Ca(2+) entry, proliferation was increased relative to mock-transfected cells. Furthermore, when HEK293 cells were transfected with a trafficking mutant (L18A/L25A) hIK1 channel, proliferation was also increased relative to control cells. The lack of functional activity of hIK1 mutants at the cell membrane was confirmed by a combination of whole cell patch-clamp electrophysiology and fura-2 imaging to assess store-operated Ca(2+) entry and cell surface immunoprecipitation assays. Moreover, in cells expressing hIK1, inhibition of ERK1/2 and JNK kinases, but not of p38 MAP kinase, reduced cell proliferation. We conclude that functional K(+) efflux at the plasma membrane and the consequent hyperpolarization and enhanced Ca(2+) entry are not necessary for hIK1-induced HEK293 cell proliferation. Rather, our data suggest that hIK1-induced proliferation occurs by a direct interaction with ERK1/2 and JNK signaling pathways.     

Impact of phosphomimetic and non‐phosphorylatable mutations of phospholemman on L‐type calcium channels gating in HEK 293T cells

Background : Phospholemman (PLM) is an important phosphorylation substrate for protein kinases A and C in the heart. Until now, the association between PLM phosphorylation status and L‐type calcium channels (LTCCs) gating has not been fully understood. We investigated the kinetics of LTCCs in HEK 293T cells expressing phosphomimetic or nonphosphorylatable PLM mutants. Methods : The LTCCs gating was measured in HEK 293T cells transfected with LTCC and wild‐type (WT) PLM, phosphomimetic or nonphosphorylatable PLM mutants: 6263AA, 6869AA, AAAA, 6263DD, 6869DD or DDDD. Results : WT PLM significantly slowed LTCCs activation and deactivation while enhanced voltage‐dependent inactivation (VDI). PLM mutants 6869DD and DDDD significantly increased the peak of the currents. 6263DD accelerated channel activation, while 6263AA slowed it more than WT PLM. 6869DD significantly enhanced PLM‐induced increase of VDI. AAAA slowed the channel activation more than 6263AA, and DDDD accelerated the channel VDI more than 6869DD. Conclusions : Our results demonstrate that phosphomimetic PLM could stimulate LTCCs and alter their dynamics, while PLM nonphosphorylatable mutant produced the opposite effects.     

Monoamine oxidase B induces ERK-dependent cell mitogenesis by hydrogen peroxide generation

The mitochondrial enzyme monoamine oxidase (MAO) A and B catalyze the oxidative deamination of various endogenous and exogenous biogenic amines. In the present study, we used human embryonic kidney 293 (HEK 293) cells stably transfected with human MAO-B cDNA to investigate the potential role of hydrogen peroxide (H(2)O(2)) produced by MAO-B isoform as an intracellular messenger involved in regulation of cell signaling and function. The MAO substrate tyramine induced tyrosine phosphorylation of Shc, ERK activation, and an increase in DNA synthesis in HEK 293 expressing MAO-B, but not in wild type HEK 293 cells, which do not express MAO. Tyramine effects were fully prevented by cell pretreatment with the MAO inhibitor pargyline or the antioxidant N-acetylcysteine. These results show that MAO-B induces MAPK/ERK activation and cell mitogenesis through H(2)O(2) production.     

Methadone but not Morphine Inhibits Lubiprostone-Stimulated Cl− Currents in T84 Intestinal Cells and Recombinant Human ClC-2, but not CFTR Cl− Currents

In clinical trials, methadone, but not morphine, appeared to prevent beneficial effects of lubiprostone, a ClC-2 Cl^? channel activator, on opioid-induced constipation. Effects of methadone and morphine on lubiprostone-stimulated Cl^? currents were measured by short circuit current (Isc) across T84 cells. Whole cell patch clamp of human ClC-2 (hClC-2) stably expressed in HEK293 cells and in a high expression cell line (HEK293EBNA) as well as human CFTR (hCFTR) stably expressed in HEK293 cells was used to study methadone and morphine effects on recombinant hClC-2 and hCFTR Cl^? currents. Methadone but not morphine inhibited lubiprostone-stimulated Isc in T84 cells with half-maximal inhibition at 100 nM. Naloxone did not affect lubiprostone stimulation or methadone inhibition of Isc. Lubiprostone-stimulated Cl^? currents in hClC-2/HEK293 cells, but not forskolin/IBMX-stimulated Cl^? currents in hCFTR/HEK293 cells, were inhibited by methadone, but not morphine. HEK293EBNA cells expressing hClC-2 showed time-dependent, voltage-activated, CdCl₂-inhibited Cl^? currents in the absence (control) and the presence of lubiprostone. Methadone, but not morphine, inhibited control and lubiprostone-stimulated hClC-2 Cl^? currents with half-maximal inhibition at 100 and 200–230 nM, respectively. Forskolin/IBMX-stimulated hClC-2 Cl^? currents were also inhibited by methadone. Myristoylated protein kinase inhibitor (a specific PKA inhibitor) inhibited forskolin/IBMX- but not lubiprostone-stimulated hClC-2 Cl^? currents. Methadone caused greater inhibition of lubiprostone-stimulated currents added before patching (66.1 %) compared with after patching (28.7 %). Methadone caused inhibition of lubiprostone-stimulated Cl^? currents in T84 cells and control; lubiprostone- and forskolin/IBMX-stimulated recombinant hClC-2 Cl^? currents may be the basis for reduced efficacy of lubiprostone in methadone-treated patients.     

Spontaneous autocrine release of protons activates ASIC-mediated currents in HEK293 cells

When examining HEK293 cells by whole-cell patch-clamp electrophysiology we found spontaneous currents, present in almost all cells. These currents were carried by Na(+) ions, were inhibited by amiloride and by cells exposure to acidic (pH 6.3) extracellular solutions. These properties (ion carrier, amiloride-sensitivity, and inactivation by constant lowering of extracellular pH) were similar to the properties of proton-activated currents measured from the same cells. Spontaneous currents required intracellular ATP, were completely inhibited by intracellular Ca(2+) buffering with BAPTA and were suppressed by intracellular administration of vesicular H(+)ATPase inhibitor bafilomycin. ATP-induced Ca(2+) influx through P2X receptors in HEK293 cells stably transfected with P2X(2), P2X(2/3) or P2X(4) purinoreceptor subunits transiently potentiated amplitude and frequency of spontaneous currents; this effect was antagonized by bafilomycin. We concluded that spontaneous currents represent activation of acid-sensitive ion channels (ASICs) by autocrine vesicular release of protons from HEK cells.     

A C-terminal lysine that controls human P2X4 receptor desensitization

Receptor desensitization can determine the time course of transmitter action and profoundly alter sensitivity to drugs. Among P2X receptors, ion currents through homomeric P2X4 receptors exhibit intermediate desensitization when compared with P2X1 and P2X3 (much faster) and P2X2 and P2X7 (slower). We recorded membrane currents in HEK293 cells transfected to express the human P2X4 receptor. The decline in current during a 4-s application of ATP (100 microm) was about 30%; this was not different during whole-cell or perforated patch recording. Alanine-scanning mutagenesis of the intracellular C terminus identified two positions with much accelerated desensitization kinetics (Lys373: 92% and Tyr374: 74%). At position 373, substitution of Arg or Cys also strongly accelerated desensitization: however, in the case of K373C the wild-type phenotype was fully restored by adding ethylammonium methanethiosulfonate. At position 374, phenylalanine could replace tyrosine. These results indicate that wild-type desensitization properties requires an aromatic moiety at position 374 and an amino rather than a guanidino group at position 373. These residues lie between previously identified motifs involved in membrane trafficking (YXXXK and YXXGL) and implicates the C-terminal also in rearrangements leading to channel closing during the presence of agonist.     

The EGFP/hERG fusion protein alter the electrophysiological properties of hERG channels in HEK293 cells

EGFP (enhanced green fluorescent protein) tagged to either the N (amino)-terminus [EGFP/hERG (human ether-a-go-go-related gene)] or C (carboxyl)-terminus (hERG/EGFP) of hERG channel is used to study mutant channel protein trafficking for several years. However, it has been reported that the process can alter hERG channel properties. The aim of the study was to determine whether EGFP tagged to N-terminus of hERG channels would alter the cellular localizations and the electrophysiological properties of hERG channels compared with untagged hERG channels. The hERG channels tagged with or without EGFP were transiently expressed in HEK (human embryonic kidney) 293 cells using a lipofectamine method. HEK 293 cells expressing pCDNA3-hERG or pEGFP-hERG were double immunolabelled with anti-hERG and anti-calnexin (an ER marker protein) followed with FITC- and TRITC (tetramethylrhodamine β-isothiocyanate)-labelled secondary antibodies, respectively. Confocal laser scanning microscope was used to observe the cellular localization of EGFP-tagged hERG channels and untagged hERG channels. Patch-clamp technique was used to record whole cell currents. We found that the EGFP/hERG fusion protein and untagged hERG channels were both expressed not only on the cell surface membrane but also in the cytoplasm of HEK293 cells. The EGFP/hERG appeared to influence the hERG channel gating properties, including reduction of the peak tail current density, more rapid inactivation process, faster recovery from inactivation and faster deactivation kinetics compared with untagged hERG channels. Our results suggest that the EGFP/hERG channel alter the electrophysiological properties of hERG channel, but it does not seem to alter the cellular location of hERG channels. Thus, EGFP tagging to N-terminus might be used for research of subcellular location of hERG channels but not for the channel electrophysiological properties.     

Functional Modulation of P2×2 Receptors by Cyclic AMP-Dependent Protein Kinase

Abstract: It is generally believed that protein phosphorylation is an important mechanism through which the functions of voltage- and ligand-gated channels are modulated. The intracellular carboxyl terminus of P_2×2 receptor contains several consensus phosphorylation sites for cyclic AMP (cAMP)-dependent protein kinase (PKA) and protein kinase C (PKC), suggesting that the function of the P_2×2 purinoceptor could be regulated by the protein phosphorylation. Whole-cell voltage-clamp recording was used to record ATP-evoked cationic currents from human embryonic kidney (HEK) 293 cells stably transfected with the cDNA encoding the rat P_2×2 receptor. Dialyzing HEK 293 cells with phorbol 12-myristate 13-acetate, a PKC activator, failed to affect the amplitude and kinetics of the ATP-induced cationic current. The role of PKA phosphorylation in modulating the function of the P_2×2 receptor was investigated by internally perfusing HEK 293 cells with 8-bromo-cAMP or the purified catalytic subunit of PKA. Both 8-bromo-cAMP and PKA catalytic subunit caused a reduction in the magnitude of the ATP-activated current without affecting the inactivation kinetics and the value of reversal potential. Site-directed mutagenesis was also performed to replace the intracellular PKA consensus phosphorylation site (Ser⁴³¹) with a cysteine residue. In HEK 293 cells expressing (S431C) mutant P_2×2 receptors, intracellular perfusion of 8-bromo-cAMP or purified PKA catalytic subunit did not affect the amplitude of the ATP-evoked current. These results suggest that as with other ligand-gated ion channels, protein phosphorylation by PKA could play an important role in regulating the function of the P_2×2 receptor and ATP-mediated physiological effects in the nervous system.     

Inherited Pain: SODIUM CHANNEL NAV1.7 A1632T MUTATION CAUSES ERYTHROMELALGIA DUE TO A SHIFT OF FAST INACTIVATION*

Inherited erythromelalgia (IEM) causes debilitating episodic neuropathic pain characterized by burning in the extremities. Inherited “paroxysmal extreme pain disorder” (PEPD) differs in its clinical picture and affects proximal body areas like the rectal, ocular, or jaw regions. Both pain syndromes have been linked to mutations in the voltage-gated sodium channel Nav1.7. Electrophysiological characterization shows that IEM-causing mutations generally enhance activation, whereas mutations leading to PEPD alter fast inactivation. Previously, an A1632E mutation of a patient with overlapping symptoms of IEM and PEPD was reported (Estacion, M., Dib-Hajj, S. D., Benke, P. J., Te Morsche, R. H., Eastman, E. M., Macala, L. J., Drenth, J. P., and Waxman, S. G. (2008) NaV1.7 Gain-of-function mutations as a continuum. A1632E displays physiological changes associated with erythromelalgia and paroxysmal extreme pain disorder mutations and produces symptoms of both disorders. J. Neurosci. 28, 11079–11088), displaying a shift of both activation and fast inactivation. Here, we characterize a new mutation of Nav1.7, A1632T, found in a patient suffering from IEM. Although transfection of A1632T in sensory neurons resulted in hyperexcitability and spontaneous firing of dorsal root ganglia (DRG) neurons, whole-cell patch clamp of transfected HEK cells revealed that Nav1.7 activation was unaltered by the A1632T mutation but that steady-state fast inactivation was shifted to more depolarized potentials. This is a characteristic normally attributed to PEPD-causing mutations. In contrast to the IEM/PEPD crossover mutation A1632E, A1632T failed to slow current decay (i.e. open-state inactivation) and did not increase resurgent currents, which have been suggested to contribute to high-frequency firing in physiological and pathological conditions. Reduced fast inactivation without increased resurgent currents induces symptoms of IEM, not PEPD, in the new Nav1.7 mutation, A1632T. Therefore, persistent and resurgent currents are likely to determine whether a mutation in Nav1.7 leads to IEM or PEPD.     

稳定表达hHCN2基因 HEK293细胞系的建立

目的：培育稳定表达hHCN2基因的细胞系,建立一种表达研究心肌离子通道的有效模型。方法：通过脂质体转染的方法,将重组pcDNA3-hHCN2真核表达载体导入人胚肾细胞（HEK293细胞）,以G418压力筛选转染细胞,并对其进行全细胞膜片钳记录。结果：经600μg／ml压力筛选后,获得抗性细胞克隆,并用全细胞膜片钳技术记录到克隆hHCN2通道编码电流。结论：本实验采用脂质体转染法成功地培育出G418抗性HEK293细胞。为进一步研究克隆离子通道结构和功能的关系奠定基础。     

Coexpression with beta(1)-subunit modifies the kinetics and fatty acid block of hH1(alpha) Na(+) channels

Voltage-gated cardiac Na(+) channels are composed of alpha- and beta(1)-subunits. In this study beta(1)-subunit was cotransfected with the alpha-subunit of the human cardiac Na(+) channel (hH1(alpha)) in human embryonic kidney (HEK293t) cells. The effects of this coexpression on the kinetics and fatty acid-induced suppression of Na(+) currents were assessed. Current density was significantly greater in HEK293t cells coexpressing alpha- and beta(1)-subunits (I(Na,alpha beta)) than in HEK293t cells expressing alpha-subunit alone (I(Na,alpha)). Compared with I(Na,alpha), the voltage-dependent inactivation and activation of I(Na,alpha beta) were significantly shifted in the depolarizing direction. In addition, coexpression with beta(1)-subunit prolonged the duration of recovery from inactivation. Eicosapentaenoic acid [EPA, C20:5(n-3)] significantly reduced I(Na,alpha beta) in a concentration-dependent manner and at 5 microM shifted the midpoint voltage of the steady-state inactivation by -22 +/- 1 mV. EPA also significantly accelerated channel transition from the resting state to the inactivated state and prolonged the recovery time from inactivation. Docosahexaenoic acid [C22:6(n-3)], alpha-linolenic acid [C18:3(n-3)], and conjugated linoleic acid [C18:2(n-6)] at 5 microM significantly inhibited both I(Na,alpha beta) and I(Na,alpha.) In contrast, saturated and monounsaturated fatty acids had no effects on I(Na,alpha beta). This finding differs from the results for I(Na,alpha), which was significantly inhibited by both saturated and unsaturated fatty acids. Our data demonstrate that functional association of beta(1)-subunit with hH1(alpha) modifies the kinetics and fatty acid block of the Na(+) channel.     

Bimodal regulation of the human H1 histamine receptor by G protein-coupled receptor kinase 2

The H1 histamine receptor (H1HR) is a member of the G protein-coupled receptor superfamily and regulates numerous cellular functions through its activation of the G(q/11) subfamily of heterotrimeric G proteins. Although the H1HR has been shown to undergo desensitization in multiple cell types, the mechanisms underlying the regulation of H1HR signaling are poorly defined. To address this issue, we examined the effects of wild type and mutant G protein-coupled receptor kinases (GRKs) on the phosphorylation and signaling of human H1HR in HEK293 cells. Overexpression of GRK2 promoted H1HR phosphorylation in intact HEK293 cells and completely inhibited inositol phosphate production stimulated by H1HR, whereas GRK5 and GRK6 had lesser effects on H1HR phosphorylation and signaling. Interestingly, catalytically inactive GRK2 (GRK2-K220R) also significantly attenuated H1HR-mediated inositol phosphate production, as did an N-terminal fragment of GRK2 previously characterized as a regulator of G protein signaling (RGS) protein for Galpha(q/11). Disruption of this RGS function in holo-GRK2 by mutation (GRK2-D110A) partially reversed the quenching effect of GRK2, whereas deletion of both the kinase activity and RGS function (GRK2-D110A/K220R) effectively relieved the inhibition of inositol phosphate generation. To evaluate the role of endogenous GRKs on H1HR regulation, we used small interfering RNAs to selectively target GRK2 and GRK5, two of the primary GRKs expressed in HEK293 cells. A GRK2-specific small interfering RNA effectively reduced GRK2 expression and resulted in a significant increase in histamine-promoted calcium flux. In contrast, knockdown of GRK5 expression was without effect on H1HR signaling. These findings demonstrate that GRK2 is the principal kinase mediating H1 histamine receptor desensitization in HEK293 cells and suggest that rapid termination of H1HR signaling is mediated by both the kinase activity and RGS function of GRK2.     

Interleukin-2 inhibits NMDA receptor-mediated currents directly and may differentially affect subtypes

Using whole-cell patch-clamp recordings, this study investigated the effects of interleukin-2 (IL-2) on N-methyl-d-aspartate (NMDA) receptor-mediated currents (I(NMDA)) in rat cultured hippocampal neurons and human embryonic kidney (HEK) 293 cells expressing recombinant NMDA receptors. We found that IL-2 (0.01-1ng/ml) immediately and significantly decreased peak I(NMDA) in cultured neurons. Interestingly, the peak I(NMDA) induced in HEK 293 cells was also inhibited by IL-2. We also found that IL-2 differentially decreased the peak amplitudes of NR2A- and NR2B-containing NMDA receptor-mediated currents (I(NR2A) and I(NR2B)) by 54+/-5% and 30+/-4%, respectively. These results provide new evidence that IL-2 induces rapid inhibition of peak currents of NMDA receptor-mediated responses with possible NR1/NR2A and NR1/NR2B subtype-differentiation, and suggest that the inhibition is mediated by direct interaction between IL-2 and NMDA receptors.