scholarly journals Effects of GABAA Receptor α3 Subunit Epilepsy Mutations on Inhibitory Synaptic Signaling

2020 ◽  
Vol 13 ◽  
Author(s):  
Parnayan Syed ◽  
Nela Durisic ◽  
Robert J. Harvey ◽  
Pankaj Sah ◽  
Joseph W. Lynch
Keyword(s):  
Expression System ◽  
Epileptic Seizures ◽  
Surface Expression ◽  
Decay Rates ◽  
Hek293 Cells ◽  
Cell Surface Expression ◽  
Missense Mutations ◽  
Decay Kinetics ◽  
Neuronal Synapses ◽  
Α3 Subunit

Missense mutations T166M, Q242L, T336M, and Y474C in the GABAA receptor (GABAAR) α3 subunit gene are associated with epileptic seizures, dysmorphic features, intellectual disability, and developmental delay. When incorporated into GABAARs expressed in oocytes, all mutations are known to reduce GABA-evoked whole-cell currents. However, their impact on the properties of inhibitory synaptic currents (IPSCs) is unknown, largely because it is difficult to establish, much less control, the stoichiometry of GABAAR expressed in native neuronal synapses. To circumvent this problem, we employed a HEK293 cell-neuron co-culture expression system that permits the recording of IPSCs mediated by a pure population of GABAARs with a defined stoichiometry. We first demonstrated that IPSCs mediated by α3-containing GABAARs (α3β3γ2) decay significantly slower than those mediated by α1-containing isoforms (α1β2γ2 or α1β3γ2). GABAAR α3 mutations did not affect IPSC peak amplitudes or 10–90% rise times, but three of the mutations affected IPSC decay. T336M significantly accelerated the IPSC decay rate whereas T166M and Y474C had the opposite effect. The acceleration of IPSC decay kinetics caused by the T366M mutation was returned to wild-type-like values by the anti-epileptic medication, midazolam. Quantification experiments in HEK293 cells revealed a significant reduction in cell-surface expression for all mutants, in agreement with previous oocyte data. Taken together, our results show that impaired surface expression and altered IPSC decay rates could both be significant factors underlying the pathologies associated with these mutations.

Abstract 1070: An Unconventional Vesicular Transport Pathway Regulates the Cell Surface Expression of hERG K + Channels

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Heather A Underkofler ◽  
Sadguna Balijepalli ◽  
Brooke M Moungey ◽  
Jessica K Slind ◽  
Jabe M Best ◽  
...  
Keyword(s):  
Cell Surface ◽  
Surface Membrane ◽  
Vesicular Transport ◽  
Surface Expression ◽  
Small Gtpases ◽  
Dominant Negative ◽  
Cell Surface Expression ◽  
Missense Mutations ◽  
Transport Pathway ◽  
Er Export

Approximately 35– 45% of patients that are genotype positive for congenital Long QT Syndrome (LQT) have mutations in the human Ether-a-go-go Related Gene ( hERG ). The purpose of this study was to elucidate the mechanisms that regulate ER export and cell surface expression of hERG channel protein, because these steps are impaired for ~90% of LQT-linked hERG missense mutations. The small GTPases Sar1 and Arf1 regulate the conventional vesicular transport (trafficking) for the ER export of proteins to the Golgi apparatus (Golgi). We generated dominant negative (DN) mutations for Sar1 and Arf1, and co-expressed these DN GTPases with hERG in HEK 293 cells. The trafficking of hERG through the Golgi can be visualized biochemically using Western blot analysis, because additional glycosylation of hERG in the Golgi (Golgi processing) increases the MW of hERG protein from 135kDa to 155kDa. Co-expression of hERG and DN-Sar1 inhibited Golgi processing, decreased hERG current (I hERG ) by 85% compared to control (n≥8 cells per group, p<0.05), and decreased the staining of hERG protein at the cell surface, while co-expression of hERG and DN-Arf1 showed no significant effect on Golgi processing or I hERG . This lack of an effect by DN-Arf1 was selective for hERG as it efficiently blocked the transport of previously reported proteins. Rab11 GTPases regulate the trafficking of proteins from endosomal compartments to the cell surface membrane and/or the Golgi. Rab11a is ubiquitously expressed, whereas Rab11b is expressed primarily in brain and heart. Co-expression of DN-Rab11a did not alter Golgi processing of hERG but reduced I hERG by 51% compared to control (n≥8 cells per group, p<0.05), whereas co-expression of DN-Rab11b inhibited Golgi processing of hERG and reduced I hERG by 79% compared to control (n=8 cells per group, p<0.05). Thus, Rab11a appears to regulate the trafficking of hERG to the cell surface after processing in the Golgi, whereas Rab11b regulates the trafficking of hERG prior to processing in the Golgi. These data suggest that hERG does not traffic via the conventional pathway from the ER to the Golgi, but rather in an unconventional pathway from the ER to endosomal compartments prior to Rab11b-mediated transport to the Golgi and subsequent delivery to the cell membrane.

Different molecular behavior of CD40 mutants causing hyper-IgM syndrome

Blood ◽  
2010 ◽  
Vol 116 (26) ◽  
pp. 5867-5874 ◽  
Author(s):  
Gaetana Lanzi ◽  
Simona Ferrari ◽  
Mauno Vihinen ◽  
Stefano Caraffi ◽  
Necil Kutukculer ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Misfolding ◽  
Cd40 Ligand ◽  
Surface Expression ◽  
Degradation Pathway ◽  
Immunoglobulin M ◽  
Cell Surface Expression ◽  
Missense Mutations ◽  
Downstream Signaling ◽  
Unfolded Protein

Abstract CD40/CD40 ligand (CD40L) cross-talk plays a key role in B-cell terminal maturation in the germinal centers. Genetic defects affecting CD40 cause a rare form of hyper-immunoglobulin M (IgM) syndrome, a disorder characterized by low or absent serum IgG and IgA, associated with recurrent infections. We previously reported on a few patients with homozygous CD40 mutations resulting in lack or severe reduction of CD40 cell surface expression. Here we characterize the 3 CD40 mutants due to missense mutations or small in-frame deletions, and show that the mutated proteins are synthesized but retained in the endoplasmic reticulum (ER), likely due to protein misfolding. Interestingly, the intracellular behavior and fate differ significantly among the mutants: progressive accumulation of the P2 mutant causes endoplasmic reticulum stress and the activation of an unfolded protein response; the mutant P4 is rather efficiently disposed by the ER-associated degradation pathway, while the P5 mutant partially negotiates transport to the plasma membrane, and is competent for CD40L binding. Interestingly, this latter mutant activates downstream signaling elements when overexpressed in transfected cells. These results give new important insights into the molecular pathogenesis of HIGM disease, and suggest that CD40 deficiency can also be regarded as an ER-storage disease.

scholarly journals Serine 129 phosphorylation of membrane-associated α-synuclein modulates dopamine transporter function in a G protein–coupled receptor kinase–dependent manner

2013 ◽  
Vol 24 (11) ◽  
pp. 1649-1660 ◽  
Author(s):  
Susumu Hara ◽  
Shigeki Arawaka ◽  
Hiroyasu Sato ◽  
Youhei Machiya ◽  
Can Cui ◽  
...  
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Dopamine Transporter ◽  
Surface Expression ◽  
Hek293 Cells ◽  
Cell Surface Expression ◽  
Receptor Kinase ◽  
Wild Type ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

Most α-synuclein (α-syn) deposited in Lewy bodies, the pathological hallmark of Parkinson disease (PD), is phosphorylated at Ser-129. However, the physiological and pathological roles of this modification are unclear. Here we investigate the effects of Ser-129 phosphorylation on dopamine (DA) uptake in dopaminergic SH-SY5Y cells expressing α-syn. Subcellular fractionation of small interfering RNA (siRNA)–treated cells shows that G protein–coupled receptor kinase 3 (GRK3), GRK5, GRK6, and casein kinase 2 (CK2) contribute to Ser-129 phosphorylation of membrane-associated α-syn, whereas cytosolic α-syn is phosphorylated exclusively by CK2. Expression of wild-type α-syn increases DA uptake, and this effect is diminished by introducing the S129A mutation into α-syn. However, wild-type and S129A α-syn equally increase the cell surface expression of dopamine transporter (DAT) in SH-SY5Y cells and nonneuronal HEK293 cells. In addition, siRNA-mediated knockdown of GRK5 or GRK6 significantly attenuates DA uptake without altering DAT cell surface expression, whereas knockdown of CK2 has no effect on uptake. Taken together, our results demonstrate that membrane-associated α-syn enhances DA uptake capacity of DAT by GRKs-mediated Ser-129 phosphorylation, suggesting that α-syn modulates intracellular DA levels with no functional redundancy in Ser-129 phosphorylation between GRKs and CK2.

scholarly journals BRET sensors unravel that Plasmodium falciparum serpentine receptor 12 (PfSR12) increases surface expression of mammalian GPCRs in HEK293 cells

2020 ◽  
Author(s):  
Pedro H. S. Pereira ◽  
Gabriela Brito ◽  
Miriam S. Moraes ◽  
Camila L. Kiyan ◽  
Charlotte Avet ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Mammalian Cells ◽  
Circulatory System ◽  
Resonance Energy ◽  
Public Health Problem ◽  
Surface Expression ◽  
Hek293 Cells ◽  
Cell Surface Expression ◽  
Thrombin Receptor

ABSTRACTMalaria causes millions of deaths worldwide and is considered a huge public health problem for underdeveloped countries. The most severe cases of malaria present complications of the host circulatory system, which may cause clogging and rupture of blood vessels, leading to death or important sequelae. Because of the previously suggested role of thrombin and platelet aggregation in Plasmodium falciparum biology, we hypothesized that one of the GPCR-like proteins identified in the genome of the parasite, P. falciparum serpentine receptor 12 (PfSR12), could be a thrombin-activated GPCR. To test this hypothesis we used a series of Bioluminescence and Bioluminescence Resonance Energy Transfer (BRET)-based biosensors to investigate the signaling activity of PfSR12. Using an Obelin based biosensor, thrombin promoted a PfSR12-dependent cytosolic Ca2+ rise in HEK293 cells. This Ca2+ mobilization was accompanied by DAG formation and PKC activation as detected using DAG and PKC BRET-based biosensors indicating a Gq/PLC/IP3 signaling pathway. The role of Gq was confirm using Gq/11 knockout HEK293 cells as well as the Gq-selective inhibitor, YM254890. Further investigation revealed that PfSR12 is not itself a thrombin receptor but rather promotes the increase of cell surface expression of an endogenous thrombin receptor. This chaperone-like effect was not selective for thrombin receptors as PfSR12 expression also promoted an increased muscarinic type 3 receptor (M3R)-promoted DAG and PKC responses. This increase response was accompanied by an increase in surface expression of M3R. Our data indicate that PfSR12 acts as a chaperone and increases the expression of several GPCRs resulting in increased responsiveness to various hormones of mammalian cells that could contribute to the deleterious effects of Plasmodium falciparum infection.

scholarly journals N-glycosylation in the protease domain of trypsin-like serine proteases mediates calnexin-assisted protein folding

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Hao Wang ◽  
Shuo Li ◽  
Juejin Wang ◽  
Shenghan Chen ◽  
Xue-Long Sun ◽  
...  
Keyword(s):  
Cell Surface ◽  
Serine Proteases ◽  
Surface Expression ◽  
Hek293 Cells ◽  
Cell Surface Expression ◽  
General Mechanism ◽  
Common Mechanism ◽  
Physiological Processes ◽  
Glycosylation Sites ◽  
Underlying Mechanisms

Trypsin-like serine proteases are essential in physiological processes. Studies have shown that N-glycans are important for serine protease expression and secretion, but the underlying mechanisms are poorly understood. Here, we report a common mechanism of N-glycosylation in the protease domains of corin, enteropeptidase and prothrombin in calnexin-mediated glycoprotein folding and extracellular expression. This mechanism, which is independent of calreticulin and operates in a domain-autonomous manner, involves two steps: direct calnexin binding to target proteins and subsequent calnexin binding to monoglucosylated N-glycans. Elimination of N-glycosylation sites in the protease domains of corin, enteropeptidase and prothrombin inhibits corin and enteropeptidase cell surface expression and prothrombin secretion in transfected HEK293 cells. Similarly, knocking down calnexin expression in cultured cardiomyocytes and hepatocytes reduced corin cell surface expression and prothrombin secretion, respectively. Our results suggest that this may be a general mechanism in the trypsin-like serine proteases with N-glycosylation sites in their protease domains.

scholarly journals Functional consequence of a novel Y129C mutation in a patient with two contradictory melanocortin-2-receptor mutations

2009 ◽  
Vol 160 (4) ◽  
pp. 705-710 ◽  
Author(s):  
Li F Chan ◽  
Teng-Teng Chung ◽  
Ahmed F Massoud ◽  
Louise A Metherell ◽  
Adrian J L Clark
Keyword(s):  
Cell Surface ◽  
Novel Mutation ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Missense Mutations ◽  
Intracellular Loop ◽  
Spastic Quadriplegia ◽  
Activating Mutation ◽  
Glucocorticoid Deficiency

ContextFamilial glucocorticoid deficiency (FGD) is a rare autosomal recessive disease, characterised by isolated glucocorticoid deficiency in the absence of mineralocorticoid deficiency. Inactivating mutations in the ACTH receptor (melanocortin-2-receptor, MC2R) are well described and account for ∼25% of cases. By contrast, activating MC2R mutations are extremely rare.PatientWe report a child of Saudi Arabian origin who was diagnosed with FGD following hypoglycaemic episodes that resulted in spastic quadriplegia.Methods and resultsMC2R gene analysis revealed an unusual combination of two homozygous missense mutations, consisting of the novel mutation Y129C and the previously described F278C activating mutation. Parents were heterozygous at both of these sites. In vitro analysis of the Y129C mutation using a fluorescent cell surface assay showed that this mutant was unable to reach the cell surface in CHO cells stably transfected with MC2R accessory protein (MRAP), despite the demonstration of an interaction with MRAP by co-immunoprecipitation. The double mutant Y129C-F278C also failed to traffic to the cell surface.ConclusionThe tyrosine residue at position 129 in the second intracellular loop is critical in MC2R folding and/or trafficking to the cell surface. Furthermore, the absence of cell surface expression of MC2R would account for the lack of activation of the receptor due to the F278C mutation located at the C-terminal tail. We provide a novel molecular explanation for a child with two opposing mutations causing severe FGD.

scholarly journals An evolutionarily conserved N-terminal Sgk1 variant with enhanced stability and improved function

2008 ◽  
Vol 295 (5) ◽  
pp. F1440-F1448 ◽  
Author(s):  
Nandita S. Raikwar ◽  
Peter M. Snyder ◽  
Christie P. Thomas
Keyword(s):  
Collecting Duct ◽  
Expression System ◽  
Bioinformatic Analysis ◽  
Surface Expression ◽  
Hek293 Cells ◽  
Alternate Transcript ◽  
Evolutionarily Conserved ◽  
Improved Stability ◽  
Improved Function ◽  
Enhanced Surface

Sgk1 is an aldosterone-induced kinase that regulates epithelial sodium channel (ENaC)-mediated Na+ transport in the collecting duct and connecting tubule of the kidney. The NH2 terminus of Sgk1 contains instability motifs that direct the ubiquitination of Sgk1 resulting in a rapidly degraded protein. By bioinformatic analysis, we identified a 5′ variant alternate transcript of human Sgk1 (Sgk1_v2) that is widely expressed, is conserved from rodent to humans, and is predicted to encode an Sgk1 isoform, Sgk1_i2, with a different NH2 terminus. When expressed in HEK293 cells, Sgk1_i2 was more abundant than Sgk1 because of an increased protein half-life and this correlated with reduced ubiquitination of Sgk1_i2 and enhanced surface expression of ENaC. Immunocytochemical studies demonstrated that in contrast to Sgk1, Sgk1_i2 is preferentially targeted to the plasma membrane. When coexpressed with ENaC subunits in FRT epithelia, Sgk1_i2 had a significantly greater effect on amiloride-sensitive Na+ transport compared with Sgk1. Together, the data demonstrate that a conserved NH2-terminal variant of Sgk1 shows improved stability, enhanced membrane association, and greater stimulation of epithelial Na+ transport in a heterologous expression system.

scholarly journals Inter-Regulation of Kv4.3 and Voltage-Gated Sodium Channels Underlies Predisposition to Cardiac and Neuronal Channelopathies

2020 ◽  
Vol 21 (14) ◽  
pp. 5057
Author(s):  
Jérôme Clatot ◽  
Nathalie Neyroud ◽  
Robert Cox ◽  
Charlotte Souil ◽  
Jing Huang ◽  
...  
Keyword(s):  
Sodium Channels ◽  
Outward Current ◽  
Surface Expression ◽  
Hek293 Cells ◽  
Cell Surface Expression ◽  
Transient Outward Current ◽  
Loss Of Function ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels ◽  
Intracellular Compartments

Background: Genetic variants in voltage-gated sodium channels (Nav) encoded by SCNXA genes, responsible for INa, and Kv4.3 channels encoded by KCND3, responsible for the transient outward current (Ito), contribute to the manifestation of both Brugada syndrome (BrS) and spinocerebellar ataxia (SCA19/22). We examined the hypothesis that Kv4.3 and Nav variants regulate each other’s function, thus modulating INa/Ito balance in cardiomyocytes and INa/I(A) balance in neurons. Methods: Bicistronic and other constructs were used to express WT or variant Nav1.5 and Kv4.3 channels in HEK293 cells. INa and Ito were recorded. Results: SCN5A variants associated with BrS reduced INa, but increased Ito. Moreover, BrS and SCA19/22 KCND3 variants associated with a gain of function of Ito, significantly reduced INa, whereas the SCA19/22 KCND3 variants associated with a loss of function (LOF) of Ito significantly increased INa. Auxiliary subunits Navβ1, MiRP3 and KChIP2 also modulated INa/Ito balance. Co-immunoprecipitation and Duolink studies suggested that the two channels interact within the intracellular compartments and biotinylation showed that LOF SCN5A variants can increase Kv4.3 cell-surface expression. Conclusion: Nav and Kv4.3 channels modulate each other’s function via trafficking and gating mechanisms, which have important implications for improved understanding of these allelic cardiac and neuronal syndromes.

scholarly journals GLUT-4 NH2 terminus contains a phenylalanine-based targeting motif that regulates intracellular sequestration.

1993 ◽  
Vol 121 (6) ◽  
pp. 1221-1232 ◽  
Author(s):  
R C Piper ◽  
C Tai ◽  
P Kulesza ◽  
S Pang ◽  
D Warnock ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
Cho Cells ◽  
Sindbis Virus ◽  
Glucose Transporter ◽  
Expression System ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Glut 4 ◽  
Intracellular Sequestration

Expression of chimeras, composed of portions of two different glucose transporter isoforms (GLUT-1 and GLUT-4), in CHO cells had indicated that the cytoplasmic NH2 terminus of GLUT-4 contains important targeting information that mediates intracellular sequestration of this isoform (Piper, R. C., C. Tai, J. W. Slot, C. S. Hahn, C. M. Rice, H. Huang, D. E. James. 1992. J. Cell Biol. 117:729-743). In the present studies, the amino acid constituents of the GLUT-4 NH2-terminal targeting domain have been identified. GLUT-4 constructs containing NH2-terminal deletions or alanine substitutions within the NH2 terminus were expressed in CHO cells using a Sindbis virus expression system. Deletion of eight amino acids from the GLUT-4 NH2 terminus or substituting alanine for phenylalanine at position 5 in GLUT-4 resulted in a marked accumulation of the transporter at the plasma membrane. Mutations at other amino acids surrounding Phe5 also caused increased cell surface expression of GLUT-4 but not to the same extent as the Phe5 mutation. GLUT-4 was also localized to clathrin lattices and this colocalization was abolished when either the first 13 amino acids were deleted or when Phe5 was changed to alanine. To ascertain whether the targeting information within the GLUT-4 NH2-terminal targeting domain could function independently of the glucose transporter structure this domain was inserted into the cytoplasmic tail of the H1 subunit of the asialoglycoprotein receptor. H1 with the GLUT-4 NH2 terminus was predominantly localized to an intracellular compartment similar to GLUT-4 and was sequestered more from the cell surface than was the wild-type H1 protein. It is concluded that the NH2 terminus of GLUT-4 contains a phenylalanine-based targeting motif that mediates intracellular sequestration at least in part by facilitating interaction of the transporter with endocytic machinery located at the cell surface.

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