BARP suppresses voltage-gated calcium channel activity and Ca2+-evoked exocytosis

Voltage-gated calcium channels (VGCCs) are key regulators of cell signaling and Ca2+-dependent release of neurotransmitters and hormones. Understanding the mechanisms that inactivate VGCCs to prevent intracellular Ca2+ overload and govern their specific subcellular localization is of critical importance. We report the identification and functional characterization of VGCC β-anchoring and -regulatory protein (BARP), a previously uncharacterized integral membrane glycoprotein expressed in neuroendocrine cells and neurons. BARP interacts via two cytosolic domains (I and II) with all Cavβ subunit isoforms, affecting their subcellular localization and suppressing VGCC activity. Domain I interacts at the α1 interaction domain–binding pocket in Cavβ and interferes with the association between Cavβ and Cavα1. In the absence of domain I binding, BARP can form a ternary complex with Cavα1 and Cavβ via domain II. BARP does not affect cell surface expression of Cavα1 but inhibits Ca2+ channel activity at the plasma membrane, resulting in the inhibition of Ca2+-evoked exocytosis. Thus, BARP can modulate the localization of Cavβ and its association with the Cavα1 subunit to negatively regulate VGCC activity.

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Congenital Lipoid Adrenal Hyperplasia: Functional Characterization of Three Novel Mutations in the STAR Gene

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2009-1176 ◽

2010 ◽

Vol 95 (3) ◽

pp. 1301-1308 ◽

Cited By ~ 20

Author(s):

Susanne Bens ◽

Angelika Mohn ◽

Bilgin Yüksel ◽

Alexandra E. Kulle ◽

Matthias Michalek ◽

...

Keyword(s):

Functional Characterization ◽

Regulatory Protein ◽

Three Dimensional ◽

Binding Pocket ◽

Adrenal Hyperplasia ◽

Turkish Patient ◽

Star Protein ◽

Sex Development ◽

Congenital Lipoid Adrenal Hyperplasia

Abstract Context: The steroidogenic acute regulatory protein (StAR) has been shown to be essential for steroidogenesis by mediating cholesterol transfer into mitochondria. Inactivating StAR mutations cause the typical clinical picture of congenital lipoid adrenal hyperplasia. Objective: The objective of the investigation was to study the functional and structural consequences of three novel StAR mutations (p.N148K in an Italian patient; p.P129fs and p.Q128R in a Turkish patient). Methods and Results: Transient in vitro expression of the mutant proteins together with P450 side-chain cleavage enzyme, adrenodoxin, and adrenodoxin reductase yielded severely diminished cholesterol conversion of the p.N148K mutant, the combined p.P129fs and p.Q128R mutant, and the p.P129fs mutant by itself. The p.Q128R mutant led to a higher cholesterol conversion than the wild-type StAR protein. As derived from three-dimensional protein modeling, the residue N148 is lining the ligand cavity of StAR. A positively charged lysine residue at position 148 disturbs the hydrophobic cluster formed by the α4-helix and the sterol binding pocket. The frame shift mutation p.P129fs truncates the StAR protein. Residue p.Q128 is situated at the surface of the molecule and is not part of any functionally characterized region of the protein. Conclusion: The mutations p.N148K and p.P129fs cause adrenal insufficiency in both cases and lead to a disorder of sex development with complete sex reversal in the 46, XY case. The mutation p.Q128R, which is not relevant for the patient’s phenotype, is the first reported variant showing a gain of function. We speculate that the substitution of hydrophilic glutamine with basic arginine at the surface of the molecule may accelerate cholesterol transfer.

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Functional Characterization Of The VAC14 Self-Interaction Domain

10.32920/ryerson.14662869.v1 ◽

2021 ◽

Author(s):

Tamadher A. Alghamdi

Keyword(s):

Functional Characterization ◽

Regulatory Protein ◽

Adaptor Protein ◽

Interaction Domain ◽

Lipid Kinase ◽

Lipid Phosphatase ◽

C Terminus ◽

Regulatory Complex ◽

Steady State Levels ◽

And Function

PtdIns(3,5)P2 is a low-abundance signaling lipid present at < 0.1 % of total PtdIns lipids in yeasts and mammals. Reduced levels of PtdIns(3,5)P2 contributes to neurodegenerative disorders in humans and vacuolar defects in yeasts. Steady-state levels of PtdIns(3,5)P2 are dependent on both its rate of synthesis and turnover. In yeast, PtdIns(3,5)P2 is produced on the vacuole membrane by phosphorylation of PtdIns(3)P at the 5 position of its inositol ring by the Fab1 lipid kinase. Cells lacking Fab1 make no PtdIns(3,5)P2 and exhibit defects in vacuole morphology and function. The lipid phosphatase Fig4 counteracts Fab1 activity by turnover of PtdIns(3,5)P2 into PtdIns(3)P. Vac14 is a regulatory protein implicated in the synthesis and turnover of PtdIns(3,5)P2. It acts as an adaptor protein that controls both of Fab1 and Fig4 proteins. In addition, Vac14 exists as a multimer that allows for self-interaction. However, multimerization state of Vac14 as well as the domain responsible for self-interaction remained unknown. This study aimed to identify the self-interaction domain to elucidate its role in the assembly of the regulatory complex of PtdIns(3,5)P2. The observations seen in this study suggested that Vac14 self-interacts via multiple conserved motifs in the C-terminus, which are crucial for interaction with Fab1 and Fig4, and the normal morphology of yeast vacuoles.

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Functional Characterization Of The VAC14 Self-Interaction Domain

10.32920/ryerson.14662869 ◽

2021 ◽

Author(s):

Tamadher A. Alghamdi

Keyword(s):

Functional Characterization ◽

Regulatory Protein ◽

Adaptor Protein ◽

Interaction Domain ◽

Lipid Kinase ◽

Lipid Phosphatase ◽

C Terminus ◽

Regulatory Complex ◽

Steady State Levels ◽

And Function

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Translocatable voltage-gated Ca2+ channel β subunits in α1–β complexes reveal competitive replacement yet no spontaneous dissociation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1809762115 ◽

2018 ◽

Vol 115 (42) ◽

pp. E9934-E9943 ◽

Cited By ~ 1

Author(s):

Jun-Hee Yeon ◽

Cheon-Gyu Park ◽

Bertil Hille ◽

Byung-Chang Suh

Keyword(s):

Plasma Membrane ◽

Channel Gating ◽

Surface Expression ◽

Cell Surface Expression ◽

Intracellular Loop ◽

Drug Induced ◽

Interaction Domain ◽

Intact Cells ◽

Voltage Gated ◽

The Stability

β subunits of high voltage-gated Ca2+ (CaV) channels promote cell-surface expression of pore-forming α1 subunits and regulate channel gating through binding to the α-interaction domain (AID) in the first intracellular loop. We addressed the stability of CaV α1B–β interactions by rapamycin-translocatable CaV β subunits that allow drug-induced sequestration and uncoupling of the β subunit from CaV2.2 channel complexes in intact cells. Without CaV α1B/α2δ1, all modified β subunits, except membrane-tethered β2a and β2e, are in the cytosol and rapidly translocate upon rapamycin addition to anchors on target organelles: plasma membrane, mitochondria, or endoplasmic reticulum. In cells coexpressing CaV α1B/α2δ1 subunits, the translocatable β subunits colocalize at the plasma membrane with α1B and stay there after rapamycin application, indicating that interactions between α1B and bound β subunits are very stable. However, the interaction becomes dynamic when other competing β isoforms are coexpressed. Addition of rapamycin, then, switches channel gating and regulation by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] lipid. Thus, expression of free β isoforms around the channel reveals a dynamic aspect to the α1B–β interaction. On the other hand, translocatable β subunits with AID-binding site mutations are easily dissociated from CaV α1B on the addition of rapamycin, decreasing current amplitude and PI(4,5)P2 sensitivity. Furthermore, the mutations slow CaV2.2 current inactivation and shift the voltage dependence of activation to more positive potentials. Mutated translocatable β subunits work similarly in CaV2.3 channels. In sum, the strong interaction of CaV α1B–β subunits can be overcome by other free β isoforms, permitting dynamic changes in channel properties in intact cells.

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Faculty Opinions recommendation of BARP suppresses voltage-gated calcium channel activity and Ca2+-evoked exocytosis.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718358255.793494421 ◽

2014 ◽

Author(s):

Geneviève Dupont

Keyword(s):

Calcium Channel ◽

Channel Activity ◽

Voltage Gated ◽

Voltage Gated Calcium Channel

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N-Terminal Acetyltransferase Naa40p Whereabouts Put into N-Terminal Proteoform Perspective

International Journal of Molecular Sciences ◽

10.3390/ijms22073690 ◽

2021 ◽

Vol 22 (7) ◽

pp. 3690

Author(s):

Veronique Jonckheere ◽

Petra Van Damme

Keyword(s):

Amino Acid ◽

Subcellular Localization ◽

Nuclear Import ◽

Ribosome Biogenesis ◽

Regulatory Protein ◽

Ectopic Expression ◽

Cytoplasmic Localization ◽

Histone H2a ◽

Histone H4 ◽

Nucleolar Proteins

The evolutionary conserved N-alpha acetyltransferase Naa40p is among the most selective N-terminal acetyltransferases (NATs) identified to date. Here we identified a conserved N-terminally truncated Naa40p proteoform named Naa40p25 or short Naa40p (Naa40S). Intriguingly, although upon ectopic expression in yeast, both Naa40p proteoforms were capable of restoring N-terminal acetylation of the characterized yeast histone H2A Naa40p substrate, the Naa40p histone H4 substrate remained N-terminally free in human haploid cells specifically deleted for canonical Naa40p27 or 237 amino acid long Naa40p (Naa40L), but expressing Naa40S. Interestingly, human Naa40L and Naa40S displayed differential expression and subcellular localization patterns by exhibiting a principal nuclear and cytoplasmic localization, respectively. Furthermore, Naa40L was shown to be N-terminally myristoylated and to interact with N-myristoyltransferase 1 (NMT1), implicating NMT1 in steering Naa40L nuclear import. Differential interactomics data obtained by biotin-dependent proximity labeling (BioID) further hints to context-dependent roles of Naa40p proteoforms. More specifically, with Naa40S representing the main co-translationally acting actor, the interactome of Naa40L was enriched for nucleolar proteins implicated in ribosome biogenesis and the assembly of ribonucleoprotein particles, overall indicating a proteoform-specific segregation of previously reported Naa40p activities. Finally, the yeast histone variant H2A.Z and the transcriptionally regulatory protein Lge1 were identified as novel Naa40p substrates, expanding the restricted substrate repertoire of Naa40p with two additional members and further confirming Lge1 as being the first redundant yNatA and yNatD substrate identified to date.

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Suramin Targets the Conserved Ligand-Binding Pocket of Human Raf1 Kinase Inhibitory Protein

Molecules ◽

10.3390/molecules26041151 ◽

2021 ◽

Vol 26 (4) ◽

pp. 1151

Author(s):

Chenyun Guo ◽

Zhihua Wu ◽

Weiliang Lin ◽

Hao Xu ◽

Ting Chang ◽

...

Keyword(s):

Side Effects ◽

Ligand Binding ◽

Mapk Pathway ◽

Regulatory Protein ◽

Therapeutic Index ◽

Binding Pocket ◽

Biological Macromolecules ◽

Inhibitory Protein ◽

Ligand Binding Pocket ◽

Raf1 Kinase

Suramin was initially used to treat African sleeping sickness and has been clinically tested to treat human cancers and HIV infection in the recent years. However, the therapeutic index is low with numerous clinical side-effects, attributed to its diverse interactions with multiple biological macromolecules. Here, we report a novel binding target of suramin, human Raf1 kinase inhibitory protein (hRKIP), which is an important regulatory protein involved in the Ras/Raf1/MEK/ERK (MAPK) signal pathway. Biolayer interference technology showed that suramin had an intermediate affinity for binding hRKIP with a dissociation constant of 23.8 µM. Both nuclear magnetic resonance technology and molecular docking analysis revealed that suramin bound to the conserved ligand-binding pocket of hRKIP, and that residues K113, W173, and Y181 play crucial roles in hRKIP binding suramin. Furthermore, suramin treatment at 160 µM could profoundly increase the ERK phosphorylation level by around 3 times. Our results indicate that suramin binds to hRKIP and prevents hRKIP from binding with hRaf1, thus promoting the MAPK pathway. This work is beneficial to both mechanistically understanding the side-effects of suramin and efficiently improving the clinical applications of suramin.

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TOK Homologue in Neurospora crassa: First Cloning and Functional Characterization of an Ion Channel in a Filamentous Fungus

Eukaryotic Cell ◽

10.1128/ec.2.1.181-190.2003 ◽

2003 ◽

Vol 2 (1) ◽

pp. 181-190 ◽

Cited By ~ 14

Author(s):

Stephen K. Roberts

Keyword(s):

Ion Channels ◽

Ion Channel ◽

Neurospora Crassa ◽

Filamentous Fungus ◽

Single Channel ◽

Functional Characterization ◽

Reversal Potential ◽

Channel Activity ◽

Biophysical Properties ◽

Patch Clamp Technique

ABSTRACT In contrast to animal and plant cells, very little is known of ion channel function in fungal physiology. The life cycle of most fungi depends on the “filamentous” polarized growth of hyphal cells; however, no ion channels have been cloned from filamentous fungi and comparatively few preliminary recordings of ion channel activity have been made. In an attempt to gain an insight into the role of ion channels in fungal hyphal physiology, a homolog of the yeast K+ channel (ScTOK1) was cloned from the filamentous fungus, Neurospora crassa. The patch clamp technique was used to investigate the biophysical properties of the N. crassa K+ channel (NcTOKA) after heterologous expression of NcTOKA in yeast. NcTOKA mediated mainly time-dependent outward whole-cell currents, and the reversal potential of these currents indicated that it conducted K+ efflux. NcTOKA channel gating was sensitive to extracellular K+ such that channel activation was dependent on the reversal potential for K+. However, expression of NcTOKA was able to overcome the K+ auxotrophy of a yeast mutant missing the K+ uptake transporters TRK1 and TRK2, suggesting that NcTOKA also mediated K+ influx. Consistent with this, close inspection of NcTOKA-mediated currents revealed small inward K+ currents at potentials negative of EK. NcTOKA single-channel activity was characterized by rapid flickering between the open and closed states with a unitary conductance of 16 pS. NcTOKA was effectively blocked by extracellular Ca2+, verapamil, quinine, and TEA+ but was insensitive to Cs+, 4-aminopyridine, and glibenclamide. The physiological significance of NcTOKA is discussed in the context of its biophysical properties.

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Functional Characterization of the obesity-linked variant of the β3-adrenergic receptor

10.20944/preprints202105.0087.v1 ◽

2021 ◽

Author(s):

Esraa Haji ◽

Saeed Al Mahri ◽

Yumna Aloraij ◽

Shuja Shafi Malik ◽

Sameer Mohammad

Keyword(s):

Cell Surface ◽

Adrenergic Receptor ◽

Functional Characterization ◽

Biochemical Properties ◽

Cell Surface Expression ◽

Cellular Distribution ◽

Hek 293 ◽

Regulatory Pathways ◽

Beta 2 ◽

Activation Behavior

Adrenergic receptor β3 (ADRβ3) is a member of the rhodopsin-like G protein-coupled receptor family. The binding of the ligand to ADRβ3 activates adenylate cyclase and increases cAMP in the cells. ADRβ3 is highly expressed in white and brown adipocytes and controls key regulatory pathways of lipid metabolism. Trp64Arg (W64R) polymorphism in the ADRβ3 has been associated with the early development of type 2 diabetes mellitus, lower resting metabolic rate, abdominal obesity, and insulin resistance. It is unclear how the substitution of W64R affects the functioning of ADRβ3. This study was initiated to functionally characterize this obesity-linked variant of ADRβ3. We evaluated in detail the expression, subcellular distribution, and post-activation behavior of the WT and W64R ADRβ3 using a single cell quantitative fluorescence microscopy. When expressed in HEK 293 cells, ADRβ3 shows a typical distribution displayed by other GPCRs with a predominant localization at the cell surface. Unlike Adrenergic receptor β2 (ADRβ2), agonist induced desensitization of ADRβ3 does not involve loss of cell surface expression. WT and W64R variant of ADRβ3 displayed comparable biochemical properties and there was no significant impact of the substitution of Tryptophan with Arginine on the expression, cellular distribution, signaling, and post-activation behavior of ADRβ3. The obesity-linked W64R variant of ADRβ3 is indistinguishable from the WT ADRβ3 in terms of expression, cellular distribution, signaling, and post-activation behavior.

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