Functional consequences of progressive cone dystrophy-associated mutations in the human cone photoreceptor cyclic nucleotide-gated channel CNGA3 subunit

2005 ◽  
Vol 289 (1) ◽  
pp. C187-C198 ◽  
Author(s):  
Chunming Liu ◽  
Michael D. Varnum
Keyword(s):  
Patch Clamp ◽  
Cyclic Nucleotide ◽  
Fluorescent Protein ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Cone Dystrophy ◽  
Cone Photoreceptor ◽  
Patch Clamp Recording ◽  
Functional Consequences ◽  
Heteromeric Channels

Progressive cone dystrophies are a genetically heterogeneous group of disorders characterized by early deterioration of visual acuity and color vision, together with psychophysical and electrophysiological evidence of abnormal cone function and cone degeneration. Recently, three mutations in the gene encoding the CNGA3 subunit of cone photoreceptor cyclic nucleotide-gated (CNG) channels have been linked to progressive cone dystrophy in humans. To investigate the functional consequences of these mutations, we expressed mutant human CNGA3 subunits in Xenopus oocytes, alone or together with human CNGB3, and studied these channels using patch-clamp recording. Compared with wild-type channels, homomeric and heteromeric channels containing CNGA3-N471S or CNGA3-R563H subunits exhibited an increase in apparent affinity for cGMP and an increase in the relative agonist efficacy of cAMP compared with cGMP. In contrast, R277C subunits did not form functional homomeric or heteromeric channels. Cell surface expression levels, determined using confocal microscopy of green fluorescent protein-tagged subunits and patch-clamp recording, were significantly reduced for both R563H and R277C but unchanged for N471S. Overall, these results suggest that the plasma membrane localization and gating properties of cone CNG channels are altered by progressive cone dystrophy-associated mutations, providing evidence that supports the pathogenicity of these mutations.

Protein kinase C induces endocytosis of the sodium taurocholate cotransporting polypeptide

2010 ◽  
Vol 299 (2) ◽  
pp. G320-G328 ◽  
Author(s):  
Claudia Stross ◽  
Angelika Helmer ◽  
Katrin Weissenberger ◽  
Boris Görg ◽  
Verena Keitel ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Bile Salt ◽  
Fluorescent Protein ◽  
Membrane Surface ◽  
Sodium Taurocholate ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Salt Uptake ◽  
Kinase C

Bile salts influence signaling and metabolic pathways. In hepatocytes, the sodium taurocholate cotransporting polypeptide (Ntcp) is a major determinant of intracellular bile salt levels. Short-term downregulation of Ntcp is not well characterized to date. FLAG and enhanced green fluorescent protein (EGFP) tags were cloned to the extra- and intracellular termini of Ntcp. Endocytosis of Ntcp in transfected HepG2 cells was visualized by fluorescence of EGFP, and membrane surface expression of Ntcp was quantified by flow cytometry with fluorochrome-labeled FLAG antibodies. Activation of protein kinase C (PKC) by phorbolester or thymeleatoxin an activator of Ca2+-dependent conventional PKCs (cPKCs), induced endocytosis of Ntcp, whereas the Na+-K+-ATPase remained in the plasma membrane. The PKC inhibitor BIM I and the cPKC-selective inhibitor Gö6976 abolished PMA-induced endocytosis. Because of this internalization, cell surface expression of Ntcp was reduced by 36 ± 7%, bile salt uptake was decreased by 25%, and taurolithocholate sulfate-induced cell toxicity was prevented. In conclusion, Ca2+-dependent PKCs induce vesicular retrieval of Ntcp, thereby reducing bile salt uptake. This mechanism may protect hepatocytes from toxic intracellular bile salt concentrations.

scholarly journals Receptor activation and 2 distinct COOH-terminal motifs control G-CSF receptor distribution and internalization kinetics

Blood ◽  
2004 ◽  
Vol 103 (2) ◽  
pp. 571-579 ◽  
Author(s):  
Lambertus H. J. Aarts ◽  
Onno Roovers ◽  
Alister C. Ward ◽  
Ivo P. Touw
Keyword(s):  
Fluorescent Protein ◽  
Surface Expression ◽  
Receptor Activation ◽  
Cell Surface Expression ◽  
Serine Threonine Kinase ◽  
Late Endosomes ◽  
Dileucine Motif ◽  
Internalization Kinetics ◽  
Green Fluorescent ◽  
Kinetics Of

Abstract We have studied the intracellular distribution and internalization kinetics of the granulocyte colony-stimulating factor receptor (G-CSF-R) in living cells using fusion constructs of wild-type or mutant G-CSF-R and enhanced green fluorescent protein (EGFP). Under steady-state conditions the G-CSF-R localized predominantly to the Golgi apparatus, late endosomes, and lysosomes, with only low expression on the plasma membrane, resulting from spontaneous internalization. Internalization of the G-CSF-R was significantly accelerated by addition of G-CSF. This ligand-induced switch from slow to rapid internalization required the presence of G-CSF-R residue Trp650, previously shown to be essential for its signaling ability. Both spontaneous and ligand-induced internalization depended on 2 distinct amino acid stretches in the G-CSF-R COOH-terminus: 749-755, containing a dileucine internalization motif, and 756-769. Mutation of Ser749 at position –4 of the dileucine motif to Ala significantly reduced the rate of ligand-induced internalization. In contrast, mutation of Ser749 did not affect spontaneous G-CSF-R internalization, suggesting the involvement of a serine-threonine kinase specifically in ligand-accelerated internalization of the G-CSF-R. COOH-terminal truncation mutants of G-CSF-R, found in severe congenital neutropenia, lack the internalization motifs and were completely defective in both spontaneous and ligand-induced internalization. As a result, these mutants showed constitutively high cell-surface expression.

Identification of the roles of conserved charged residues in the extracellular domain of an epithelial sodium channel (ENaC) subunit by alanine mutagenesis

2011 ◽  
Vol 300 (4) ◽  
pp. F887-F897 ◽  
Author(s):  
Oded Edelheit ◽  
Israel Hanukoglu ◽  
Nathan Dascal ◽  
Aaron Hanukoglu
Keyword(s):  
Cell Surface ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Xenopus Laevis Oocytes ◽  
Epithelial Sodium Channels ◽  
Charged Residues ◽  
Inhibition Control ◽  
Two Parameters

Epithelial sodium channels (ENaC) are composed of three homologous subunits whose extracellular domains (ECD) form a funnel that directs ions from the lumen into the pore of ENaC. To examine the roles of conserved charged residues (Asp, Glu, Arg, and Lys) on ECD, we mutated 16 residues in human α-ENaC to alanine. The modified cRNAs were expressed in Xenopus laevis oocytes together with wild-type β- and γ-ENaC. The effect of each mutation was examined on three parameters: amiloride-sensitive Na+ conductance (assayed by the two-electrode voltage-clamp method), Na+-dependent self-inhibition of ENaC, and oocyte cell surface expression of ENaC (quantitated by confocal microscopy of yellow fluorescent protein linked to γ-ENaC). Mutation of 13 of 16 residues reduced the ENaC Na+ conductance (to 40–80% of WT). Mutation of only six residues showed a significant effect on the Na+ self-inhibition time constant (τ). All 16 mutants showed a strong correlation between ENaC activity and oocyte surface expression ( r = 0.62). Exclusion of four mutants showing the greatest effect on self-inhibition kinetics (Glu250 and Arg350 with τ = ∼30% of WT, and Asp393 and Glu530 with τ = ∼170% of WT) increased the correlation to r = 0.87. In the ASIC1 homotrimeric model, the homologs of α-ENaC Asp400 and Asp446 are exposed on the protein surface far from the other two chains. The mutations of these two residues showed the strongest effect on cell surface expression but had no effect on self-inhibition. Control mutations to a homologous charged residue (e.g., Asp to Glu) did not significantly affect ENaC activity. Changes in the two parameters, Na+ self-inhibition and oocyte surface expression level, accounted for the magnitude of reduction in ENaC activity as a result of the mutation to Ala. These results establish that while some conserved charged residues are part of the structure responsible for Na+ self-inhibition, most are essential for transport to the oocyte cell surface.

scholarly journals Expressional and functional interactions of two Apis cerana cerana olfactory receptors

2018 ◽  
Author(s):  
Lina Guo ◽  
Huiting Zhao ◽  
Yusuo Jiang
Keyword(s):  
Fluorescent Protein ◽  
Apis Cerana ◽  
Olfactory Receptors ◽  
Surface Expression ◽  
Apis Cerana Cerana ◽  
Cell Surface Expression ◽  
Protein Levels ◽  
Green Fluorescent ◽  
Insight Into

Apis cerana cerana relies on the sensitive olfactory system to perform the foraging activities in the surrounding environment. Olfactory receptors (ORs) are a primary requirement for odorant recognition and coding. However, the molecular recognition of volatile with olfactory receptor in Apis cerana cerana is still not clear. Hence, in the present study, we achieved transient transfection and cell surface expression of Apis cerana cerana ORs (AcerOr1 and AcerOr2; AcerOr2 is orthologous to the co-receptor) in Spodoptera frugiperda Sf9 cells. The results showed that both mRNA and protein levels of AcerOr1 and AcerOr2 were drastically reduced when treated with their respective double stranded (ds) RNA compared to those in the control and double-stranded green fluorescent protein (dsGFP)-treated cells. The response to Ca2+ using 33 volatile odorants indicated that the molecular receptive range of AcerOr2 narrowly responded to N-(4-ethylphenyl)-2-((4-ethyl-5-(3-pyridinyl)-4H-1, 2, 4- triazol-3-yl) thio) acetamide (VUAA1) whereas AcerOr1 was sensitive to eugenol, lauric acid, ocimene, 1-nonanol, linolenic acid, hexyl acetate, undecanoic acid, 1-octyl alcohol, and nerol, and it revealed distinct changes in the dose-response curve. We discovered ligands that were useful for probing receptor activity during odor stimulation and validated three of them using an electroantennography (EAG) assay. The response increased with the concentration of the odorant. Further, both AcerOr1 and AcerOr2 knockdowns exhibited significantly reduced intracellular Ca2+ levels in response to the corresponding ligands in vitro. Overall, the present study provides insight into the mechanism of olfactory discrimination in Apis cerana cerana.

scholarly journals Expressional and functional interactions of two Apis cerana cerana olfactory receptors

2018 ◽  
Author(s):  
Lina Guo ◽  
Huiting Zhao ◽  
Yusuo Jiang
Keyword(s):  
Fluorescent Protein ◽  
Apis Cerana ◽  
Olfactory Receptors ◽  
Surface Expression ◽  
Apis Cerana Cerana ◽  
Cell Surface Expression ◽  
Protein Levels ◽  
Green Fluorescent ◽  
Insight Into

Apis cerana cerana relies on the sensitive olfactory system to perform the foraging activities in the surrounding environment. Olfactory receptors (ORs) are a primary requirement for odorant recognition and coding. However, the molecular recognition of volatile with olfactory receptor in Apis cerana cerana is still not clear. Hence, in the present study, we achieved transient transfection and cell surface expression of Apis cerana cerana ORs (AcerOr1 and AcerOr2; AcerOr2 is orthologous to the co-receptor) in Spodoptera frugiperda Sf9 cells. The results showed that both mRNA and protein levels of AcerOr1 and AcerOr2 were drastically reduced when treated with their respective double stranded (ds) RNA compared to those in the control and double-stranded green fluorescent protein (dsGFP)-treated cells. The response to Ca2+ using 33 volatile odorants indicated that the molecular receptive range of AcerOr2 narrowly responded to N-(4-ethylphenyl)-2-((4-ethyl-5-(3-pyridinyl)-4H-1, 2, 4- triazol-3-yl) thio) acetamide (VUAA1) whereas AcerOr1 was sensitive to eugenol, lauric acid, ocimene, 1-nonanol, linolenic acid, hexyl acetate, undecanoic acid, 1-octyl alcohol, and nerol, and it revealed distinct changes in the dose-response curve. We discovered ligands that were useful for probing receptor activity during odor stimulation and validated three of them using an electroantennography (EAG) assay. The response increased with the concentration of the odorant. Further, both AcerOr1 and AcerOr2 knockdowns exhibited significantly reduced intracellular Ca2+ levels in response to the corresponding ligands in vitro. Overall, the present study provides insight into the mechanism of olfactory discrimination in Apis cerana cerana.

scholarly journals Surface μ Heavy Chain Signals Down-Regulation of the V(D)J-Recombinase Machinery in the Absence of Surrogate Light Chain Components

2004 ◽  
Vol 199 (11) ◽  
pp. 1523-1532 ◽  
Author(s):  
Gunther R. Galler ◽  
Cornelia Mundt ◽  
Mathew Parker ◽  
Roberta Pelanda ◽  
Inga-Lill Mårtensson ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Fluorescent Protein ◽  
Cell Receptor ◽  
Surface Expression ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Cell Surface Expression ◽  
Allelic Exclusion ◽  
Down Regulation ◽  
Green Fluorescent

Early B cell development is characterized by stepwise, ordered rearrangement of the immunoglobulin (Ig) heavy (HC) and light (LC) chain genes. Only one of the two alleles of these genes is used to produce a receptor, a phenomenon referred to as allelic exclusion. It has been suggested that pre–B cell receptor (pre-BCR) signals are responsible for down-regulation of the VDJH-recombinase machinery (Rag1, Rag2, and terminal deoxynucleotidyl transferase [TdT]), thereby preventing further rearrangement on the second HC allele. Using a mouse model, we show that expression of an inducible μHC transgene in Rag2−/− pro–B cells induces down-regulation of the following: (a) TdT protein, (b) a transgenic green fluorescent protein reporter reflecting endogenous Rag2 expression, and (c) Rag1 primary transcripts. Similar effects were also observed in the absence of surrogate LC (SLC) components, but not in the absence of the signaling subunit Ig-α. Furthermore, in wild-type mice and in mice lacking either λ5, VpreB1/2, or the entire SLC, the TdT protein is down-regulated in μHC+LC− pre–B cells. Surprisingly, μHC without LC is expressed on the surface of pro–/pre–B cells from λ5−/−, VpreB1−/−VpreB2−/−, and SLC−/− mice. Thus, SLC or LC is not required for μHC cell surface expression and signaling in these cells. Therefore, these findings offer an explanation for the occurrence of HC allelic exclusion in mice lacking SLC components.

scholarly journals Dynamics of Ca2+-Calmodulin–dependent Inhibition of Rod Cyclic Nucleotide-gated Channels Measured by Patch-clamp Fluorometry

2004 ◽  
Vol 124 (3) ◽  
pp. 211-223 ◽  
Author(s):  
Matthew C. Trudeau ◽  
William N. Zagotta
Keyword(s):  
Patch Clamp ◽  
Cyclic Nucleotide ◽  
Time Course ◽  
Physiological Role ◽  
Terminal Region ◽  
Patch Clamp Recording ◽  
Sensory Stimuli ◽  
Channel Inhibition ◽  
Relative Rearrangement ◽  
Dependent Inhibition

Cyclic nucleotide-gated (CNG) ion channels mediate cellular responses to sensory stimuli. In vertebrate photoreceptors, CNG channels respond to the light-induced decrease in cGMP by closing an ion-conducting pore that is permeable to cations, including Ca2+ ions. Rod CNG channels are directly inhibited by Ca2+-calmodulin (Ca2+/CaM), but the physiological role of this modulation is unknown. Native rod CNG channels comprise three CNGA1 subunits and one CNGB1 subunit. The single CNGB1 subunit confers several key properties on heteromeric channels, including Ca2+/CaM-dependent modulation. The molecular basis for Ca2+/CaM inhibition of rod CNG channels has been proposed to involve the binding of Ca2+/CaM to a site in the NH2-terminal region of the CNGB1 subunit, which disrupts an interaction between the NH2-terminal region of CNGB1 and the COOH-terminal region of CNGA1. Here, we test this mechanism for Ca2+/CaM-dependent inhibition of CNGA1/CNGB1 channels by simultaneously monitoring protein interactions with fluorescence spectroscopy and channel function with patch-clamp recording. Our results show that Ca2+/CaM binds directly to CNG channels, and that binding is the rate-limiting step for channel inhibition. Further, we show that the NH2- and COOH-terminal regions of CNGB1 and CNGA1 subunits, respectively, are in close proximity, and that Ca2+/CaM binding causes a relative rearrangement or separation of these regions. This motion occurs with the same time course as channel inhibition, consistent with the notion that rearrangement of the NH2- and COOH-terminal regions underlies Ca2+/CaM-dependent inhibition.

scholarly journals The HCN domain is required for HCN channel cell-surface expression and couples voltage- and cAMP-dependent gating mechanisms

2020 ◽  
Vol 295 (24) ◽  
pp. 8164-8173
Author(s):  
Ze-Jun Wang ◽  
Ismary Blanco ◽  
Sebastien Hayoz ◽  
Tinatin I. Brelidze
Keyword(s):  
Cell Surface ◽  
Cyclic Nucleotide ◽  
Rhythmic Activity ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Hcn Channels ◽  
Hcn Channel ◽  
Structural Element ◽  
Functional Link ◽  
Membrane Hyperpolarization

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are major regulators of synaptic plasticity and rhythmic activity in the heart and brain. Opening of HCN channels requires membrane hyperpolarization and is further facilitated by intracellular cyclic nucleotides (cNMPs). In HCN channels, membrane hyperpolarization is sensed by the membrane-spanning voltage sensor domain (VSD), and the cNMP-dependent gating is mediated by the intracellular cyclic nucleotide-binding domain (CNBD) connected to the pore-forming S6 transmembrane segment via the C-linker. Previous functional analysis of HCN channels has suggested a direct or allosteric coupling between the voltage- and cNMP-dependent activation mechanisms. However, the specifics of this coupling remain unclear. The first cryo-EM structure of an HCN1 channel revealed that a novel structural element, dubbed the HCN domain (HCND), forms a direct structural link between the VSD and C-linker–CNBD. In this study, we investigated the functional significance of the HCND. Deletion of the HCND prevented surface expression of HCN2 channels. Based on the HCN1 structure analysis, we identified Arg237 and Gly239 residues on the S2 of the VSD that form direct interactions with Ile135 on the HCND. Disrupting these interactions abolished HCN2 currents. We also identified three residues on the C-linker–CNBD (Glu478, Gln482, and His559) that form direct interactions with residues Arg154 and Ser158 on the HCND. Disrupting these interactions affected both voltage- and cAMP-dependent gating of HCN2 channels. These findings indicate that the HCND is necessary for the cell-surface expression of HCN channels and provides a functional link between voltage- and cAMP-dependent mechanisms of HCN channel gating.

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