Regulation of a cloned epithelial Na+ channel by its β- and γ-subunits

1997 ◽  
Vol 273 (6) ◽  
pp. C1889-C1899 ◽  
Author(s):  
Mouhamed S. Awayda ◽  
Albert Tousson ◽  
Dale J. Benos
Keyword(s):  
Fluorescence Intensity ◽  
Expression System ◽  
Fold Increase ◽  
Na Channel ◽  
Β Subunit ◽  
Open Probability ◽  
Animal Pole ◽  
Α Subunit ◽  
Amino Acid Peptide ◽  
Xenopus Oocyte Expression

Using the Xenopus oocyte expression system, we examined the mechanisms by which the β- and γ-subunits of an epithelial Na+channel (ENaC) regulate α-subunit channel activity and the mechanisms by which β-subunit truncations cause ENaC activation. Expression of α-ENaC alone produced small amiloride-sensitive currents (−43 ± 10 nA, n = 7). These currents increased >30-fold with the coexpression of β- and γ-ENaC to −1,476 ± 254 nA ( n = 20). This increase was accompanied by a 3.1- and 2.7-fold increase of membrane fluorescence intensity in the animal and vegetal poles of the oocyte, respectively, with use of an antibody directed against the α-subunit of ENaC. Truncation of the last 75 amino acids of the β-subunit COOH terminus, as found in the original pedigree of individuals with Liddle’s syndrome, caused a 4.4-fold ( n = 17) increase of the amiloride-sensitive currents compared with wild-type αβγ-ENaC. This was accompanied by a 35% increase of animal pole membrane fluorescence intensity. Injection of a 30-amino acid peptide with sequence identity to the COOH terminus of the human β-ENaC significantly reduced the amiloride-sensitive currents by 40–50%. These observations suggest a tonic inhibitory role on the channel’s open probability ( P o) by the COOH terminus of β-ENaC. We conclude that the changes of current observed with coexpression of the β- and γ-subunits or those observed with β-subunit truncation are likely the result of changes of channel density in combination with large changes of P o.

scholarly journals Translational Fusion of Two β-Subunits of Human Chorionic Gonadotropin Results in Production of a Novel Antagonist of the Hormone

Endocrinology ◽  
2007 ◽  
Vol 148 (8) ◽  
pp. 3977-3986 ◽  
Author(s):  
Satarupa Roy ◽  
Sunita Setlur ◽  
Rupali A. Gadkari ◽  
H. N. Krishnamurthy ◽  
Rajan R. Dighe
Keyword(s):  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Expression System ◽  
Biologically Active ◽  
Chain Molecule ◽  
Dependent Manner ◽  
Β Subunit ◽  
Single Chain ◽  
Α Subunit ◽  
Lh Receptor

The strategy of translationally fusing the α- and β-subunits of human chorionic gonadotropin (hCG) into a single-chain molecule has been used to produce novel analogs of hCG. Previously we reported expression of a biologically active single-chain analog hCGαβ expressed using Pichia expression system. Using the same expression system, another analog, in which the α-subunit was replaced with the second β-subunit, was expressed (hCGββ) and purified. hCGββ could bind to LH receptor with an affinity three times lower than that of hCG but failed to elicit any response. However, it could inhibit response to the hormone in vitro in a dose-dependent manner. Furthermore, it inhibited response to hCG in vivo indicating the antagonistic nature of the analog. However, it was unable to inhibit human FSH binding or response to human FSH, indicating the specificity of the effect. Characterization of hCGαβ and hCGββ using immunological tools showed alterations in the conformation of some of the epitopes, whereas others were unaltered. Unlike hCG, hCGββ interacts with two LH receptor molecules. These studies demonstrate that the presence of the second β-subunit in the single-chain molecule generated a structure that can be recognized by the receptor. However, due to the absence of α-subunit, the molecule is unable to elicit response. The strategy of fusing two β-subunits of glycoprotein hormones can be used to produce antagonists of these hormones.

scholarly journals Specific and Nonspecific Effects of Protein Kinase C on the Epithelial Na + Channel

2000 ◽  
Vol 115 (5) ◽  
pp. 559-570 ◽  
Author(s):  
Mouhamed S. Awayda
Keyword(s):  
Membrane Trafficking ◽  
Expression System ◽  
Na Channel ◽  
Specific Effects ◽  
Nonspecific Effects ◽  
Oocyte Expression ◽  
Xenopus Oocyte Expression ◽  
Oocyte Expression System ◽  
Baseline Values ◽  
Epithelial Na Channel

The Xenopus oocyte expression system was used to explore the mechanisms of inhibition of the cloned rat epithelial Na+ channel (rENaC) by PKC (Awayda, M.S., I.I. Ismailov, B.K. Berdiev, C.M. Fuller, and D.J. Benos. 1996. J. Gen. Physiol. 108:49–65) and to determine whether human ENaC exhibits similar regulation. Effects of PKC activation on membrane and/or channel trafficking were determined using impedance analysis as an indirect measure of membrane area. hENaC-expressing oocytes exhibited an appreciable activation by hyperpolarizing voltages. This activation could be fit with a single exponential, described by a time constant (τ) and a magnitude (ΔI V). A similar but smaller magnitude of activation was also observed in oocytes expressing rENaC. This activation likely corresponds to the previously described effect of hyperpolarizing voltage on gating of the native Na+ channel (Palmer, L.G., and G. Frindt. 1996. J. Gen. Physiol. 107:35–45). Stimulation of PKC with 100 nM PMA decreased ΔIV in hENaC-expressing oocytes to a plateau at 57.1 ± 4.9% (n = 6) of baseline values at 20 min. Similar effects were observed in rENaC-expressing oocytes. PMA decreased the amiloride-sensitive hENaC slope conductance (gNa) to 21.7 ± 7.2% (n = 6) of baseline values at 30 min. This decrease was similar to that previously reported for rENaC. This decrease of g Na was attributed to a decrease of membrane capacitance (C m), as well as the specific conductance (gm/Cm ). The effects on gm/Cm reached a plateau within 15 min, at ∼60% of baseline values. This decrease is likely due to the specific ability of PKC to inhibit ENaC. On the other hand, the decrease of Cm was unrelated to ENaC and is likely an effect of PKC on membrane trafficking, as it was observed in ENaC-expressing as well as control oocytes. At lower PMA concentrations (0.5 nM), smaller changes of Cm were observed in rENaC- and hENaC-expressing oocytes, and were preceded by larger changes of gm and by changes of gm/Cm, indicating specific effects on ENaC. These findings indicate that PKC exhibits multiple and specific effects on ENaC, as well as nonspecific effects on membrane trafficking. Moreover, these findings provide the electrophysiological basis for assessing channel-specific effects of PKC in the Xenopus oocyte expression system.

Heterologous expression of Na+-K+-ATPase in insect cells: intracellular distribution of pump subunits

2001 ◽  
Vol 281 (3) ◽  
pp. C982-C992 ◽  
Author(s):  
Craig Gatto ◽  
Scott M. McLoud ◽  
Jack H. Kaplan
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Membrane Protein ◽  
Atpase Activity ◽  
Insect Cells ◽  
Expression System ◽  
Β Subunit ◽  
Α Subunit ◽  
Enzyme Preparations ◽  
High Five Cells

The Na+-K+-ATPase is a heterodimeric plasma membrane protein responsible for cellular ionic homeostasis in nearly all animal cells. It has been shown that some insect cells (e.g., High Five cells) have no (or extremely low) Na+-K+-ATPase activity. We expressed sheep kidney Na+-K+-ATPase α- and β-subunits individually and together in High Five cells via the baculovirus expression system. We used quantitative slot-blot analyses to determine that the expressed Na+-K+-ATPase comprises between 0.5% and 2% of the total membrane protein in these cells. Using a five-step sucrose gradient (0.8–2.0 M) to separate the endoplasmic reticulum, Golgi apparatus, and plasma membrane fractions, we observed functional Na+ pump molecules in each membrane pool and characterized their properties. Nearly all of the expressed protein functions normally, similar to that found in purified dog kidney enzyme preparations. Consequently, the measurements described here were not complicated by an abundance of nonfunctional heterologously expressed enzyme. Specifically, ouabain-sensitive ATPase activity, [3H]ouabain binding, and cation dependencies were measured for each fraction. The functional properties of the Na+-K+-ATPase were essentially unaltered after assembly in the endoplasmic reticulum. In addition, we measured ouabain-sensitive 86Rb+ uptake in whole cells as a means to specifically evaluate Na+-K+-ATPase molecules that were properly folded and delivered to the plasma membrane. We could not measure any ouabain-sensitive activities when either the α-subunit or β-subunit were expressed individually. Immunostaining of the separate membrane fractions indicates that the α-subunit, when expressed alone, is degraded early in the protein maturation pathway (i.e., the endoplasmic reticulum) but that the β-subunit is processed normally and delivered to the plasma membrane. Thus it appears that only the α-subunit has an oligomeric requirement for maturation and trafficking to the plasma membrane. Furthermore, assembly of the α-β heterodimer within the endoplasmic reticulum apparently does not require a Na+pump-specific chaperone.

scholarly journals Functional Coupling of the β1 Subunit to the Large Conductance Ca2+-Activated K+ Channel in the Absence of Ca2+

2000 ◽  
Vol 115 (6) ◽  
pp. 719-736 ◽  
Author(s):  
Crina M. Nimigean ◽  
Karl L. Magleby
Keyword(s):  
Fold Increase ◽  
Bk Channels ◽  
Burst Duration ◽  
K Channel ◽  
Functional Coupling ◽  
Open Probability ◽  
Α Subunit ◽  
Gating Kinetics ◽  
Leftward Shift ◽  
Wide Range

Coexpression of the β1 subunit with the α subunit (mSlo) of BK channels increases the apparent Ca2+ sensitivity of the channel. This study investigates whether the mechanism underlying the increased Ca2+ sensitivity requires Ca2+, by comparing the gating in 0 Ca2+i of BK channels composed of α subunits to those composed of α+β1 subunits. The β1 subunit increased burst duration ∼20-fold and the duration of gaps between bursts ∼3-fold, giving an ∼10-fold increase in open probability (Po) in 0 Ca2+i. The effect of the β1 subunit on increasing burst duration was little changed over a wide range of Po achieved by varying either Ca2+i or depolarization. The effect of the β1 subunit on increasing the durations of the gaps between bursts in 0 Ca2+i was preserved over a range of voltage, but was switched off as Ca2+i was increased into the activation range. The Ca2+-independent, β1 subunit-induced increase in burst duration accounted for 80% of the leftward shift in the Po vs. Ca2+i curve that reflects the increased Ca2+ sensitivity induced by the β1 subunit. The Ca2+-dependent effect of the β1 subunit on the gaps between bursts accounted for the remaining 20% of the leftward shift. Our observation that the major effects of the β1 subunit are independent of Ca2+i suggests that the β1 subunit mainly alters the energy barriers of Ca2+-independent transitions. The changes in gating induced by the β1 subunit differ from those induced by depolarization, as increasing Po by depolarization or by the β1 subunit gave different gating kinetics. The complex gating kinetics for both α and α+β1 channels in 0 Ca2+i arise from transitions among two to three open and three to five closed states and are inconsistent with Monod-Wyman-Changeux type models, which predict gating among only one open and one closed state in 0 Ca2+i.

Multiple epithelial Na+ channel domains participate in subunit assembly

2003 ◽  
Vol 285 (4) ◽  
pp. F600-F609 ◽  
Author(s):  
James B. Bruns ◽  
Baofeng Hu ◽  
Yoon J. Ahn ◽  
Shaohu Sheng ◽  
Rebecca P. Hughey ◽  
...  
Keyword(s):  
Transmembrane Protein ◽  
Expression System ◽  
Functional Expression ◽  
Full Length ◽  
Na Channel ◽  
Xenopus Laevis Oocyte ◽  
Subunit Interactions ◽  
Α Subunit ◽  
Hydrophobic Domain ◽  
Membrane Spanning

Epithelial sodium channels (ENaCs) are composed of three structurally related subunits that form a tetrameric channel. The Xenopus laevis oocyte expression system was used to identify regions within the ENaC α-subunit that confer a dominant negative phenotype on functional expression of αβγ-ENaC to define domains that have a role in subunit-subunit interactions. Coexpression of full-length mouse αβγ-ENaC with either 1) the α-subunit first membrane-spanning domain and short downstream hydrophobic domain (α-M1H1); 2) α-M1H1 and its downstream hydrophilic extracellular loop (α-M1H1-ECL); 3) the membrane-spanning domain of a control type 2 transmembrane protein (glutamyl transpeptidase; γ-GT) fused to the α-ECL (γ-GT-α-ECL); 4) the extracellular domain of a control type 1 transmembrane protein (Tac) fused to the α-subunit second membrane-spanning domain and short upstream hydrophobic domain (Tac-α-H2M2); or 5) the α-subunit cytoplasmic COOH terminus (α-Ct) significantly reduced amiloride-sensitive Na+ currents in X. laevis oocytes. Functional expression of Na+ channels was not inhibited when full-length αβγ-ENaC was coexpressed with either 1) the α-ECL lacking a signal-anchor sequence, 2) α-M1H1 and α-Ct expressed as a fusion protein, 3) full-length γ-GT, or 4) full-length Tac. Furthermore, the expression of ROMK channels was not inhibited when full-length ROMK was coexpressed with either α-M1H1-ECL or α-Ct. Full-length FLAG-tagged α-, β-, or γ-ENaC coimmunoprecipitated with myc-tagged α-M1H1-ECL, whereas wild-type γ-GT did not. These data suggest that multiple sites within the α-subunit participate in subunit-subunit interactions that are required for proper assembly of the heterooligomeric ENaC complex.

scholarly journals Gain-of-function variant of the human epithelial sodium channel

2013 ◽  
Vol 304 (2) ◽  
pp. F207-F213 ◽  
Author(s):  
Jingxin Chen ◽  
Thomas R. Kleyman ◽  
Shaohu Sheng
Keyword(s):  
Single Channel ◽  
Expression System ◽  
Surface Expression ◽  
Wild Type ◽  
Open Probability ◽  
Gain Of Function ◽  
Human Disorders ◽  
Human Genome Sequencing ◽  
Xenopus Oocyte Expression ◽  
Salt Sensitive Hypertension

Epithelial Na+ channel (ENaC) mutations are associated with several human disorders, underscoring the importance of these channels in human health. Recent human genome sequencing projects have revealed a large number of ENaC gene variations, several of which have been found in individuals with salt-sensitive hypertension, cystic fibrosis, and other disorders. However, the functional consequences of most variants are unknown. In this study, we used the Xenopus oocyte expression system to examine the functional properties of a human ENaC variant. Oocytes expressing αβγL511Q human ENaCs showed 4.6-fold greater amiloride-sensitive currents than cells expressing wild-type channels. The γL511Q variant did not significantly alter channel surface expression. Single channel recordings revealed that the variant had fourfold higher open probability than wild type. In addition, γL511Q largely eliminated the Na+ self-inhibition response, which reflects a downregulation of ENaC open probability by extracellular Na+. Moreover, γL511Q diminished chymotrypsin-induced activation of the mutant channel. We conclude that γL511Q is a gain-of-function human ENaC variant. Our results suggest that γL511Q enhances ENaC activity by increasing channel open probability and dampens channel regulation by extracellular Na+ and proteases.

scholarly journals Regulation of Nav1.6 and Nav1.8 peripheral nerve Na+ channels by auxiliary β-subunits

2011 ◽  
Vol 106 (2) ◽  
pp. 608-619 ◽  
Author(s):  
Juan Zhao ◽  
Michael E. O'Leary ◽  
Mohamed Chahine
Keyword(s):  
Current Density ◽  
Small Diameter ◽  
Fold Increase ◽  
Β Subunit ◽  
Drg Neurons ◽  
Α Subunit ◽  
Terminal Domain ◽  
Steady State Inactivation ◽  
Hyperpolarizing Shift ◽  
Auxiliary Β Subunits

Voltage-gated Na+ (Nav) channels are composed of a pore-forming α-subunit and one or more auxiliary β-subunits. The present study investigated the regulation by the β-subunit of two Na+ channels (Nav1.6 and Nav1.8) expressed in dorsal root ganglion (DRG) neurons. Single cell RT-PCR was used to show that Nav1.8, Nav1.6, and β1–β3 subunits were widely expressed in individually harvested small-diameter DRG neurons. Coexpression experiments were used to assess the regulation of Nav1.6 and Nav1.8 by β-subunits. The β1-subunit induced a 2.3-fold increase in Na+ current density and hyperpolarizing shifts in the activation (−4 mV) and steady-state inactivation (−4.7 mV) of heterologously expressed Nav1.8 channels. The β4-subunit caused more pronounced shifts in activation (−16.7 mV) and inactivation (−9.3 mV) but did not alter the current density of cells expressing Nav1.8 channels. The β3-subunit did not alter Nav1.8 gating but significantly reduced the current density by 31%. This contrasted with Nav1.6, where the β-subunits were relatively weak regulators of channel function. One notable exception was the β4-subunit, which induced a hyperpolarizing shift in activation (−7.6 mV) but no change in the inactivation or current density of Nav1.6. The β-subunits differentially regulated the expression and gating of Nav1.8 and Nav1.6. To further investigate the underlying regulatory mechanism, β-subunit chimeras containing portions of the strongly regulating β1-subunit and the weakly regulating β2-subunit were generated. Chimeras retaining the COOH-terminal domain of the β1-subunit produced hyperpolarizing shifts in gating and increased the current density of Nav1.8, similar to that observed for wild-type β1-subunits. The intracellular COOH-terminal domain of the β1-subunit appeared to play an essential role in the regulation of Nav1.8 expression and gating.

scholarly journals Exogenous expression of glucagon-like peptide 1 receptor and human insulin in AtT-20 corticotrophs confers cAMP-mediated gene transcription and insulin secretion

2005 ◽  
Vol 187 (3) ◽  
pp. 419-427 ◽  
Author(s):  
K K Sidhu ◽  
R C Fowkes ◽  
R H Skelly ◽  
J M Burrin
Keyword(s):  
Insulin Secretion ◽  
Cell Line ◽  
Gene Transcription ◽  
Expression System ◽  
Fold Increase ◽  
Receptor Expression ◽  
Glycoprotein Hormone ◽  
Α Subunit ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1

The insulinotrophic effects of glucagon-like peptide 1 (GLP-1) are mediated by its seven-transmembrane receptor (GLP-1R) in pancreatic β-cells. We have transiently transfected the GLP-1R and a proopiomelanocortin (POMC) promoter-driven human preproinsulin gene vector (pIRES) into the AtT-20 pituitary corticotrophic cell line, to investigate the possibility of creating a regulated, insulin-expressing cell line. Receptor expression was confirmed by RT-PCR and functionality was demonstrated by measuring changes in cAMP levels in response to GLP-1. Rapid (5 min) stimulation of cAMP production was observed with 100 nM GLP-1, 24 h after transfection of 2 μg GLP-1R DNA. AtT-20 cells co-transfected with GLP-1R and human glycoprotein hormone α-subunit or rat POMC promoters revealed GLP-1-stimulated cAMP activation of transcription. Co-transfection of the pIRES vector with the GLP-1R resulted in GLP-1-stimulated activation of POMC promoter-driven preproinsulin gene transcription but insulin secretion was not detected. However, using an adenoviral expression system to infect AtT-20 cells with GLP-1R and the preproinsulin gene (including 120 bp of its own promoter) resulted in a 6.4 ± 0.6-fold increase in cAMP and a 4.9 ± 0.8-fold increase in insulin secretion in response to 100 nM GLP-1. These results demonstrate, for the first time, functional GLP-1R-mediated preproinsulin gene transcription and secretion in a transplantable cell line.

scholarly journals The β Subunit Increases the Ca2+ Sensitivity of Large Conductance Ca2+-activated Potassium Channels by Retaining the Gating in the Bursting States

1999 ◽  
Vol 113 (3) ◽  
pp. 425-440 ◽  
Author(s):  
Crina M. Nimigean ◽  
Karl L. Magleby
Keyword(s):  
Single Channel ◽  
Bk Channels ◽  
Bk Channel ◽  
Β Subunit ◽  
Open Probability ◽  
Α Subunit ◽  
Closed Intervals ◽  
Single Channel Analysis ◽  
Channel Analysis ◽  
The Mean

Coexpression of the β subunit (KV,Caβ) with the α subunit of mammalian large conductance Ca2+- activated K+ (BK) channels greatly increases the apparent Ca2+ sensitivity of the channel. Using single-channel analysis to investigate the mechanism for this increase, we found that the β subunit increased open probability (Po) by increasing burst duration 20–100-fold, while having little effect on the durations of the gaps (closed intervals) between bursts or on the numbers of detected open and closed states entered during gating. The effect of the β subunit was not equivalent to raising intracellular Ca2+ in the absence of the beta subunit, suggesting that the β subunit does not act by increasing all the Ca2+ binding rates proportionally. The β subunit also inhibited transitions to subconductance levels. It is the retention of the BK channel in the bursting states by the β subunit that increases the apparent Ca2+ sensitivity of the channel. In the presence of the β subunit, each burst of openings is greatly amplified in duration through increases in both the numbers of openings per burst and in the mean open times. Native BK channels from cultured rat skeletal muscle were found to have bursting kinetics similar to channels expressed from alpha subunits alone.

scholarly journals H,K-ATPase α subunit C-terminal membrane topology: epitope tags in the insect cell expression system

1999 ◽  
Vol 340 (3) ◽  
pp. 601-611
Author(s):  
Adam J. SMOLKA ◽  
Kellie A. LARSEN ◽  
Clifford W. SCHWEINFEST ◽  
Charles E. HAMMOND
Keyword(s):  
Expression System ◽  
Recombinant Baculovirus ◽  
Transmembrane Domains ◽  
Terminal Region ◽  
Structural Basis ◽  
Sf9 Cells ◽  
Β Subunit ◽  
Α Subunit ◽  
Baculovirus Infection ◽  
P Type

The H,K-ATPase responsible for gastric acidification is a heterodimeric (α and β subunit) P-type ATPase, an integral protein of parietal cell apical membranes, which promotes the electroneutral exchange of K+ for protons, is stimulated by K+ and is inhibited by 2-methyl-8-(phenylmethoxy)imidazo[1,2-α]pyridine-3-acetonitrile (SCH 28080). Hydropathy analysis of the catalytic α subunit has been interpreted in terms of four N-terminal transmembrane domains, a cytoplasmically oriented segment containing ATP binding and phosphorylation sites, and a C-terminal region with four or six putative transmembrane domains. Several lines of evidence implicate the C-terminal region of P-type ATPases in cation-binding and occlusion, conformational changes, and interactions with the β subunit (HKβ), making the definition of topology a prerequisite for understanding the structural basis of these functions. Influenza haemagglutinin epitopes (YPYDVPDYA; flu tag) were inserted in predicted hydrophilic segments of the α subunit (HKα) to establish the membrane orientation of two amino acids with different predicted topologies in the C-terminal four- and six-transmembrane models. Wild-type and mutated HKα and HKβ cDNA species were expressed in insect cells (Sf9) via recombinant baculovirus infection, and expression of H,K-ATPase was verified by immunoblotting with HKα- and HKβ-specific and flu-tag-specific antibodies. Functional assays showed K+-stimulated, SCH 28080-sensitive ATPase activity, confirming neo-native topology in H,K-ATPase heterodimers expressed in Sf9 cells. The topology of flu tags was determined by microsomal protease protection assays in Sf9 cells and immunolabelling of HKα and HKβ in intact and permeabilized Sf9 cells. In addition, MS of native H,K-ATPase tryptic peptides identified cytoplasmically oriented HKα residues. The results indicated cytoplasmic exposure of Leu844 and Phe996, and luminal exposure of Pro898, leading to a revised secondary structure model of the C-terminal third of HKα.

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