scholarly journals The N terminus of α-ENaC mediates ENaC cleavage and activation by furin

2018 ◽  
Vol 150 (8) ◽  
pp. 1179-1187 ◽  
Author(s):  
Pradeep Kota ◽  
Martina Gentzsch ◽  
Yan L. Dang ◽  
Richard C. Boucher ◽  
M. Jackson Stutts
Keyword(s):  
Xenopus Oocytes ◽  
Feedback Inhibition ◽  
Brefeldin A ◽  
Open Probability ◽  
Α Subunit ◽  
Naturally Occurring ◽  
Epithelial Na Channels ◽  
N Terminus ◽  
Oocyte Membrane ◽  
Pore Blocker

Epithelial Na+ channels comprise three homologous subunits (α, β, and γ) that are regulated by alternative splicing and proteolytic cleavage. Here, we determine the basis of the reduced Na+ current (INa) that results from expression of a previously identified, naturally occurring splice variant of the α subunit (α-ENaC), in which residues 34–82 are deleted (αΔ34–82). αΔ34–82-ENaC expression with WT β and γ subunits in Xenopus oocytes produces reduced basal INa, which can largely be recovered by exogenous trypsin. With this αΔ34–82-containing ENaC, both α and γ subunits display decreased cleavage fragments, consistent with reduced processing by furin or furin-like convertases. Data using MTSET modification of a cysteine, introduced into the degenerin locus in β-ENaC, suggest that the reduced INa of αΔ34–82-ENaC arises from an increased population of uncleaved, near-silent ENaC, rather than from a reduced open probability spread uniformly across all channels. After treatment with brefeldin A to disrupt anterograde trafficking of channel subunits, INa in oocytes expressing αΔ34–82-ENaC is reestablished more slowly than that in oocytes expressing WT ENaC. Overnight or acute incubation of oocytes expressing WT ENaC in the pore blocker amiloride increases basal ENaC proteolytic stimulation, consistent with relief of Na+ feedback inhibition. These responses are reduced in oocytes expressing αΔ34–82-ENaC. We conclude that the α-ENaC N terminus mediates interactions that govern the delivery of cleaved and uncleaved ENaC populations to the oocyte membrane.

scholarly journals Feedback inhibition of epithelial Na+ channels in Xenopus oocytes does not require G0 or Gi2 proteins

FEBS Letters ◽  
1999 ◽  
Vol 459 (3) ◽  
pp. 443-447 ◽  
Author(s):  
M. Hübner ◽  
R. Schreiber ◽  
A. Boucherot ◽  
A. Sanchez-Perez ◽  
P. Poronnik ◽  
...  
Keyword(s):  
Xenopus Oocytes ◽  
Feedback Inhibition ◽  
Na Channels ◽  
Epithelial Na Channels

scholarly journals Protease Modulation of the Activity of the Epithelial Sodium Channel Expressed in Xenopus Oocytes

1998 ◽  
Vol 111 (1) ◽  
pp. 127-138 ◽  
Author(s):  
Ahmed Chraïbi ◽  
Véronique Vallet ◽  
Dmitri Firsov ◽  
Solange Kharoubi Hess ◽  
Jean-Daniel Horisberger
Keyword(s):  
Sodium Channel ◽  
Xenopus Oocytes ◽  
Epithelial Sodium Channel ◽  
Cell Surface Expression ◽  
K Channel ◽  
Na Channel ◽  
Extracellular Proteases ◽  
Open Probability ◽  
Α Subunit ◽  
Channel Protein

We have investigated the effect of extracellular proteases on the amiloride-sensitive Na+ current (INa) in Xenopus oocytes expressing the three subunits α, β, and γ of the rat or Xenopus epithelial Na+ channel (ENaC). Low concentrations of trypsin (2 μg/ml) induced a large increase of INa within a few minutes, an effect that was fully prevented by soybean trypsin inhibitor, but not by amiloride. A similar effect was observed with chymotrypsin, but not with kallikrein. The trypsin-induced increase of INa was observed with Xenopus and rat ENaC, and was very large (∼20-fold) with the channel obtained by coexpression of the α subunit of Xenopus ENaC with the β and γ subunits of rat ENaC. The effect of trypsin was selective for ENaC, as shown by the absence of effect on the current due to expression of the K+ channel ROMK2. The effect of trypsin was not prevented by intracellular injection of EGTA nor by pretreatment with GTP-γS, suggesting that this effect was not mediated by G proteins. Measurement of the channel protein expression at the oocyte surface by antibody binding to a FLAG epitope showed that the effect of trypsin was not accompanied by an increase in the channel protein density, indicating that proteolysis modified the activity of the channel present at the oocyte surface rather than the cell surface expression. At the single channel level, in the cell-attached mode, more active channels were observed in the patch when trypsin was present in the pipette, while no change in channel activity could be detected when trypsin was added to the bath solution around the patch pipette. We conclude that extracellular proteases are able to increase the open probability of the epithelial sodium channel by an effect that does not occur through activation of a G protein-coupled receptor, but rather through proteolysis of a protein that is either a constitutive part of the channel itself or closely associated with it.

scholarly journals Cftr

2001 ◽  
Vol 118 (4) ◽  
pp. 433-446 ◽  
Author(s):  
Xuehong Liu ◽  
Stephen S. Smith ◽  
Fang Sun ◽  
David C. Dawson
Keyword(s):  
Plasma Membrane ◽  
Xenopus Oocytes ◽  
Brefeldin A ◽  
Covalent Modification ◽  
Half Time ◽  
Open Probability ◽  
Major Mechanism ◽  
Electrode Voltage ◽  
On Line ◽  
Percent Decrease

Some studies of CFTR imply that channel activation can be explained by an increase in open probability (Po), whereas others suggest that activation involves an increase in the number of CFTR channels (N) in the plasma membrane. Using two-electrode voltage clamp, we tested for changes in N associated with activation of CFTR in Xenopus oocytes using a cysteine-substituted construct (R334C CFTR) that can be modified by externally applied, impermeant thiol reagents like [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET+). Covalent modification of R334C CFTR with MTSET+ doubled the conductance and changed the I-V relation from inward rectifying to linear and was completely reversed by 2-mercaptoethanol (2-ME). Thus, labeled and unlabeled channels could be differentiated by noting the percent decrease in conductance brought about by exposure to 2-ME. When oocytes were briefly (20 s) exposed to MTSET+ before CFTR activation, the subsequently activated conductance was characteristic of labeled R334C CFTR, indicating that the entire pool of CFTR channels activated by cAMP was accessible to MTSET+. The addition of unlabeled, newly synthesized channels to the plasma membrane could be monitored on-line during the time when the rate of addition was most rapid after cRNA injection. The addition of new channels could be detected as early as 5 h after cRNA injection, occurred with a half time of ∼24–48 h, and was disrupted by exposing oocytes to Brefeldin A, whereas activation of R334C CFTR by cAMP occurred with a half time of tens of minutes, and did not appear to involve the addition of new channels to the plasma membrane. These findings demonstrate that in Xenopus oocytes, the major mechanism of CFTR activation by cAMP is by means of an increase in the open probability of CFTR channels.

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 The N Terminus of GTPγS-activated Transducin α-Subunit Interacts with the C Terminus of the cGMP Phosphodiesterase γ-Subunit

2006 ◽  
Vol 281 (10) ◽  
pp. 6194-6202 ◽  
Author(s):  
Jennifer E. Grant ◽  
Lian-Wang Guo ◽  
Martha M. Vestling ◽  
Kirill A. Martemyanov ◽  
Vadim Y. Arshavsky ◽  
...  
Keyword(s):  
Α Subunit ◽  
Cgmp Phosphodiesterase ◽  
Γ Subunit ◽  
C Terminus ◽  
N Terminus

Inhibin immunoneutralization by antibodies raised against synthetic peptide sequences of inhibin α subunit: effects on gonadotrophin concentrations and ovulation rate in sheep

1990 ◽  
Vol 124 (1) ◽  
pp. 167-176 ◽  
Author(s):  
J. H. M. Wrathall ◽  
B. J. McLeod ◽  
R. G. Glencross ◽  
A. J. Beard ◽  
P. G. Knight
Keyword(s):  
Plasma Levels ◽  
Synthetic Peptide ◽  
Plasma Concentrations ◽  
Ovulation Rate ◽  
Control Groups ◽  
Α Subunit ◽  
Blood Samples ◽  
Antigenic Properties ◽  
N Terminus ◽  
The Mean

ABSTRACT Two experiments were conducted to explore the effectiveness of synthetic peptide-based vaccines for active and passive autoimmunization of sheep against inhibin. In the first experiment, adult Romney ewes (n = 20) were actively immunized against a synthetically produced peptide that corresponded to the N-terminus of the α-subunit of bovine inhibin (bIα(1–29)-Tyr30). This peptide was conjugated to tuberculin purified protein derivative (PPD) to increase its antigenic properties. Control groups comprised non-immunized (n = 10) and PPD-immunized (n = 10) ewes. Primary immunization (400 μg conjugate/ewe) was followed by two booster immunizations (200 μg conjugate/ewe), given 5 and 8 weeks later. Following synchronization of oestrus using progestagen sponges, ovulation rates were assessed by laparoscopy. Weekly blood samples were taken throughout the experiment. All inhibin-immunized ewes produced antibodies which bound 125I-labelled bovine inhibin (Mr 32 000), and ovulation rate in inhibin-immunized ewes (2·15 ± 0·22; mean ± s.e.m.) was significantly (P<0·01) greater than in both non-immunized (0·90 ± 0·23) and PPD-immunized (1·20 ± 0·13) control groups. Immunization against the peptide, but not against PPD alone, resulted in a modest rise in plasma FSH, with mean levels after the second boost being significantly (P<0·025) higher (22%) than those before immunization. Moreover, when blood samples were taken (2-h intervals) from randomly selected groups of control (n = 7) and inhibin-immunized (n = 7) ewes for an 84-h period following withdrawal of progestagen sponges, the mean plasma concentration of FSH during the 48 h immediately before the preovulatory LH surge was 37% greater (P< 0·025) in immunized than in control animals. However, more frequent blood sampling (every 15 min for 12 h) during follicular and mid-luteal phases of the oestrous cycle revealed no significant differences between treatment groups in mean plasma concentrations of FSH. In addition, neither mean concentrations of LH nor the frequency and amplitude of LH episodes differed between immunized and control ewes. However, the mean response of LH to a 2 μg bolus of gonadotrophin-releasing hormone, given during the luteal phase, was significantly (P<0·05) less in immunized than in control ewes. These findings indicate that active immunization of Romney ewes against a synthetic fragment of inhibin can promote a controlled increase in ovulation rate, but this response cannot be unequivocally related to an increase in plasma levels of FSH. In the second experiment, passive immunization of seasonally anoestrous ewes (mule × Suffolk crossbred; n = 6 per group) against inhibin, using an antiserum raised in sheep against a synthetic peptide corresponding to the N-terminus of the α-subunit of human inhibin promoted a rapid (<3 h), dose-dependent rise in plasma levels of FSH which remained increased (2·5-fold; P<0·001) for up to 30 h. Plasma concentrations of LH, however, were unaffected by treatment with the antiserum. It is deduced from this observation that, even in the seasonally anoestrous ewe, the ovary secretes physiologically active levels of inhibin, which exert an inhibitory action on the synthesis and secretion of FSH. Journal of Endocrinology (1990) 124, 167–176

scholarly journals The neurosteroid allopregnanolone sulfate inhibits Nav1.3 α subunit-containing voltage-gated sodium channels, expressed in Xenopus oocytes

2018 ◽  
Vol 137 (1) ◽  
pp. 93-97 ◽  
Author(s):  
Takafumi Horishita ◽  
Nobuyuki Yanagihara ◽  
Susumu Ueno ◽  
Dan Okura ◽  
Reiko Horishita ◽  
...  
Keyword(s):  
Sodium Channels ◽  
Xenopus Oocytes ◽  
Α Subunit ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels

Identification of a highly conserved sequence at the N-terminus of the epithelial Na + channel α subunit involved in gating

1999 ◽  
Vol 438 (5) ◽  
pp. 709-715 ◽  
Author(s):  
S. Gründer ◽  
N. Fowler Jaeger ◽  
I. Gautschi ◽  
L. Schild ◽  
B.C. Rossier
Keyword(s):  
Na Channel ◽  
Α Subunit ◽  
Conserved Sequence ◽  
N Terminus ◽  
Epithelial Na Channel

scholarly journals A Novel Bradykinin-Related Peptide, RVA-Thr6-BK, from the Skin Secretion of the Hejiang Frog; Ordorrana hejiangensis: Effects of Mammalian Isolated Smooth Muscle

Toxins ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 376 ◽  
Author(s):  
Yue Wu ◽  
Daning Shi ◽  
Xiaoling Chen ◽  
Lei Wang ◽  
Yuan Ying ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Single Site ◽  
Skin Secretion ◽  
Rat Uterus ◽  
Rat Ileum ◽  
Related Peptide ◽  
Naturally Occurring ◽  
N Terminus ◽  
The Difference

A novel naturally-occurring bradykinin-related peptide (BRP) with an N-terminal extension, named RVA-Thr6-Bradykinin (RVA-Thr6-BK), was here isolated and identified from the cutaneous secretion of Odorrana hejiangensis (O. hejiangensis). Thereafter, in order to evaluate the difference in myotropic actions, a leucine site-substitution variant from Amolops wuyiensis skin secretion, RVA-Leu1, Thr6-BK, was chemically synthesized. Myotropic studies indicated that single-site arginine (R) replacement by leucine (L) at position-4 from the N-terminus, altered the action of RVA-Thr6-BK from an agonist to an antagonist of BK actions on rat ileum smooth muscle. Additionally, both BK N-terminal extended derivatives (RVA-Thr6-BK and RVA-Leu1, Thr6-BK) exerted identical myotropic actions to BK, such as increasing the frequency of contraction, contracting and relaxing the rat uterus, bladder and artery preparations, respectively.

Single-channel basis for conductance increase induced by isoflurane in Shaker H4 IR K+ channels

2001 ◽  
Vol 280 (5) ◽  
pp. C1130-C1139 ◽  
Author(s):  
Jichang Li ◽  
Ana M. Correa
Keyword(s):  
Time Course ◽  
Xenopus Oocytes ◽  
Single Channel ◽  
Noise Analysis ◽  
Single Channels ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Open Probability ◽  
Slowing Down ◽  
Conductance Increase

Volatile anesthetics modulate the function of various K+ channels. We previously reported that isoflurane induces an increase in macroscopic currents and a slowing down of current deactivation of Shaker H4 IR K+ channels. To understand the single-channel basis of these effects, we performed nonstationary noise analysis of macroscopic currents and analysis of single channels in patches from Xenopus oocytes expressing Shaker H4 IR. Isoflurane (1.2% and 2.5%) induced concentration-dependent, partially reversible increases in macroscopic currents and in the time course of tail currents. Noise analysis of currents (70 mV) revealed an increase in unitary current (∼17%) and maximum open probability (∼20%). Single-channel conductance was larger (∼20%), and opening events were more stable, in isoflurane. Tail-current slow time constants increased by 41% and 136% in 1.2% and 2.5% isoflurane, respectively. Our results show that, in a manner consistent with stabilization of the open state, isoflurane increased the macroscopic conductance of Shaker H4 IR K+ channels by increasing the single-channel conductance and the open probability.

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