The activity of the thiazide-sensitive Na+–Cl– cotransporter is regulated by protein phosphatase PP4

2010 ◽  
Vol 88 (10) ◽  
pp. 986-995 ◽  
Author(s):  
Mark Glover ◽  
Annie Mercier Zuber ◽  
Nikki Figg ◽  
Kevin M. O’Shaughnessy
Keyword(s):  
Membrane Trafficking ◽  
Expression System ◽  
Surface Membrane ◽  
Physiological Role ◽  
Dynamic Regulation ◽  
Regulatory Pathways ◽  
Wnk Kinases ◽  
Xenopus Oocyte Expression ◽  
Oocyte Expression System

Cation transport in the distal mammalian nephron relies on the SLC12 family of membrane cotransporters that include the thiazide-sensitive Na+–Cl– cotransporter (NCC). NCC is regulated through a scaffold of interacting proteins, including the WNK kinases, WNK 1 and WNK 4, which are mutated in the hypertensive Gordon’s syndrome. Dynamic regulation of NCC function by kinases must involve dephosphorylation by phosphatases, as illustrated by the role of PP1 and PP2B in the regulation of KCC members of the SLC12 family. There are 2 phosphorylation-controlled regulatory pathways for NCC: type 1, mediated by WNK4 and affecting trafficking to the surface membrane, and type 2, affecting intrinsic transporter kinetics by phosphorylation of conserved N-terminal S/T amino acids. Using the Xenopus oocyte expression system, we show that PP4 inhibits NCC activity — but not trafficking to the surface membrane — by a mechanism that requires phosphatase activity and a conserved N-terminal amino acid of NCC, threonine 58. This action is distinct from WNK4 regulation of membrane trafficking. In the mouse kidney, PP4 is selectively expressed in the distal nephron, including cells of the distal convoluted tubule cells, suggesting that PP4 may have a physiological role in regulating NCC and hence NaCl reabsorption in vivo.

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.

[28] In vivo incorporation of unnatural amino acids into ion channels in Xenopus oocyte expression system

1998 ◽  
pp. 504-529 ◽  
Author(s):  
Mark W. Nowak ◽  
Justin P. Gallivan ◽  
Scott K. Silverman ◽  
Cesar G. Labarca ◽  
Dennis A. Dougherty ◽  
...  
Keyword(s):  
Amino Acids ◽  
Ion Channels ◽  
Unnatural Amino Acids ◽  
Xenopus Oocyte ◽  
Expression System ◽  
Oocyte Expression ◽  
Xenopus Oocyte Expression ◽  
Oocyte Expression System

Bisphosphonates stimulate an endogenous nonselective cation channel in Xenopus oocytes: potential mechanism of action

2005 ◽  
Vol 289 (2) ◽  
pp. C248-C256 ◽  
Author(s):  
Weijian Shao ◽  
Roy C. Orlando ◽  
Mouhamed S. Awayda
Keyword(s):  
Tyrosine Phosphatase ◽  
Expression System ◽  
Mechanisms Of Action ◽  
Gastrointestinal Complications ◽  
Cation Conductance ◽  
Xenopus Oocyte Expression ◽  
Oocyte Expression System ◽  
Small Inhibition ◽  
Kinase Pathway ◽  
Stimulation Of

The mechanisms of action of bisphosphonates (BPs) have been poorly determined. Besides their actions on osteoclasts, these agents exhibit gastrointestinal complications. They have also recently been described as affecting various preparations that express an epithelial Na+ channel (ENaC). To understand the effects of BP on ion channels and the ENaC in particular, we used the Xenopus oocyte expression system. Alendronate, and similarly risedronate, two aminobisphosphonates, caused a large stimulation of an endogenous nonselective cation conductance (NSCC). This stimulation averaged 63 ± 12 μS ( n = 18) 60 min after the addition of 2 mM alendronate. The effects on the endogenous NSCC were blocked by extracellular acidification to pH 6.4. On the other hand, alendronate caused a small inhibition of ENaC conductance at pH 7.4 and 6.4, but the effects at pH 6.4 were more readily observed in the absence of changes of the endogenous conductance. The effects on membrane capacitance were also markedly different, with a clear decrease at pH 6.4 and no consistent changes at pH 7.4. The effects on the endogenous channel were further augmented by genistein and were inhibited by a tyrosine phosphatase inhibitor, indicating the involvement of the tyrosine kinase pathway. Stimulation of NSCC with BP is expected to cause membrane depolarization and may explain, in part, its mechanisms of action in inhibiting osteoclasts.

scholarly journals Characterization of mouse amino acid transporter B0AT1 (slc6a19)

2005 ◽  
Vol 389 (3) ◽  
pp. 745-751 ◽  
Author(s):  
Christoph Böhmer ◽  
Angelika Bröer ◽  
Michael Munzinger ◽  
Sonja Kowalczuk ◽  
John E. J. Rasko ◽  
...  
Keyword(s):  
Amino Acids ◽  
Membrane Potential ◽  
Amino Acid ◽  
Expression System ◽  
Complex Function ◽  
Aromatic Amino Acids ◽  
Inward Currents ◽  
Xenopus Oocyte Expression ◽  
Oocyte Expression System

The mechanism of the mouse (m)B0AT1 (slc6a19) transporter was studied in detail using two electrode voltage-clamp techniques and tracer studies in the Xenopus oocyte expression system. All neutral amino acids induced inward currents at physiological potentials, but large neutral non-aromatic amino acids were the preferred substrates of mB0AT1. Substrates were transported with K0.5 values ranging from approx. 1 mM to approx. 10 mM. The transporter mediates Na+–amino acid co-transport with a stoichiometry of 1:1. No other ions were involved in the transport mechanism. An increase in the extracellular Na+ concentration reduced the K0.5 for leucine, and vice versa. Moreover, the K0.5 values and Vmax values of both substrates varied with the membrane potential. As a result, K0.5 and Vmax values are a complex function of the concentration of substrate and co-substrate and the membrane potential. A model is presented assuming random binding order and a positive charge associated with the ternary [Na+–substrate–transporter] complex, which is consistent with the experimental data.

scholarly journals Novel Mutations in the TMPRSS3 Gene May Contribute to Taiwanese Patients with Nonsyndromic Hearing Loss

2020 ◽  
Vol 21 (7) ◽  
pp. 2382
Author(s):  
Swee-Hee Wong ◽  
Yung-Chang Yen ◽  
Shuan-Yow Li ◽  
Jiann-Jou Yang
Keyword(s):  
Hearing Loss ◽  
Serine Protease ◽  
Xenopus Oocyte ◽  
Expression System ◽  
Missense Mutations ◽  
Nonsyndromic Hearing Loss ◽  
Oocyte Expression ◽  
Xenopus Oocyte Expression ◽  
Oocyte Expression System ◽  
Transmembrane Serine Protease

A previous study indicated that mutations in the transmembrane protease serine 3 (TMPRSS3) gene, which encodes a transmembrane serine protease, cause nonsyndromic hearing loss (NSHL). This was the first description of a serine protease involved in hearing loss (HL). In Taiwan, however, data on the TMPRSS3 gene’s association with NSHL is still insufficient. In this study, we described 10 mutations of TMPRSS3 genes found in 14 patients after screening 230 children with NSHL. The prevalence of the TMPRSS3 mutation appeared to be 6.09% (14/230). Of the 10 mutations, three were missense mutations: c.239G>A (p.R80H), c.551T>C (p.L184S), and 1253C>T (p.A418V); three were silent mutations, and four were mutations in introns. To determine the functional importance of TMPRSS3 mutations, we constructed plasmids carrying TMPRSS3 mutations of p.R80H, p.L184S, and p.A418V. TMPRSS3 function can be examined by secretory genetic assay for site-specific proteolysis (sGASP) and Xenopus oocyte expression system. Our results showed that p.R80H, p.L184S, and p.A418V TMPRSS3 mutations gave ratios of 19.4%, 13.2%, and 27.6%, respectively, via the sGASP system. Moreover, these three TMPRSS3 mutations failed to activate the epithelial sodium channel (ENaC) in the Xenopus oocyte expression system. These results indicate that the p.R80H, p.L184S, and p.A418V missense mutations of TMPRSS3 resulted in greatly diminishing the proteolytic activity of TMPRSS3. Our study provides information for understanding the importance of TMPRSS3 in the NSHL of Taiwanese children and provides a novel molecular explanation for the role of TMPRSS3 in HL.

scholarly journals Signal transduction of rodent opioid μ, δ, κ-receptor in Xenopus oocyte expression system.

1994 ◽  
Vol 64 ◽  
pp. 129
Author(s):  
Takeaki Miyamae ◽  
Nobuyuki Fukushima ◽  
Yoshimi Misu ◽  
Hiroshi Takeshima ◽  
Kazuhiko Fukuda ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Xenopus Oocyte ◽  
Expression System ◽  
Oocyte Expression ◽  
Xenopus Oocyte Expression ◽  
Oocyte Expression System ◽  
Κ Receptor

Lipid mediators modulate NMDA receptor currents in a Xenopus oocyte expression system

1997 ◽  
Vol 237 (1) ◽  
pp. 13-16 ◽  
Author(s):  
Sadaharu Tabuchi ◽  
Kazuhiko Kume ◽  
Makoto Aihara ◽  
Satoshi Ishii ◽  
Masayoshi Mishina ◽  
...  
Keyword(s):  
Nmda Receptor ◽  
Xenopus Oocyte ◽  
Expression System ◽  
Lipid Mediators ◽  
Oocyte Expression ◽  
Xenopus Oocyte Expression ◽  
Oocyte Expression System
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