Single-channel analysis of functional epithelial sodium channel (ENaC) stability at the apical membrane of A6 distal kidney cells

Yu, Ling; Helms, My N.; Yue, Qiang; Eaton, Douglas C.

doi:10.1152/ajprenal.00605.2007

Cited by 25 publications

(15 citation statements)

References 41 publications

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“…Because, in the present study, H 2 O 2 was applied outside the patch pipette (not to ENaC in the patch), it remains to be determined whether extracellular H 2 O 2 could also directly regulate ENaC by oxidizing cysteine and/or histidine residues in the extracellular loops of ENaC subunits. Compared with previously published studies from us and others (23,34), ENaC mean P o presented in this study is relatively low. In previous reports, ENaC mean P o was calculated according to the number of active channels in the patch estimated under resting conditions.…”

Section: Discussioncontrasting

confidence: 90%

Hydrogen Peroxide Stimulates the Epithelial Sodium Channel through a Phosphatidylinositide 3-Kinase-dependent Pathway

2011

Journal of Biological Chemistry

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ENaC 2 in the distal nephron plays an important role in maintaining Na ϩ homeostasis and consequently controls systemic blood pressure. Excess sodium reabsorption due to enhanced ENaC activity can lead to hypertension, as is seen in Liddle syndrome (1, 2) and other genetic diseases (3). Surprisingly, whether high salt intake induces hypertension by targeting ENaC has not been examined. High salt intake can elevate both superoxide (O 2 . ) (4, 5) and H 2 O 2 (6) in the kidney by stimulating NAD(P)H oxidase (7-9), and O 2 . can increase aldosterone-me- , stimulate the non-receptor tyrosine kinase, c-Src (15). c-Src is known to be directly associated with p85, the regulatory subunit of PI3K (16). Tyrosine phosphorylation of p85 reduces its inhibitory effect on PI3K (17). These studies indicate that ROS may stimulate PI3K and consequently promote the synthesis of PI(3,4,5)P 3 . We (18 -20) and others (21,22) have previously shown that PI(3,4,5)P 3 stimulates ENaC. Taken together, these studies suggest that H 2 O 2 may stimulate ENaC by elevating PI(3,4,5)P 3 through activation of PI3K. In the present study, using patch clamp techniques combined with confocal microscopy analysis of apical PI(3,4,5)P 3 levels, we show that H 2 O 2 stimulates ENaC in A6 distal nephron cells via PI3K-dependent elevation of PI(3,4,5)P 3 in the apical membrane. MATERIALS AND METHODSCell Culture-Xenopus A6 distal nephron cells were purchased from the American Type Culture Collection (Manassas, VA). Under previously used culture conditions, basal activity of ENaC in A6 cells is relatively high (23). It is difficult to see and analyze any stimulatory effect when basal ENaC activity is high. Therefore, in the present study, we modified the culture medium to reduce basal ENaC activity. The medium contained 2 parts of DMEM/F-12 (1:1) medium (Invitrogen), 1 part of H 2 O, 15 mM NaHCO 3 (total Na ϩ ϭ 101 mM, which is ideal for amphibian A6 cells), 2 mM L-glutamine, 10% fetal bovine serum (Invitrogen), 25 units/ml penicillin, 25 units/ml streptomycin. A6 cells were cultured in plastic flasks in the presence of 1 M aldosterone at 26°C and 4% CO 2 . After the cells became 70% * This work was supported, in whole or in part, by National Institutes of Health Grant 5R01-DK067110 (to H.-P. M.

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Section: Discussioncontrasting

confidence: 90%

Hydrogen Peroxide Stimulates the Epithelial Sodium Channel through a Phosphatidylinositide 3-Kinase-dependent Pathway

2011

Journal of Biological Chemistry

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“…This means that some functional quality control of ENaCs is conducted after retrieval of ENaCs by regulating relative amounts of recycling vs. degradation of retrieved ENaC depending on whether the γ ENaC subunit is cleaved or not. Our observation on how long an individual ENaC stays at the apical membrane during its whole life-time period before degradation T CTAM shown in Table 3 was 5.61 h. Using T CTAM of 5.61 h, we calculated the half life-time of ENaC staying at the apical membrane, which was 3.89 h. This value is similar to that (3 ~ 3.5 h) reported by Gonzalez-Montelongo et al [46] and Yu et al [47]. Further, this value is not so different from that of the half-life time of cell surface β-ENaC, approximately 6 h [48], although Weisz et al [48] report the half-lives of α-and γ-ENaC subunits at the apical cell surface are more than 24 h and Lu et al [49] suggest the half-life time of β-ENaC is about 30 min.…”

Section: Discussionsupporting

confidence: 82%

Analysis of Aprotinin, a Protease Inhibitor, Action on the Trafficking of Epithelial Na+ Channels (ENaC) in Renal Epithelial Cells Using a Mathematical Model

Sasamoto¹,

Marunaka²,

Niisato

et al. 2017

Cell Physiol Biochem

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Background/Aim: Epithelial Na⁺ channels (ENaC) play a crucial role in control of blood pressure by regulating renal Na⁺ reabsorption. Intracellular trafficking of ENaC is one of the key regulators of ENaC function, but a quantitative description of intracellular recycling of endogenously expressed ENaC is unavailable. We attempt here to provide a model for intracellular recycling after applying a protease inhibitor under hypotonic conditions. Methods: We simulated the ENaC-mediated Na⁺ transport in renal epithelial A6 cells measured as short-circuit currents using a four-state mathematical ENaC trafficking model. Results: We developed a four-state mathematical model of ENaC trafficking in the cytosol of renal epithelial cells that consists of: an insertion state of ENaC that can be trafficked to the apical membrane state (insertion rate); an apical membrane state of ENaC conducting Na⁺ across the apical membrane; a recycling state containing ENaC that are retrieved from the apical membrane state (endocytotic rate) and then to the insertion state (recycling rate) communicating with the apical membrane state or to a degradation state (degradation rate). We studied the effect of aprotinin (a protease inhibitor) blocking protease-induced cleavage of the extracellular loop of γ ENaC subunit on the rates of intracellular ENaC trafficking using the above-defined four-state mathematical model of ENaC trafficking and the recycling number relative to ENaC staying in the apical membrane. We found that aprotinin significantly reduced the insertion rate of ENaC to the apical membrane by 40%, the recycling rate of ENaC by 81%, the cumulative time of an individual ENaC staying in the apical membrane by 32%, the cumulative life-time after the first endocytosis of ENaC by 25%, and the cumulative Na⁺ absorption by 31%. The most interesting result of the present study is that cleavage of ENaC affects the intracellular ENaC trafficking rate and determines the residency time of ENaC, indicating that more active cleaved ENaCs stay longer at the apical membrane contributing to transcellular Na⁺ transport via an increase in recycling of ENaC to the apical membrane. Conclusion: The extracellular protease-induced cleavage of the extracellular loop of γ ENaC subunit increases transcellular epithelial Na⁺ transport by elevating the recycling rate of ENaC due to an increase in the recycling rate of ENaCs associated with increases in the insertion rate of ENaC.

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“…The reasons for these differences are unclear. Finally, measurements of the lifetime of functional channels in the plasma membrane using the same cell line after inhibition of protein synthesis or membrane insertion suggested a halflife of about 3.5 h [43]. This value could be influenced by the turnover of regulatory proteins as well as of the channel subunits themselves, but should provide a lower limit for the surface lifetimes.…”

Section: Regulation Of Channel Traffickingmentioning

confidence: 94%

Regulation and dysregulation of epithelial Na+ channels

2011

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Epithelial Na(+) channels (ENaC) form a highly regulated pathway for the reabsorption of Na(+) from urine. This regulation can take place at a number of different levels, including synthesis of channel protein, trafficking of the protein between the surface and internal membranes, proteolytic cleavage and channel gating. This article reviews the role of these different modes of regulation under physiological conditions and considers the possible contributions of dysregulation of these processes in disease states, particularly hypertension.

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Single-channel analysis of functional epithelial sodium channel (ENaC) stability at the apical membrane of A6 distal kidney cells

Abstract: Yu L, Helms MN, Yue Q, Eaton DC. Single-channel analysis of functional epithelial sodium channel (ENaC) stability at the apical membrane of A6 distal kidney cells.

Cited by 25 publications

References 41 publications

Hydrogen Peroxide Stimulates the Epithelial Sodium Channel through a Phosphatidylinositide 3-Kinase-dependent Pathway

Hydrogen Peroxide Stimulates the Epithelial Sodium Channel through a Phosphatidylinositide 3-Kinase-dependent Pathway

Analysis of Aprotinin, a Protease Inhibitor, Action on the Trafficking of Epithelial Na+ Channels (ENaC) in Renal Epithelial Cells Using a Mathematical Model

Regulation and dysregulation of epithelial Na+ channels

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