Single-channel analysis of functional epithelial sodium channel (ENaC) stability at the apical membrane of A6 distal kidney cells.
ABSTRACT Epithelial sodium channels (ENaC) play an essential role in maintaining total body fluid and electrolyte homeostasis. As such, abnormal expression of ENaC at the cell surface is linked to several important human diseases. Although the stability of ENaC subunits has been extensively studied by protein biochemical analysis, the half-life of the functional channel in the apical membrane remains controversial. Because the functional stability of the multisubunit channel may be more physiologically relevant than the stability of individual subunit proteins, we performed studies of functional ENaC channels using A6 epithelial cells, a Xenopus laevis distal nephron cell line. We recorded single-channel activity in over 400 cells with the translation blockers cycloheximide (CHX) or puromycin, as well as the intracellular protein trafficking inhibitors brefeldin A (BFA) or nocodazole. Our cell-attached, single-channel recordings allow us to quantify the channel density in the apical membrane, as well as to determine channel open probability (Po) from control (untreated) cells and from cells at different times of drug treatment. The data suggest that the half-life of ENaC channels is approximately 3.5 h following puromycin, BFA, and nocodazole treatment. Furthermore, these three drugs had no significant effect on the Po of ENaC for at least 6 h after exposure. A decrease in apical channel number and Po was observed following 2 h of CHX inhibition of protein synthesis, and the apparent channel half-life was closer to 1.5 h following CHX treatment. Treatment of cells with the translation inhibitors does not alter the expression of the protease furin, and therefore changes in protease activity cannot explain changes in ENaC Po. Confocal images show that BFA and nocodazole both disrupt most of the Golgi apparatus after 1-h exposure. In cells with the Golgi totally disrupted by overnight exposure to BFA, 20% of apical ENaC channels remained functional. This result suggests that ENaC is delivered to the apical membrane via a pathway that might bypass the Golgi vesicular trafficking pathway, or that there might be two pools of channels with markedly different half-lives in the apical membrane.
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ABSTRACT: Ion channels play key roles in physiology. They function as protein transducers able to transform stimuli and chemical gradients into electrical signals. They also are critical for cell signaling and play a particularly important role in epithelial transport acting as gateways for the movement of electrolytes across epithelial cell membranes. Experimental limitations, though, have hampered the recording of ion channel activity in many types of tissue. This has slowed progress in understanding the cellular and physiological function of these channels with most function inferred from in vitro systems and cell culture models. In many cases, such inferences have clouded rather than clarified the picture. Here, we describe a contemporary method for isolating and patch-clamping renal tubules for ex vivo analysis of ion channel function in native tissue. Focus is placed on quantifying the activity of the epithelial Na(+) channel (ENaC) in the aldosterone--sensitive distal nephron (ASDN). This isolated, split-open tubule preparation enables recording of renal ion channels in the close-to-native environment under the control of native cell signaling pathways and receptors. When combined with complementary measurements of organ and system function, and contemporary molecular genetics and pharmacology used to manipulate function and regulation, patch-clamping renal channels in the isolated, split-open tubule enables understanding to emerge about the physiological function of these key proteins from the molecule to the whole animal.Methods in molecular biology (Clifton, N.J.) 01/2013; 998:341-53. · 1.29 Impact Factor
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ABSTRACT: Redundancies in both the ubiquitin and epithelial sodium transport regulatory pathways allude to the importance of proteolytic degradation and ion transport in maintaining normal cell function. The classical pathway implicated in ubiquitination of ENaC typically involves Nedd4 2 regulation of sodium channel subunit expression, and has been extensively described in (1,2,3,4,5,6), whereas less attention has been given to the role of Nedd8, a Ubl protein, in terms of regulating ion transport. Herein, we show that Nedd8 plays an important role in the ubiquitination of ENaC in alveolar epithelial cells. We report that the Nedd8 pathway is redox sensitive and that under oxidizing conditions neddylation of ENaC (i.e. Nedd8 conjugation to its E2 enzyme and subsequent activation of E3 ubiquitin protein ligase) is attenuated resulting in greater surface expression of α-ENaC. This observation was confirmed in our electrophysiology studies in which we inhibited Nedd8 activating enzyme (NAE) using MLN4924 [a specific NAE inhibitor (7)] and observed a marked increase in ENaC activity, measured as the product of the number of channels (N) and the open probability (Po) of a channel. These results suggest that ubiquitination of lung ENaC is redox sensitive and may have significant implications on our understanding of the role of ENaC in pulmonary conditions where oxidative stress occurs, such as pulmonary edema and acute lung injury.Journal of Biological Chemistry 01/2013; · 4.65 Impact Factor
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ABSTRACT: Nadph oxidase 4 is an important cellular source of reactive oxygen species (ROS) generation in the kidney. Novel anti-oxidant drugs, such as Nox4-inhibitor compounds, are being developed. There is however, very little experimental evidence for the biological role and regulation of Nadph oxidase isoforms in the kidney. Herein, we show that Fulvene-5 is an effective inhibitor of Nox-generated ROS and report the role of Nox isoforms in activating epithelial sodium channels (ENaC) in A6 distal nephron cells via oxidant signaling and cell stretch activation. Using single channel patch clamp analysis, we report that Fulvene-5 blocked the increase in ENaC activity that is typically observed with H2O2 treatment of A6 cells: average ENaC NPo values decreased from a baseline level of 1.04±0.18 (mean±SE) to 0.25±0.08 following Fulvene-5 treatment. H2O2 treatment failed to increase ENaC activity in the presence of Fulvene-5. Moreover, Fulvene-5 treatment of A6 cells blocked the osmotic-cell stretch response of A6 cells; indicating that stretch activation of Nox-derived ROS plays an important role in ENaC regulation. Together, these findings indicate that Fulvene-5, and perhaps other classes of antioxidant inhibitors, may represent a novel class of compounds useful for the treatment of pathological disorders stemming from inappropriate ion channel activity, such as hypertention.AJP Renal Physiology 07/2013; · 4.42 Impact Factor