Cytochalasin E alters the cytoskeleton and decreases ENaC activity in Xenopus 2F3 cells

Abstract

Numerous reports have linked cytoskeleton-associated proteins with the regulation of epithelial Na(+) channel (ENaC) activity. The purpose of the present study was to determine the effect of actin cytoskeleton disruption by cytochalasin E on ENaC activity in Xenopus 2F3 cells. Here, we show that cytochalasin E treatment for 60 min can disrupt the integrity of the actin cytoskeleton in cultured Xenopus 2F3 cells. We show using single channel patch-clamp experiments and measurements of short-circuit current that ENaC activity, but not its density, is altered by cytochalasin E-induced disruption of the cytoskeleton. In nontreated cells, 8 of 33 patches (24%) had no measurable ENaC activity, whereas in cytochalasin E-treated cells, 17 of 32 patches (53%) had no activity. Analysis of those patches that did contain ENaC activity showed channel open probability significantly decreased from 0.081 ± 0.01 in nontreated cells to 0.043 ± 0.01 in cells treated with cytochalasin E. Transepithelial current from mpkCCD cells treated with cytochalasin E, cytochalasin D, or latrunculin B for 60 min was decreased compared with vehicle-treated cells. The subcellular expression of fodrin changed significantly, and several protein elements of the cytoskeleton decreased at least twofold after 60 min of cytochalasin E treatment. Cytochalasin E treatment disrupted the association between ENaC and myristoylated alanine-rich C-kinase substrate. The results presented here suggest disruption of the actin cytoskeleton by different compounds can attenuate ENaC activity through a mechanism involving changes in the subcellular expression of fodrin, several elements of the cytoskeleton, and destabilization of the ENaC-myristoylated alanine-rich C-kinase substrate complex.

Keywords: actin; cytoskeleton; epithelial Na+ channels.

Fig. 1.

Single channel characteristics. A: representative single channel recordings from Xenopus 2F3 cells treated with vehicle (0.1% DMSO; left) or 1.5 μM cytochalasin E (Cyto E; right). As shown, there are two open levels (O₁ and O₂) in the vehicle-treated control group but only one open level (O₂) in the Cyto E-treated group. C, closed state. B–D: single channel characteristics from cells pretreated with vehicle or 1.5 μM Cyto E for 60 min. B: epithelial Na⁺ channel (ENaC) activity (NP_o). C: average ENaC channel number (N). D: ENaC open probability (P_o) decreased after Cyto E treatment compared with control. Values are means ± SE; n = 19 observations for vehicle and 13 for Cyto E. NS, not significant. *P < 0.05.

Fig. 2.

Effect of Cyto E on the surface expression of ENaC in Xenopus 2F3 cells. Cyto E treatment (1.5 μM) for 60 min did not change the surface expression of α- or γ-ENaC. A: control experiments demonstrating that the biotinylation protocol/technique was successful. In these experiments, cells were either treated with biotin (+B) or borate buffer alone (−B). As expected, when cells were probed for γ-ENaC, a much stronger signal emanated from the lane loaded with cells initially treated with biotin compared with borate buffer alone, suggesting that biotinylation was successful. In these control experiments, values are normalized so that the signal emanating from the biotin lane is 100%. B and C: cells were pretreated with either vehicle (0.1% DMSO; V) or 1.5 μM Cyto E for 60 min. There were no statistical differences between the surface expression levels of α-ENaC (B) and γ-ENaC (C). In these experiments, values are normalized so that the signal in the vehicle lane is 100%. Left: representative blots; right: summary of densitometry analysis. Values are means ± SE. *P < 0.05.

Fig. 3.

Effect of Cyto E on actin organization in Xenopus 2F3 and mpkCCD cells. Cyto E treatment disrupted actin organization. A and C: 2F3 cells (A) or mpkCCD cells (C) were pretreated with vehicle (0.1% DMSO). B and D: 2F3 cells (B) or mpkCCD cells (D) were pretreated with 1.5 μM Cyto E for 60 min. Increased magnification of the area in the white square is shown on the right. Cells were fixed and stained with Alexa fluor 568 phalloidin (actin, red) and 4′,6-diamidino-2-phenylindole (nucleus, blue). Cyto E treatment induced actin “clumping” (see arrowheads) that was absent in vehicle-treated cells. Z-stack images were taken using an Olympus F1000 confocal microscope.

Fig. 4.

Sucrose density gradient analysis of actin cytoskeleton-associated proteins in Xenopus 2F3 cells after Cyto E treatment. A and B: Western blot (WB) analysis showed there was no appreciable change in the relative level of spectrin protein expression (A) or ezrin/moesin protein expression (B) in any of the isolated sucrose gradient fractions after pretreatment with Cyto E (1.5 μM) compared with vehicle (0.1% DMSO) for 60 min. WCL, whole cell lystate. C: WB analysis showing the relative level of fodrin protein expression changes in the medium to heavy sucrose gradient fractions after Cyto E treatment compared with vehicle. D: densitometric analysis showing that there was a statistically significant change in fodrin protein expression between fractions 6 and 10 for the Cyto E-treated group compared with the control group. Values are means ± SE. *P < 0.05.

Fig. 5.

Proteomic analysis of proteins expressed in Xenopus 2F3 cells after Cyto E treatment. Xenopus 2F3 cells were pretreated with vehicle (0.1% DMSO; A) or 1.5 μM Cyto E for 60 min (B) before proteins were fractionated by sucrose density gradient ultracentrifugation. Cytoplasmic and membrane fractions were subject to two-dimensional gel electrophoresis analysis to further separate the proteins by their isoelectric point (pI) in the horizontal direction and then by their molecular mass in the vertical direction. A and B: Coomassie-stained gels showing membrane proteins separated by two-dimensional gel electrophoresis after mock treatment (A) or after Cyto E treatment (B).C: mass spectrometry analysis of proteins identified in A and B. D: protein elements of the cytoskeleton identified by mass spectrometry. IEF, isoelectric focusing.

Fig. 6.

Immunoprecipitation (IP)-WB [immunoblot (IB)] analysis of the effect of Cyto E treatment on the association between ENaC and myristoylated alanine-rich C-kinase substrate (MARCKS). Xenopus 2F3 cells were pretreated with vehicle (0.1% DMSO; mock) or 1.5 μM Cyto E for 60 min before cells were lysed. A–C: antibodies against α-ENaC (A), β-ENaC (B), or γ-ENaC (C) were used to immunoprecipitate ENaC-binding proteins. Separate blots were probed for α-, β-, and γ-ENaC after IP with each antibody. The elutant was subject to SDS-PAGE before blots were probed for MARCKS protein. ENaC alpha 59, beta 60, and gamma 2102 represent specific antibodies used for IP of the ENaC subunits and associated proteins from the cellular lysate. Arrows indicate MARCKS (top) and ENaC (bottom).

Fig. 7.

Transepithelial current measurements in mpkCCD epithelia after exposure to Cyto E, Cyto D, and latrunculin B. mpkCCD cells cultured on permeable supports for 10 days to allow for the formation of tight junctions were treated with Cyto E, Cyto D, and latrunculin B for 0-, 15-, 30-, or 60-min intervals before transepithelial voltage and resistance were measured. To normalize the current to amiloride, 1 μM amiloride was added after transepithelial voltage and resistance were measured. There was no observable difference in current between groups after the application of amiloride. Data are means ± SE. In some cases, the error bars are smaller than the symbol.*P < 0.05.