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.

Exosomal GAPDH from Proximal Tubule Cells Regulate ENaC Activity

“…Polyclonal antibodies specific for each ENaC subunit were used to immunoprecipitate ENaC and pull-down associated proteins. These antibodies were previously characterized [28] and it was demonstrated that they can be used to enrich for each specific ENaC subunit [23]. The blots were then probed for GAPDH to identify an association between ENaC subunits and GAPDH.…”

“…ENaC interacts with several actin cytoskeleton associated proteins including MARCKS, filamin, and fodrin [19, 23, 28]. The actin cytoskeleton serves as an organizing center to keep ENaC and key regulators including other proteins and lipids in close proximity.…”

Effects of cytochalasin E onParacoccidioides brasiliensis

“…In a study on Xenopus cells, the treatment with cytochalasin at a concentration of 1·5 μ mol l −1 changed the shape of the cells from polygonal to flattened (Reifenberger et al . ).…”

“…This inhibition of actin polymerization occurs because cytochalasin E binds to the ends of actin filaments and prevents their elongation and shortening (Reifenberger et al . ).…”

Lipidomic and proteomic analysis of exosomes from mouse cortical collecting duct cells

“…Proteins that have been shown to regulate trafficking of AQP2 to the apical plasma membrane include F-actin, Hsc70 (heat shock constitutive), and A kinase anchoring proteins (11). ENaC directly interacts with the myristoylated alaninerich C kinase substrate (12)(13)(14), filamin (13,15), 14-3-3 proteins (16)(17)(18), actin (19)(20)(21), and actin binding proteins (22,23). We recently showed ENaC activity in cultured mouse cortical collecting duct cells is regulated by exosomal GAPDH (24).…”