The activity of the amiloride-sensitive epithelial sodium channel (ENaC) is modulated by F-actin. However, it is unknown if there is a direct interaction between ␣-ENaC and actin. We have investigated the hypothesis that the actin cytoskeleton directly binds to the carboxyl terminus of ␣-ENaC using a combination of confocal microscopy, co-immunoprecipitation, and protein binding studies. Confocal microscopy of Madin-Darby canine kidney cell monolayers stably transfected with wild type, rat isoforms of ␣-, ␤-, and ␥-ENaC revealed co-localization of ␣-ENaC with the cortical F-actin cytoskeleton both at the apical membrane and within the subapical cytoplasm. F-actin was found to co-immunoprecipitate with ␣-ENaC from whole cell lysates of this cell line. Gel overlay assays demonstrated that F-actin specifically binds to the carboxyl terminus of ␣-ENaC. A direct interaction between F-actin and the COOH terminus of ␣-ENaC was further corroborated by F-actin co-sedimentation studies. This is the first study to report a direct and specific biochemical interaction between F-actin and ENaC.The amiloride-sensitive epithelial sodium channel (ENaC) 2 is a member of the degenerin/epithelial sodium channel superfamily of ion channels. ENaC is expressed at the apical surface of polarized epithelia and is in part responsible for maintaining proper salt and water homeostasis in the body. A great deal of information is known about the biophysical properties of ENaC once it is inserted into the apical surface of an epithelial cell plasma membrane. However, less is known about the proteins that interact with ENaC. Data from the literature indicate an interaction between ENaC and components of the apical membrane cytoskeleton. A partially purified ENaC complex from bovine renal epithelia copurifies with ankyrin, spectrin, and actin (1), suggesting that these cytoskeletal proteins may be associated with ENaC. In addition, ␣-rENaC has been shown to bind to ␣-spectrin, and this is mediated through direct interaction between the ␣-spectrin Src homology 3 domain and the second proline-rich region in the COOH terminus of ␣-rENaC (2). Electrophysiological data provide further support for an interaction between ENaC and the actin-based cytoskeleton. In cellattached patches of A6 renal epithelial cells treated with the actin filament disrupter cytochalasin D, an induction of ENaC activity was observed (3), thereby suggesting that changes in the actin cytoskeleton affect the activity of ENaC. ENaC activation was also observed when short F-actin filaments were added to excised patches, and this effect was increased with the addition of cytochalasin D and/or ATP. These effects were reversed by the addition of the G-actin binding protein, DNase I. In planar lipid bilayers, short F-actin filaments were demonstrated to increase the open probability of rENaC (4), whereas application of DNase I prevented the activation of rENaC. The application of gelsolin, a Ca 2ϩ -activated protein that severs actin filaments and caps the plus end of the actin fi...

The interaction of ion channels with the actin-based cytoskeleton in epithelial cells not only maintains the polarized expression of ion channels within specific membrane domains, it also functions in the intracellular trafficking and regulation of channel activity. Initial evidence supporting an interaction between epithelial ion channels and the actin-based cytoskeleton came from patch-clamp studies examining the effects of cytochalasins on channel activity. Cytochalasins were shown to either activate or inactivate epithelial ion channels. An interaction between the actin-based cytoskeleton and epithelial ion channels was further supported by the fact that the addition of monomeric or filamentous actin to excised patches had an effect on channel activity comparable to that of cytochalasins. Through the recent application of molecular and proteomic approaches, we now know that the interactions between epithelial ion channels and actin can either be direct or indirect, the latter being mediated through scaffolding or actin-binding proteins that serve as links between the channels and the actin-based cytoskeleton. This review discusses recent advances in our understanding of the interactions between epithelial ion channels and the actin-based cytoskeleton, and the roles these interactions play in regulating the cell surface expression, activity, and intracellular trafficking of epithelial ion channels.

Endoplasmic reticulum (ER) degradation pathways can selectively route proteins away from folding and maturation. Both soluble and integral membrane proteins can be targeted from the ER to proteasomal degradation in this fashion. The cystic fibrosis transmembrane conductance regulator (CFTR) is an integral, multidomain membrane protein localized to the apical surface of epithelial cells that functions to facilitate Cl ؊ transport. CFTR was among the first membrane proteins for which a role of the proteasome in ER-related degradation was described. However, the signals that route CFTR to ubiquitination and subsequent degradation are not known. Moreover, limited information is available concerning the subcellular localization of polyubiquitinated CFTR or mechanisms underlying retrograde dislocation of CFTR from the ER membrane to the proteasome either before or after ubiquitination. In the present study, we show that proteasome inhibition with clasto-lactacystin ␤-lactone (4 M, 1 h) stabilizes the presence of a deglycosylated CFTR intermediate for up to 5 h without increasing the core glycosylated (band B) form of CFTR. Deglycosylated CFTR is present under the same conditions that result in accumulation of polyubiquitinated CFTR. Moreover, the deglycosylated form of both wild type and ⌬F508 CFTR can be found in the cytosolic fraction. Both the level and stability of cytosolic, deglycosylated CFTR are increased by proteasome blockade. During retrograde translocation from the ER to the cytosol, CFTR associates with the Sec61 trimeric complex. Sec61 is the key component of the mammalian co-translational protein translocation system and has been proposed to function as a two way channel that transports proteins both into the ER and back to the cytosol for degradation. We show that the level of the Sec61⅐CFTR complexes are highest when CFTR degradation proceeds at the greatest rate (approximately 90 min after pulse labeling). Quantities of Sec61⅐CFTR complexes are also increased by inhibition of the proteasome. Based on these results, we propose a model in which complex membrane proteins such as CFTR are transported through the Sec61 trimeric complex back to the cytosol, escorted by the ␤ subunit of Sec61, and degraded by the proteasome or by other proteolytic systems.

Whether changes in neuronal excitability can cause neurodegenerative disease in the absence of other factors such as protein aggregation is unknown. Mutations in the Kv3.3 voltage-gated K+ channel cause spinocerebellar ataxia type-13 (SCA13), a human autosomal dominant disease characterized by locomotor impairment and the death of cerebellar neurons. Kv3.3 channels facilitate repetitive, high-frequency firing of action potentials, suggesting that pathogenesis in SCA13 is triggered by changes in electrical activity in neurons. To investigate whether SCA13 mutations alter excitability in vivo, we expressed the human dominant negative R420H mutant subunit in zebrafish. The disease-causing mutation specifically suppressed the excitability of Kv3.3-expressing, fast-spiking motor neurons during evoked firing and fictive swimming and, in parallel, decreased the precision and amplitude of the startle response. The dominant negative effect of the mutant subunit on K+ current amplitude was directly responsible for the reduced excitability and locomotor phenotype. Our data provide strong evidence that changes in excitability initiate pathogenesis in SCA13 and establish zebrafish as an excellent model system for investigating how changes in neuronal activity impair locomotor control and cause cell death.

There are multiple macromolecules that cross into the lumen of the prosthesis. We believe spontaneous autoinflation is occurring more often than is believed or reported.