In striated muscle, the plasma membrane forms tubular invaginations (transverse tubules or T-tubules) that function in depolarization-contraction coupling. Caveolin-3 and amphiphysin were implicated in their biogenesis. Amphiphysin isoforms have a putative role in membrane deformation at endocytic sites. An isoform of amphiphysin 2 concentrated at T-tubules induced tubular plasma membrane invaginations when expressed in nonmuscle cells. This property required exon 10, a phosphoinositide-binding module. In developing myotubes, amphiphysin 2 and caveolin-3 segregated in tubular and vesicular portions of the T-tubule system, respectively. These findings support a role of the bilayer-deforming properties of amphiphysin at T-tubules and, more generally, a physiological role of amphiphysin in membrane deformation.

Epithelial sodium channels (ENaC) are expressed in the apical membrane of high resistance Na ؉ transporting epithelia and have a key role in regulating extracellular fluid volume and the volume of airway surface liquids. Maturation and activation of ENaC subunits involves furin-dependent cleavage of the ectodomain at two sites in the ␣ subunit and at a single site within the ␥ subunit. We now report that the serine protease prostasin further activates ENaC by inducing cleavage of the ␥ subunit at a site distal to the furin cleavage site. Dual cleavage of the ␥ subunit is predicted to release a 43-amino acid peptide. Channels with a ␥ subunit lacking this 43-residue tract have increased activity due to a high open probability. A synthetic peptide corresponding to the fragment cleaved from the ␥ subunit is a reversible inhibitor of endogenous ENaCs in mouse corticalcollecting duct cells and in primary cultures of human airway epithelial cells. Our results suggest that multiple proteases cleave ENaC ␥ subunits to fully activate the channel.Epithelial sodium channels (ENaC) 3 are expressed at the apical plasma membrane of cells lining the distal nephron, airway and alveoli, and distal colon, where they play a key role in the regulation of extracellular fluid volume, blood pressure, and airway surface liquid volume. These channels are composed of three homologous subunits, termed ␣, ␤, and ␥. Each subunit has cytosolic amino and carboxyl termini and two membranespanning domains separated by a large ectodomain (1-3). The second membrane-spanning domain and the preceding region of each subunit are predicted to form the channel pore (4 -7). Proteolysis of ENaC subunit extracellular domains at specific sites has a key role in modulating channel gating (8 -10). Maturation of ENaC subunits in Xenopus oocytes, Madin-Darby canine kidney (MDCK) cells, and Chinese hamster ovary cells involves furin-dependent cleavage at two sites in the extracellular loop of the ␣ subunit and at a single site within the extracellular loop of the ␥ subunit (8). Channels that lack proteolytic processing exhibit markedly reduced activity and enhanced inhibition by external Na ϩ , a process referred to as Na ϩ selfinhibition (9). ENaC subunit cleavage by furin or exogenous trypsin relieves channels from inhibition by external Na ϩ (9, 11). We previously proposed that furin-dependent proteolysis of the ␣ subunit activates ENaC by disassociating an inhibitory domain (␣Asp-206 -Arg-231) from its effector site within the channel complex (10).Endogenous proteases other than furin likely have a role in the processing and activation of ENaC. A number of serine proteases, referred to as "channel activating proteases," have been identified that increase ENaC activity when co-expressed with ENaC in heterologous expression systems (12-14). Furthermore, selective serine protease inhibitors that do not block furin, such as aprotinin and bikunin, reduce ENaC activity (14 -20). Prostasin is an aprotinin-sensitive "channel activating (serine) protease" that inc...

In the early days of epithelial cell biology, researchers working with kidney and/or intestinal epithelial cell lines and with hepatocytes described the biosynthetic and recycling routes followed by apical and basolateral plasma membrane (PM) proteins. They identified the trans-Golgi network and recycling endosomes as the compartments that carried out apical-basolateral sorting. They described complex apical sorting signals that promoted association with lipid rafts, and simpler basolateral sorting signals resembling clathrin-coated-pit endocytic motifs. They also noticed that different epithelial cell types routed their apical PM proteins very differently, using either a vectorial (direct) route or a transcytotic (indirect) route. Although these original observations have generally held up, recent studies have revealed interesting complexities in the routes taken by apically destined proteins and have extended our understanding of the machinery required to sustain these elaborate sorting pathways. Here, we critically review the current status of apical trafficking mechanisms and discuss a model in which clustering is required to recruit apical trafficking machineries. Uncovering the mechanisms responsible for polarized trafficking and their epithelial-specific variations will help understand how epithelial functional diversity is generated and the pathogenesis of many human diseases.

Newly synthesized basolateral markers can traverse recycling endosomes en route to the surface of Madin-Darby canine kidney cells; however, the routes used by apical proteins are less clear. Here, we functionally inactivated subsets of endocytic compartments and examined the effect on surface delivery of the basolateral marker vesicular stomatitis virus glycoprotein (VSV-G), the raft-associated apical marker influenza hemagglutinin (HA), and the non-raft-associated protein endolyn. Inactivation of transferrin-positive endosomes after internalization of horseradish peroxidase (HRP)-containing conjugates inhibited VSV-G delivery, but did not disrupt apical delivery. In contrast, inhibition of protein export from apical recycling endosomes upon expression of dominant-negative constructs of myosin Vb or Sec15 selectively perturbed apical delivery of endolyn. Ablation of apical endocytic components accessible to HRP-conjugated wheat germ agglutinin (WGA) disrupted delivery of HA but not endolyn. However, delivery of glycosylphosphatidylinositolanchored endolyn was inhibited by 450% under these conditions, suggesting that the biosynthetic itinerary of a protein is dependent on its targeting mechanism. Our studies demonstrate that apical and basolateral proteins traverse distinct endocytic intermediates en route to the cell surface, and that multiple routes exist for delivery of newly synthesized apical proteins.