Bacterial pathogens modulate host cell apoptosis to establish a successful infection. Pore-forming toxins (PFTs) secreted by pathogenic bacteria are major virulence factors and have been shown to induce various forms of cell death in infected cells. Here we demonstrate that the highly conserved caspase-2 is required for PFT-mediated apoptosis. Despite being the second mammalian caspase to be identified, the role of caspase-2 during apoptosis remains enigmatic. We show that caspase-2 functions as an initiator caspase during Staphylococcus aureus a-toxin-and Aeromonas aerolysinmediated apoptosis in epithelial cells. Downregulation of caspase-2 leads to a strong inhibition of PFT-mediated apoptosis. Activation of caspase-2 is PIDDosome-independent, and endogenous caspase-2 is recruited to a highmolecular-weight complex in a-toxin-treated cells. Interestingly, prevention of PFT-induced potassium efflux inhibits the formation of caspase-2 complex, leading to its inactivation, thus resisting apoptosis. These results revealed a thus far unknown, obligatory role for caspase-2 as an initiator caspase during PFT-mediated apoptosis.

Aldosterone binds to the mineralocorticoid receptor (MR) and exerts fine control over Na ϩ absorption in renal collecting duct cells (CCDs). Many natural and synthetic steroids can also bind to the MR to produce agonist or antagonist effects. Here, we investigate whether androgenic hormones act as MR agonist or antagonist ligands in CCDs. Testosterone (T), dihydrotestosterone (DHT), and methyltrienolone (R1881), a synthetic androgen agonist, all bind to the MR. R1881 displayed the same affinity for MR as aldosterone. Androgens did not activate the MR transiently expressed in human embryonic kidney 293T cells but did antagonize aldosterone-induced MR trans-activation activity (R1881ϾDHTϾT). Short-circuit current (I sc ) experiments, used to measure transepithelial Na ϩ transport, revealed that 10 Ϫ5 M T and DHT or R1881 prevented the increase in the amiloride-sensitive component of I sc caused by aldosterone in mouse mpkCCD cl4 collecting duct cells partially and totally, respectively. In contrast, androgens had no effect on stimulated I sc elicited by the specific glucocorticoid agonist 11␤,17␤-dihydroxy-17␣-(1-propynyl)androst-1,4,6-trien-3-one (RU26988). Docking of steroids within the crystal structure of the ligandbinding domain of MR, together with trans-activation studies, revealed that the contacts between the 17␤-hydroxyl group of androgens and the Asn770, Cys942, and Thr945 residues of the ligand-binding cavity stabilize ligand binding complexes but are not strong enough to keep the receptor in its active state. Altogether, these findings indicate that androgen ligands, particularly R1881, act as MR antagonists in aldosterone target cells and provide new insights into the requirements for MR activation to occur and for the designing of new selective MR antagonists.

The putative selectivity filter of the epithelial sodium channel (ENaC) comprises a three-residue sequence G/SXS, but it remains uncertain whether the backbone atoms of this sequence or whether their side chains are lining the pore. It has been reported that the S589C mutation in the selectivity filter of ␣ENaC renders the channel sensitive to block by externally applied Cd 2؉ ; this was interpreted as evidence for Cd 2؉ coordination with the thiol group of the side chain of ␣589C, pointing toward the pore lumen. Because the ␣S589C mutation alters the monovalent to divalent cation selectivity ratio of ENaC and because internally applied Cd 2؉ blocks wild-type ENaC with high affinity, we hypothesized that the inhibition of ␣S589C ENaC by Cd 2؉ results rather from the coordination of this cation with native cysteine residues located in the internal pore of ENaC. We show here that Cd 2؉ inhibits not only ENaC ␣S589C and ␣S589D but also ␣S589N mutants and that Ca 2؉ weakly interacts with the S589D mutant. The block of ␣S589C, -D, and -N mutants is characterized by a slow on-rate, is nearly irreversible, is voltage-dependent, and can be prevented by amiloride. The C546S mutation in the second transmembrane helix of ␥ subunit in the background of the ENaC ␣S589C, -D, or -N mutants reduces the sensitivity to block by Cd 2؉ and renders the block rapidly reversible. We conclude therefore that the block by Cd 2؉ of the ␣S589C, -D, and -N mutants results from the trapping of Cd 2؉ ions in the internal pore of the channel and involves Cys-546 in the second transmembrane helix of the ␥ENaC subunit.The epithelial sodium channel (ENaC) 3 mediates the Na ϩ ion influx across the apical membrane of tight epithelia such as the aldosterone-sensitive distal nephron, the distal colon, or the airways epithelium (1). In principal cells of the aldosteronesensitive distal nephron, the ENaC-mediated influx of Na ϩ ions constitutes the apical step of the transepithelial Na ϩ absorption and is regulated by aldosterone (2). The aldosterone-sensitive distal nephron plays an important role in the maintenance of Na ϩ homeostasis, the regulation of extracellular volume, and blood pressure (3). In the lungs, ENaC together with cystic fibrosis transmembrane conductance regulator controls the height of the airway surface liquid that forms a thin layer coating the airways epithelium, which contributes to the clearance of particles and microbes (4).Functional ENaC at the cell plasma membrane is a heteromeric protein made of homologous ␣, ␤, and ␥ subunits arranged pseudosymetrically around a central channel pore, presumably in a 2 ␣, 1 ␤, and 1 ␥ configuration (5, 6). Each ENaC subunit is made of two transmembrane helices (TM1 and TM2), a large extracellular loop that represents more than half of the mass of the protein, and intracellular N and C termini. The ENaC subunits belong to the ENaC/degenerin superfamily of ion channels but share no sequence homology with other tetrameric channels also made of two transmembrane domains, such as inward rect...