Signaling to the mechanistic target of rapamycin (mTOR) regulates diverse cellular processes, including protein translation, cellular proliferation, metabolism, and autophagy. These effects are mediated in part by the mTOR targets S6 kinase (S6K) and eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). Most models place Akt upstream of the best-studied mTOR complex, mTORC1; however, studies have called into question whether Akt is necessary for this pathway, at least in T cells. We found that the adaptor protein Carma1 [caspase recruitment domain (CARD)-containing membrane-associated protein 1 (Carma1)] and at least one of its associated proteins, the paracaspase MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1), were required for optimal activation of mTOR in T cells in response to stimulation of the T cell receptor (TCR) and the coreceptor CD28. However, another common binding partner of Carma1 and MALT1, Bcl10, was not required for TCR-dependent activation of the mTOR pathway. Consistent with these findings, MALT1 activity was required for the proliferation of CD4 + T cells, but not early TCR-dependent activation events. Also consistent with an effect on mTOR, MALT1 activity was required for the increased metabolic flux in activated CD4 + T cells. Together, our data suggest that Carma1 and MALT1 play previously unappreciated roles in the activation of mTOR signaling in T cells after engagement of the TCR.

We demonstrate that the C-terminal truncation of hIK1 results in a loss of functional channels. This could be caused by either (i) a failure of the channel to traffic to the plasma membrane or (ii) the expression of nonfunctional channels. To delineate among these possibilities, a hemagglutinin epitope was inserted into the extracellular loop between transmembrane domains S3 and S4. Surface expression and channel function were measured by immunofluorescence, cell surface immunoprecipitation, and whole-cell patch clamp techniques. Although deletion of the last 14 amino acids of hIK1 (L414STOP) had no effect on plasma membrane expression and function, deletion of the last 26 amino acids (K402STOP) resulted in a complete loss of membrane expression. Mutation of the leucine heptad repeat ending at Leu 406 (L399A/L406A) completely abrogated membrane localization. Additional mutations within the heptad repeat (L385A/L392A, L392A/L406A) or of the a positions (I396A/L403A) resulted in a near-complete loss of membrane-localized channel. In contrast, mutating individual leucines did not compromise channel trafficking or function. Both membrane localization and function of L399A/L406A could be partially restored by incubation at 27°C. Co-immunoprecipitation studies demonstrated that leucine zipper mutations do not compromise multimer formation. In contrast, we demonstrated that the leucine zipper region of hIK1 is capable of co-assembly and that this is dependent upon an intact leucine zipper. Finally, this leucine zipper is conserved in another member of the gene family, SK3. However, mutation of the leucine zipper in SK3 had no effect on plasma membrane localization or function. In conclusion, we demonstrate that the C-terminal leucine zipper is critical to facilitate correct folding and plasma membrane trafficking of hIK1, whereas this function is not conserved in other gene family members.

Counter-regulation afforded by specialized regulatory cell populations and immunosuppressive cytokines is critical for balancing immune outcome. The inhibitory potential of the established suppressive cytokines, IL-10 and TGFβ, has been well elucidated in diverse inflammatory scenarios in conjunction with their key roles in Treg development and function. Despite the early predictions for an immunomodulatory role for the Ebi3/p35 heterodimer in placental trophoblasts, IL-35 biology remained elusive until 2007 when it was established as a Treg-restricted inhibitory cytokine. Since then, Treg-derived IL-35 has been shown to exhibit its suppressive activities in a range of autoimmune diseases and cancer models. Recent studies are beginning to explore other cellular sources of IL-35, such as Bregs and CD8(+) Tregs. Despite these new cellular sources and targets, the mode of IL-35 suppression remains restricted to inhibition of proliferation and induction of an IL-35-producing induced regulatory T cell population referred to as iTr35. In this review, we explore the early beginnings, status quo, and future prospects of IL-35 biology. The unparalleled opportunity of targeting multiple immunosuppressive populations (Tregs, Bregs, CD8(+) Tregs) through IL-35 is highly exciting and offers tremendous promise from a translational standpoint, particularly for cancer immunotherapies.

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The intermediate conductance, Ca2+-activated K+ channel (KCa3.1) targets to the basolateral (BL) membrane in polarized epithelia where it plays a key role in transepithelial ion transport. However, there are no studies defining the anterograde and retrograde trafficking of KCa3.1 in polarized epithelia. Herein, we utilize Biotin Ligase Acceptor Peptide (BLAP)-tagged KCa3.1 to address these trafficking steps in polarized epithelia, using MDCK, Caco-2 and FRT cells. We demonstrate that KCa3.1 is exclusively targeted to the BL membrane in these cells when grown on filter supports. Following endocytosis, KCa3.1 degradation is prevented by inhibition of lysosomal/proteosomal pathways. Further, the ubiquitylation of KCa3.1 is increased following endocytosis from the BL membrane and PR-619, a deubiquitylase inhibitor, prevents degradation, indicating KCa3.1 is targeted for degradation by ubiquitylation. We demonstrate that KCa3.1 is targeted to the BL membrane in polarized LLC-PK1 cells which lack the μ1B subunit of the AP-1 complex, indicating BL targeting of KCa3.1 is independent of μ1B. As Rabs 1, 2, 6 and 8 play roles in ER/Golgi exit and trafficking of proteins to the BL membrane, we evaluated the role of these Rabs in the trafficking of KCa3.1. In the presence of dominant negative Rab1 or Rab8, KCa3.1 cell surface expression was significantly reduced, whereas Rabs 2 and 6 had no effect. We also co-immunoprecipitated KCa3.1 with both Rab1 and Rab8. These results suggest these Rabs are necessary for the anterograde trafficking of KCa3.1. Finally, we determined whether KCa3.1 traffics directly to the BL membrane or through recycling endosomes in MDCK cells. For these studies, we used either recycling endosome ablation or dominant negative RME-1 constructs and determined that KCa3.1 is trafficked directly to the BL membrane rather than via recycling endosomes. These results are the first to describe the anterograde and retrograde trafficking of KCa3.1 in polarized epithelia cells.

We report here the first evidence in intact epithelial cells of unit conductance events from an amiloride-sensitive Na+ channel. The events were observed when patch-clamp recordings were made from the apical surface of cultured epithelial kidney cells (A6). The channel characteristics are as follows. Single-channel conductance ranged between 7 and 10 pS (mean = 8.4 +/- 1.3), the current-voltage (I-V) relationship displayed little if any nonlinearity over a range of +/- 80 mV (with respect to the patch pipette), and the channel Na+/K+ selectivity was approximately 3-4:1. Amiloride, a cationic blocker of the channel, reduced channel mean open time and increased channel mean closed time as the voltage of the cell interior was made more negative. Amiloride induced channel flickering at increased negative potentials (intracellular potential with respect to the patch) but did not alter the single-channel conductance or the I-V relationship from that observed in control patches.

The role of the NH 2 -terminal leucine zipper and dileucine motifs of hIK1 in the assembly, trafficking, and function of the channel was investigated using cell surface immunoprecipitation, co-immunoprecipitation (Co-IP), immunoblot, and whole-cell patch clamp techniques. Mutation of the NH 2 -terminal leucine zipper at amino acid positions 18 and 25 (L18A/L25A) resulted in a complete loss of steady-state protein expression, cell surface expression, and whole-cell current density. Inhibition of proteasomal degradation with lactacystin restored L18A/L25A protein expression, although this channel was not expressed at the cell surface as assessed by cell surface immunoprecipitation and wholecell patch clamp. In contrast, inhibitors of lysosomal degradation (leupeptin/pepstatin) and endocytosis (chloroquine) had little effect on L18A/L25A protein expression or localization. Further studies confirmed the rapid degradation of this channel, having a time constant of 19.0 ؎ 1.3 min compared with 3.2 ؎ 0.8 h for wild type hIK1. Co-expression studies demonstrated that the L18A/L25A channel associates with wild type channel, thereby attenuating its expression at the cell surface. Co-IP studies confirmed this association. However, L18A/L25A channels failed to form homotetrameric channels, as assessed by Co-IP, suggesting the NH 2 terminus plays a role in tetrameric channel assembly. As with the leucine zipper, mutation of the dileucine motif to alanines, L18A/L19A, resulted in a near complete loss in steady-state protein expression with the protein being similarly targeted to the proteasome for degradation. In contrast to our results on the leucine zipper, however, both chloroquine and growing the cells at the permissive temperature of 27°C restored expression of L18A/L19A at the cell surface, suggesting that the defect in the channel trafficking is the result of a subtle folding error. In conclusion, we demonstrate that the NH 2 terminus of hIK1 contains overlapping leucine zipper and dileucine motifs essential for channel assembly and trafficking to the plasma membrane.The KCNN gene family is composed of four members, including the small conductance Ca 2ϩ -activated K ϩ channels (SK1, SK2, and SK3) 1 and the intermediate conductance Ca 2ϩ -activated K ϩ channel (IK1 or SK4). While the IK1 and SK channels share roughly 40% homology, their expression patterns and pharmacology are widely disparate, consistent with their unique physiological functions (1). The human IK1 channel (hIK1) is widely expressed, being found in salivary gland, colon, bladder, stomach, lung, smooth muscle, red blood cells, T-cells, and placenta (2, 3) where it plays a crucial role in a variety of physiological functions. Indeed hIK1 plays a seminal role in modulating Ca 2ϩ -dependent transepithelial ion transport (4, 5), has recently been confirmed to be the Gardos channel of red blood cells (6), and has been proposed to play a role in both vascular remodeling (7) and in modulating vascular tone (8). Because of these critical physiological roles, the ...

The major function of epithelial tissues is to maintain proper ion, solute, and water homeostasis. The tubule of the renal nephron has an amazingly simple structure, lined by epithelial cells, yet the segments (i.e., proximal tubule vs. collecting duct) of the nephron have unique transport functions. The functional differences are because epithelial cells are polarized and thus possess different patterns (distributions) of membrane transport proteins in the apical and basolateral membranes of the cell. K(+) channels play critical roles in normal physiology. Over 90 different genes for K(+) channels have been identified in the human genome. Epithelial K(+) channels can be located within either or both the apical and basolateral membranes of the cell. One of the primary functions of basolateral K(+) channels is to recycle K(+) across the basolateral membrane for proper function of the Na(+)-K(+)-ATPase, among other functions. Mutations of these channels can cause significant disease. The focus of this review is to provide an overview of the basolateral K(+) channels of the nephron, providing potential physiological functions and pathophysiology of these channels, where appropriate. We have taken a "K(+) channel gene family" approach in presenting the representative basolateral K(+) channels of the nephron. The basolateral K(+) channels of the renal epithelia are represented by members of the KCNK, KCNJ, KCNQ, KCNE, and SLO gene families.