In mammals, the extracellular calcium concentration is maintained within a narrow range despite large variations in daily dietary input and body demand. The small intestine and kidney constitute the influx pathways into the extracellular Ca 2؉ pool and, therefore, play a primary role in Ca 2؉ homeostasis. We identified an apical Ca 2؉ influx channel, which is expressed in proximal small intestine, the distal part of the nephron and placenta. This novel epithelial Ca 2؉ channel (ECaC) of 730 amino acids contains six putative membranespanning domains with an additional hydrophobic stretch predicted to be the pore region. ECaC resembles the recently cloned capsaicin receptor and the transient receptor potential-related ion channels with respect to its predicted topology but shares less than 30% sequence homology with these channels. In kidney, ECaC is abundantly present in the apical membrane of Ca 2؉ transporting cells and colocalizes with 1,25-dihydroxyvitamin D 3 -dependent calbindin-D 28K . ECaC expression in Xenopus oocytes confers Ca 2؉ influx with properties identical to those observed in distal renal cells. Thus, ECaC has the expected properties for being the gatekeeper of 1,25-dihydroxyvitamin D 3 -dependent active transepithelial Ca 2؉ transport.

TRPV5 and TRPV6 constitute the Ca 2+ in¯ux pathway in a variety of epithelial cells. Here, we identi®ed S100A10 as the ®rst auxiliary protein of these epithelial Ca 2+ channels using yeast two-hybrid and GST pull-down assays. This S100 protein forms a heterotetrameric complex with annexin 2 and associates speci®cally with the conserved sequence VATTV located in the C-terminal tail of TRPV5 and TRPV6. Of these ®ve amino acids, the ®rst threonine plays a crucial role since the corresponding mutants (TRPV5 T599A and TRPV6 T600A) exhibited a diminished capacity to bind S100A10, were redistributed to a subplasma membrane area and did not display channel activity. Using GST pull-down and co-immunoprecipitation assays we demonstrated that annexin 2 is part of the TRPV5±S100A10 complex. Furthermore, the S100A10±annexin 2 pair colocalizes with the Ca 2+ channels in TRPV5-expressing renal tubules and TRPV6-expressing duodenal cells. Importantly, downregulation of annexin 2 using annexin 2-speci®c small interfering RNA inhibited TRPV5 and TRPV6-mediated currents in transfected HEK293 cells. In conclusion, the S100A10±annexin 2 complex plays a crucial role in routing of TRPV5 and TRPV6 to plasma membrane.

Lipid overload and adipocyte dysfunction are key to the development of insulin resistance and can be induced by a high-fat diet. CD1d-restricted invariant natural killer T (iNKT) cells have been proposed as mediators between lipid overload and insulin resistance, but recent studies found decreased iNKT cell numbers and marginal effects of iNKT cell depletion on insulin resistance under high-fat diet conditions. Here, we focused on the role of iNKT cells under normal conditions. We showed that iNKT cell-deficient mice on a low-fat diet, considered a normal diet for mice, displayed a distinctive insulin resistance phenotype without overt adipose tissue inflammation. Insulin resistance was characterized by adipocyte dysfunction, including adipocyte hypertrophy, increased leptin, and decreased adiponectin levels. The lack of liver abnormalities in CD1d-null mice together with the enrichment of CD1d-restricted iNKT cells in both mouse and human adipose tissue indicated a specific role for adipose tissue-resident iNKT cells in the development of insulin resistance. Strikingly, iNKT cell function was directly modulated by adipocytes, which acted as lipid antigen-presenting cells in a CD1d-mediated fashion. Based on these findings, we propose that, especially under low-fat diet conditions, adipose tissue-resident iNKT cells maintain healthy adipose tissue through direct interplay with adipocytes and prevent insulin resistance.

Our data suggest that IgG1-mediated pro-inflammatory autoreactivity against annexin A11 in patients with IgG4-RD may be attenuated by formation of annexin A11-specific IgG4 antibodies supporting an anti-inflammatory role of IgG4 in IgG4-RD.

Members of the P 4 family of P-type ATPases (P 4 -ATPases) are believed to function as phospholipid flippases in complex with CDC50 proteins. Mutations in the human class 1 P 4 -ATPase gene ATP8B1 cause a severe syndrome characterized by impaired bile flow (intrahepatic cholestasis), often leading to end-stage liver failure in childhood. In this study, we determined the specificity of human class 1 P 4 -ATPase interactions with CDC50 proteins and the functional consequences of these interactions on protein abundance and localization of both protein classes. ATP8B1 and ATP8B2 co-immunoprecipitated with CDC50A and CDC50B, whereas ATP8B4, ATP8A1, and ATP8A2 associated only with CDC50A. ATP8B1 shifted from the endoplasmic reticulum (ER) to the plasma membrane upon coexpression of CDC50A or CDC50B. ATP8A1 and ATP8A2 translocated from the ER to the Golgi complex and plasma membrane upon coexpression of CDC50A, but not CDC50B. ATP8B2 and ATP8B4 already displayed partial plasma membrane localization in the absence of CDC50 coexpression but displayed a large increase in plasma membrane abundance upon coexpression of CDC50A. ATP8B3 did not bind CDC50A and CDC50B and was invariably present in the ER. Our data show that interactions between CDC50 proteins and class 1 P 4 -ATPases are essential for ER exit and stability of both subunits. Furthermore, the subcellular localization of the complex is determined by the P 4 -ATPase, not the CDC50 protein. The interactions of CDC50A and CDC50B with multiple members of the human P 4 -ATPase family suggest that these proteins perform broader functions in human physiology than thus far assumed.