Herein, we demonstrated by Ussing chamber technique that male mice administered 1 g/kg 1,25(OH) 2D3 sc daily for 3 days exhibited increased duodenal calcium absorption, which was abolished by concurrent intravenous injection of recombinant mouse FGF-23. This FGF-23 administration had no effect on the background epithelial electrical properties, i.e., short-circuit current, transepithelial potential difference, and resistance. Immunohistochemical evidence of protein expressions of FGFR isoforms 1-4 in mouse duodenal epithelial cells suggested a possible direct effect of FGF-23 on the intestine. This was supported by the findings that FGF-23 directly added to the serosal compartment of the Ussing chamber and completely abolished the 1,25(OH) 2D3-induced calcium absorption in the duodenal tissues taken from the 1,25(OH) 2D3-treated mice. However, direct FGF-23 exposure did not decrease the duodenal calcium absorption without 1,25(OH) 2D3 preinjection. The observed FGF-23 action was mediated by MAPK/ERK, p38 MAPK, and PKC. Quantitative real-time PCR further showed that FGF-23 diminished the 1,25(OH) 2D3-induced upregulation of TRPV5, TRPV6, and calbindin-D 9k, but not PMCA1b expression in the duodenal epithelial cells. In conclusion, besides being a phosphatonin, FGF-23 was shown to be a novel calcium-regulating hormone that acted directly on the mouse intestine, thereby compromising the 1,25(OH) 2D3-induced calcium absorption.calbindin-D 9k; fibroblast growth factor receptor; Klotho; transient receptor potential vanilloid type 6; Ussing chamber FIBROBLAST GROWTH FACTOR (FGF)-23 has been recognized as the osteoblast/osteocyte-derived phosphate-regulating hormone, a phosphatonin with phosphaturic and hypophosphatemic action (19,42,45 (29). Some hereditary and acquired diseases, e.g., autosomal dominant hypophosphatemic rickets/osteomalacia, tumor-induced osteomalacia, and X-linked hypophosphatemic rickets, result from abnormally high circulating FGF-23 activity. Dysregulation of FGF-23 action is also evident in various pathological conditions, such as chronic metabolic acidosis and chronic kidney disease (11,20).Once secreted from osteoblasts and osteocytes, FGF-23 exerts its phosphaturic action in the renal proximal tubular cells via the FGF receptor (FGFR)/Klotho coreceptor complex, thereby downregulating Na ϩ -dependent phosphate transporter (NPT)-2a and NPT-2c expression (14). Its intracellular signaling in renal epithelial cells is conveyed through a number of pathways, e.g., mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), p38 MAPK, phosphoinositide 3-kinase (PI3K)/Akt, and protein kinase C (PKC) (12,13,43). FGF-23 also downregulates renal 25-hydroxyvitamin D 1␣-hydroxylase (1-OHase; also known as Cyp27b1) and upregulates 24-hydroxylase (24-OHase, Cyp24a1), which are important enzymes for production and inactivation of 1,25(OH) 2 D 3 , respectively, thereby reducing circulating levels of 1,25(OH) 2 D 3 (35, 42). Since 1,25(OH) 2 D 3 is the cardinal regulator of intestinal cal...

Key pointsr Malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR), a gated pathway for chloride movement, causes the common life-shortening genetic disease cystic fibrosis (CF).r Towards the development of a sheep model of CF, we have investigated the function of sheep CFTR.r We found that sheep CFTR was noticeably more active than human CFTR, while the most common CF mutation, F508del, had reduced impact on sheep CFTR function.r Our results demonstrate that subtle changes in protein structure have marked effects on CFTR function and the consequences of the CF mutation F508del.Abstract Cross-species comparative studies are a powerful approach to understanding the epithelial Cl − channel cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in the genetic disease cystic fibrosis (CF). Here, we investigate the single-channel behaviour of ovine CFTR and the impact of the most common CF mutation, F508del-CFTR, using excised inside-out membrane patches from transiently transfected CHO cells. Like human CFTR, ovine CFTR formed a weakly inwardly rectifying Cl − channel regulated by PKA-dependent phosphorylation, inhibited by the open-channel blocker glibenclamide. However, for three reasons, ovine CFTR was noticeably more active than human CFTR. First, single-channel conductance was increased. Second, open probability was augmented because the frequency and duration of channel openings were increased. Third, with enhanced affinity and efficacy, ATP more strongly stimulated ovine CFTR channel gating. Consistent with these data, the CFTR modulator phloxine B failed to potentiate ovine CFTR Cl − currents. Similar to its impact on human CFTR, the F508del mutation caused a temperature-sensitive folding defect, which disrupted ovine CFTR protein processing and reduced membrane stability. However, the F508del mutation had reduced impact on ovine CFTR channel gating in contrast to its marked effects on human CFTR. We conclude that ovine CFTR forms a regulated Cl − channel with enhanced conductance and ATP-dependent channel gating. This phylogenetic analysis of CFTR structure and function demonstrates that subtle changes in structure have pronounced effects on channel function and the consequences of the CF mutation F508del.

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