Segmental differences in Slc26a3-dependent Cl− absorption and HCO3− secretion in the mouse large intestine in vitro in Ussing chambers

The anion exchanger slc26a3 (DRA), which is mutated in congenital chloride-losing diarrhea, is expressed in the apical membrane of the cecum and middle-distal colon but not in the proximal colon of rodent large intestines. To elucidate the functional roles of DRA, we measured unidirectional 36Cl− and 22Na+ fluxes and HCO3− secretion in vitro in each of these segments using DRA-KO mice. Robust Cl− absorption, which was largely abolished after DRA deficiency, was present in the cecum and middle-distal colon but absent in the proximal colon. Na+ absorption was present in all three segments in both the control and DRA-KO mice. The luminal-Cl−-dependent HCO3− secretions in the cecum and middle-distal colon were abolished in the DRA-KO mice. In conclusion, DRA mediates Cl− absorption and HCO3− secretion in the mouse cecum and middle-distal colon, and may have roles in H2O absorption and luminal acid/base regulation in these segments.

A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome

We have developed Cappable-seq that specifically captures primary RNA transcripts by enzymatically modifying the 5' triphosphorylated end of RNA with a selectable tag. We first applied Cappable-seq to E. coli , achieving up to 50 fold enrichment of primary transcripts and identifying an unprecedented 16539 transcription start sites (TSS) genome-wide at single base resolution. We also applied Cappable-seq to a mouse cecum sample and for the first time identified TSS in a microbiome. Furthermore, Cappable-seq universally depletes ribosomal RNA and reduces the complexity of the transcriptome to a single quantifiable tag per TSS enabling digital profiling of gene expression in any microbiome.

Sulfate secretion and chloride absorption are mediated by the anion exchanger DRA (Slc26a3) in the mouse cecum

Inorganic sulfate (SO42−) is essential for a multitude of physiological processes. The specific molecular pathway has been identified for uptake from the small intestine but is virtually unknown for the large bowel, although there is evidence for absorption involving Na+-independent anion exchange. A leading candidate is the apical chloride/bicarbonate (Cl−/HCO3−) exchanger DRA (down-regulated in adenoma; Slc26a3), primarily linked to the Cl− transporting defect in congenital chloride diarrhea. The present study set out to characterize transepithelial 35SO42− and 36Cl− fluxes across the isolated, short-circuited cecum from wild-type (WT) and knockout (KO) mice and subsequently to define the contribution of DRA. The cecum demonstrated simultaneous net SO42− secretion (−8.39 ± 0.88 nmol·cm−2·h−1) and Cl− absorption (10.85 ± 1.41 μmol·cm−2·h−1). In DRA-KO mice, SO42− secretion was reversed to net absorption via a 60% reduction in serosal to mucosal SO42− flux. Similarly, net Cl− absorption was abolished and replaced by secretion, indicating that DRA represents a major pathway for transcellular SO42− secretion and Cl− absorption. Further experiments including the application of DIDS (500 μM), bumetanide (100 μM), and substitutions of extracellular Cl− or HCO3−/CO2 helped to identify specific ion dependencies and driving forces and suggested that additional anion exchangers were operating at both apical and basolateral membranes supporting SO42− transport. In conclusion, DRA contributes to SO42− secretion via DIDS-sensitive HCO3−/SO42− exchange, in addition to being the principal DIDS-resistant Cl−/HCO3− exchanger. With DRA linked to the pathogenesis of other gastrointestinal diseases extending its functional characterization offers a more complete picture of its role in the intestine.