Abstract
SLC26A6 (also known as putative anion transporter 1 [PAT1]) is a Cl–/HCO3– exchanger expressed at the luminal membrane of enterocytes where it facilitates intestinal Cl– and fluid absorption. Here, high-throughput screening of 50,000 synthetic small molecules in cells expressing PAT1 and a halide-sensing fluorescent protein identified several classes of inhibitors. The most potent compound, the pyrazolo-pyrido-pyrimidinone PAT1inh-B01, fully inhibited PAT1-mediated anion exchange (IC50 ~350 nM), without inhibition of the related intestinal transporter SLC26A3 (also known as DRA). In closed midjejunal loops in mice, PAT1inh-B01 inhibited fluid absorption by 50%, which increased to >90% when coadministered with DRA inhibitor DRAinh-A270. In ileal loops, PAT1inh-B01 blocked fluid absorption by >80%, whereas DRAinh-A270 was without effect. In colonic loops, PAT1inh-B01 was without effect, whereas DRAinh-A270 completely blocked fluid absorption. In a loperamide constipation model, coadministration of PAT1inh-B01 with DRAinh-A270 increased stool output compared with DRAinh-A270 alone. These results provide functional evidence for complementary and region-specific roles of PAT1 and DRA in intestinal fluid absorption, with PAT1 as the predominant anion exchanger in mouse ileum. We believe that PAT1inh-B01 is a novel tool to study intestinal ion and fluid transport and perhaps a drug candidate for small intestinal hyposecretory disorders such as cystic fibrosis–related meconium ileus and distal intestinal obstruction syndrome.
Highlights
Electroneutral NaCl absorption in the small intestine, which drives fluid absorption, is mediated by the parallel actions of Na+/H+ and Cl–/HCO3– exchangers
A selective inhibitor of PAT1-mediated anion exchange was identified and characterized in order to investigate the relative contributions of PAT1 and DRA to fluid absorption in different regions of mouse intestine
An early study showed strong PAT1 mRNA expression throughout the small intestine in mice, with no expression in the colon [5], whereas DRA mRNA was highly expressed in the colon with low-level expression throughout the small intestine
Summary
Electroneutral NaCl absorption in the small intestine, which drives fluid absorption, is mediated by the parallel actions of Na+/H+ and Cl–/HCO3– exchangers. Due to the lack of selective inhibitors, current understanding of the functional roles of PAT1 and DRA in intestinal fluid transport is mainly from studies in knockout mice. Some earlier studies suggested PAT1 as the predominant anion exchanger in small intestine [4, 5], while another study reported DRA expression in small intestine and loss of small intestinal Cl– absorption in DRA-knockout mice [6]. This difference may be the consequence of a combination of factors, including the lack of selective inhibitors, confounding effects of compensatory mechanisms in knockout mice, and possible species/strain differences
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