Introduction: 

Congenital chloride diarrhea is a rare disease endemic in Finland. It is due to loss of function of the anion exchanger DRA (down regulated in adenoma, SLC26A3), which is involved in electroneutral NaCl absorption in the ileum and colon. Inhibition of NaCl absorption occurs in other diarrheal diseases and may involve elevated [Ca]_i. It is unknown whether DRA is inhibited by [Ca]_i or whether its function follows the inhibition of NHE3, to which it is functionally and structurally coupled

Aim: 

Is DRA inhibited by elevated [Ca]_i? Does this involve changes in cell surface expression? What is the role of the C-terminal PDZ interaction motif ETKF?

Methods: 

In EGFP-DRA or EGFP-DRA-ETKFminus transfected HEK and Caco-2/BBE cells [Ca]_i was elevated by 4Br-A23187 or by 100 mM UTP. Surface expression was quantified by biotinylation.

Results: 

In DRA-transfected HEK and Caco-2/BBE cells DRA is inhibited by elevated [Ca]_i (100 sec 4Br-A23187) independently of its PDZ interaction (45 %, P< 0.05). With a time delay surface expression is reduced (0, 10, 30 min: 34 %, 22 %, 10 %, p< 0.05). In DRA but not in DRA-ETKFminus transfected Caco-2/BBE cells UTP inhibits DRA (15 %, p < 0.05). HEK cells have a lower expression of the PDZ proteins E3KARP and PDZK1 than Caco-2 cells. Transient transfection of HEK/EGFP-DRA cells with PDZK1 establishes inhibition by UTP.

Conclusion: 

At non-physiologic elevations of [Ca]_i by 4Br-A23187 DRA is inhibited and its surface expression is reduced independently of its PDZ interaction. Physiologic elevation of [Ca]_i by UTP causes PDZ dependent inhibition of DRA in Caco-2/BBE cells. This can be recapitulated in HEK cells that are transfected with PDZK1 suggesting that this adapter protein plays an important role in the transduction of the Ca signal.