Abstract

The Na/H exchanger 3 (NHE3) and the Cl/HCO(3) exchanger down-regulated in adenoma (DRA) together facilitate intestinal electroneutral NaCl absorption. Elevated Ca(2+)(i) inhibits NHE3 through mechanisms involving the PDZ domain proteins NHE3 kinase A regulatory protein (E3KARP) or PDZ kidney 1 (PDZK1). DRA also possesses a PDZ-binding motif, but the roles of interactions with E3KARP or PDZK1 and Ca(2+)(i) in DRA regulation are unknown. Wild type DRA and a mutant lacking the PDZ interaction motif (DRA-ETKFminus) were expressed constitutively in human embryonic kidney (HEK) and inducibly in Caco-2/BBE cells. DRA-mediated Cl/HCO(3) exchange was measured as intracellular pH changes. Ca(2+)(i) was assessed fluorometrically. DRA was induced 8-16-fold and was delivered to the apical surface of polarized Caco-2 cells. Putative anion transporter 1 and cystic fibrosis transmembrane regulator did not contribute to Cl/HCO(3) exchange in transfected Caco-2 cells. The calcium ionophore 4Br-A23187 inhibited DRA and DRA-ETKFminus in HEK cells, but only full-length DRA was inhibited in Caco-2 cells. In contrast, 100 microm UTP, which increased Ca(2+)(i), inhibited full-length DRA but not DRA-ETKFminus in Caco-2 and HEK cells. In HEK cells, which express little PDZK1, additional transfection of PDZK1 was required for UTP to inhibit DRA. As HEK cells do not express cystic fibrosis transmembrane regulator or NHE3, the data indicate that Ca(2+)(i)-dependent DRA inhibition is not because of modulation of other transport activities. In polarized epithelium, this inhibition requires interaction of DRA with PDZK1. Together with data from PDZK1(-/-) mice, these data underscore the prominent role of PDZK1 in Ca(2+)(i)-mediated inhibition of colonic NaCl absorption.

Highlights

  • 1066/3-2 and La 1066/3-3. 1 To whom correspondence should be addressed

  • In human embryonic kidney (HEK) Cells Increased Intracellular Calcium Inhibits down-regulated in adenoma (DRA) Independently of Its PDZ Interaction Motif—Calcium-dependent short term regulation of Na/H exchanger 3 (NHE3) has been studied in transfected PS120 cells [19, 22]

  • This study was undertaken to test whether DRA is regulated by elevated intracellular calcium and to determine whether this involves interactions with PDZ adapter proteins

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Summary

Introduction

1066/3-2 and La 1066/3-3. 1 To whom correspondence should be addressed. Tel.: 49-7071-2987108; Fax: SLC9A3) and DRA (down-regulated in adenoma; SLC26A3) are the primary transporters responsible for these events [1, 2]. In HEK Cells Increased Intracellular Calcium Inhibits DRA Independently of Its PDZ Interaction Motif—Calcium-dependent short term regulation of NHE3 has been studied in transfected PS120 cells [19, 22].

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