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J. Biol. Chem., Vol. 261, Issue 2, 704-712, Jan, 1986

Characterization of a cyclic AMP-activated Cl-transport pathway in the apical membrane of a human colonic epithelial cell line

KG Mandel, K Dharmsathaphorn and JA McRoberts

This report describes a Cl- transport pathway in confluent monolayer cultures of the T84 human colonic carcinoma cell line which is: 1) activated by vasoactive intestinal polypeptide, or other agents which induce or mimic cAMP; 2) independent of extracellular Na+ or K+; 3) refractory to inhibition by 0.1 mM bumetanide and 1 mM 4-acetamido-4'- isothiocyanostilbene-2,-2'-disulfonic acid; 4) competitively inhibited by NO3-, I-, SCN-, and Br-; 5) inhibited in a noncompetitive-complex manner by the putative Cl- channel-blocking agent, N-phenylanthranilic acid; and 6) localized to the apical membrane of confluent monolayers. This Cl- transport system is, therefore, distinct from the bumetanide- sensitive, basolateral membrane-localized, Na+, K+, Cl- cotransport system previously described in these cells (Dharmsathaphorn, K., Mandel, K., Masui, H., and McRoberts, J.A. (1985) J. Clin. Invest. 75, 462-471). Kinetic studies revealed that Cl- transport by this pathway fit simple Michaelis-Menten kinetics with an apparent Km for Cl- of about 6 mM. Activation by vasoactive intestinal polypeptide increased the Vmax but did not alter the apparent Km. We discuss the possibility that this transport system is a Cl- channel which is intimately involved in hormonally mediated, electrogenic Cl- secretion across T84 cell monolayers.
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