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MUCOSAL BIOLOGY

pCLCA1 lacks inherent chloride channel activity in an epithelial colon carcinoma cell line

¹Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5B4; ²Department of Physiology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E5; and ³Department of Pediatrics, University of North Carolina, Chapel Hill, North Carolina 27599-7020

Submitted 15 January 2004 ; accepted in final form 18 February 2004

The effects of CLCA protein expression on the regulation of Cl^– conductance by intracellular Ca²⁺ and cAMP have been studied previously in nonepithelial cell lines chosen for low backgrounds of endogenous Cl^– conductance. However, CLCA proteins have been cloned from, and normally function in, differentiated epithelial cells. In this study, we examine the effects of differentiation of the Caco-2 epithelial colon carcinoma cell line on modulation of Cl^– conductance by pCLCA1 protein expression. Cl^– transport was measured as ³⁶Cl^– efflux, as transepithelial short-circuit currents, and as whole cell patch-clamp current-voltage relations. The rate of ³⁶Cl^– efflux and amplitude of currents in patch-clamp studies after the addition of the Ca²⁺ ionophore A-23187 were increased significantly by pCLCA1 expression in freshly passaged Caco-2 cells. However, neither endogenous nor pCLCA1-dependent Ca²⁺-sensitive Cl^– conductance could be detected in 14-day-postpassage cells. In contrast to Ca²⁺-sensitive Cl^– conductance, endogenous cAMP-dependent Cl^– conductance does not disappear on Caco-2 differentiation. cAMP-dependent Cl^– conductance was modulated by pCLCA1 expression in Caco-2 cells, and this modulation was observed in freshly passaged and in mature 14-day-postpassage Caco-2 cultures. pCLCA1 mRNA expression, antigenic pCLCA1 protein epitope expression, and pCLCA1 function as a modulator of cAMP-dependent Cl^– conductance were retained through differentiation in Caco-2 cells, whereas Ca²⁺-dependent Cl^– conductance disappeared. We conclude that pCLCA1 expression may increase the sensitivity of preexisting endogenous Cl^– channels to Ca²⁺ and cAMP agonists but apparently lacks inherent Cl^– channel activity under growth conditions where endogenous channels are not expressed.

cystic fibrosis transmembrane conductance regulator; cyclic adenosine 5'-monophosphate; ionomycin; calcium

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