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

Abstract

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.

Structure and Function of CLCA Proteins

“…In an interesting aside, hCLCA1 expression was not detected by RT-PCR in a human pancreatic duct adenocarcinoma cell line that had an active calcium-dependent chloride conductance (56). This negative finding is consistent with observations by others that CLCA expression does not always correlate with the presence of calcium-activated chloride currents (114).…”

“…Expression of pCLCA1 changed the chloride conductance activated by calcium ionophore or PKA agonists in Caco-2 epithelial cells to a time-dependent outwardly rectifying current (114). However, the endogenous Ca 2ϩ -activated chloride conductance is lost as Caco-2 cells mature, and the ability of pCLCA1 to activate Ca 2ϩ -dependent chloride conductance also disappears (114).…”

“…However, the endogenous Ca 2ϩ -activated chloride conductance is lost as Caco-2 cells mature, and the ability of pCLCA1 to activate Ca 2ϩ -dependent chloride conductance also disappears (114). Again, the endogenous chloride channel activity in the expression system influences the properties of the chloride channels observed upon CLCA expression.…”

Murine mCLCA6 Is an Integral Apical Membrane Protein of Non-goblet Cell Enterocytes and Co-localizes With the Cystic Fibrosis Transmembrane Conductance Regulator

“…For example, murine mCLCA1 and 2 have been found only in the mouse without any orthologs in the human genome (Ritzka et al 2003). Also, different functional properties have been reported for direct orthologs of different species (Gruber et al 1998a;Loewen et al 2004). Therefore, caution is warranted when results on CLCA family members are translated to another species including the model proposed here for the intestine (Figure 8).…”

Genomic, Tissue Expression, and Protein Characterization of pCLCA1, a Putative Modulator of Cystic Fibrosis in the Pig

“…Our detailed analyses of the genomic organization of porcine CLCA genes failed to identify any significant differences between the pCLCA1 gene and the genes encoding its human and murine orthologs, hCLCA1 and mCLCA3 alias gob-5 (Gruber et al 1998a;Ritzka et al 2003). The lack of two amino acids in both the cloned cDNA sequence and the genomic BAC clones at positions 525 and 526 when compared with the published mRNA sequence of pCLCA1 (GenBank accession number NM_214148; Loewen et al 2004) is probably due to allelic variations that have previously been described for hCLCA1 and eCLCA1 in humans and horses, respectively (Kamada et al 2004;Anton et al 2005). Whether these allelic variations are accompanied by functional differences, as has been suggested for the allelic variants of hCLCA1 and hCLCA4, remains to be established (Ritzka et al 2004).…”