ClC-2 Contributes to Native Chloride Secretion by a Human Intestinal Cell Line, Caco-2

Abstract

It has been previously determined that ClC-2, a member of the ClC chloride channel superfamily, is expressed in certain epithelial tissues. These findings fueled speculation that ClC-2 can compensate for impaired chloride transport in epithelial tissues affected by cystic fibrosis and lacking the cystic fibrosis transmembrane conductance regulator. However, direct evidence linking ClC-2 channel expression to epithelial chloride secretion was lacking. In the present studies, we show that ClC-2 transcripts and protein are present endogenously in the Caco-2 cell line, a cell line that models the human small intestine. Using an antisense strategy we show that ClC-2 contributes to native chloride currents in Caco-2 cells measured by patch clamp electrophysiology. Antisense ClC-2-transfected monolayers of Caco-2 cells exhibited less chloride secretion (monitored as iodide efflux) than did mock transfected monolayers, providing the first direct molecular evidence that ClC-2 can contribute to chloride secretion by the human intestinal epithelium. Further, examination of ClC-2 localization by confocal microscopy revealed that ClC-2 contributes to secretion from a unique location in this epithelium, from the apical aspect of the tight junction complex. Hence, these studies provide the necessary rationale for considering ClC-2 as a possible therapeutic target for diseases affecting intestinal chloride secretion such as cystic fibrosis.

Evidence for a Functional Interaction between the ClC-2 Chloride Channel and the Retrograde Motor Dynein Complex

“…Constructs and Antibodies-The antisense ClC-2 construct was made by cloning the rat ClC-2 open reading frame into pCDNA 3.1(Ϫ) vector so that the reversed restriction sites on this vector would reverse the orientation of the open reading frame to create the antisense plasmid (10). The constructs for the expression of GFP 1 -tagged dynamitin, and DSRed-tagged CC1 were kindly provided by Dr. T. Schroer (The Johns Hopkins University).…”

“…Patch Clamp Studies of COS7 Cells-COS7 membrane currents were measured using conventional whole cell patch clamp technique as described previously (10). Briefly, currents were measured using an Axopatch 200A patch clamp amplifier (Axon Instruments, Foster City, CA) and were filtered at 100 Hz with a six-pore Bessel Filter.…”

“…Localization of ClC-2 to the apical membrane of fetal lung epithelia suggests a role in regulating airway surface fluid (7,8). In addition, ClC-2 channel function has been implicated in mediating intestinal fluid secretion, as it has been localized close to the apical tight junctions of intestinal epithelia (9,10).…”

Loss of Hyperpolarization-activated Cl− Current in Salivary Acinar Cells from Clcn2 Knockout Mice

“…It is unclear whether this barrier function is related to the regulation of ClC-2 activity by extracellular pH (13,14) or cell swelling (15). The apical location of the ClC-2 channel in rat small intestine, renal, and airway epithelia further suggests that ClC-2 plays a role in regulating fluid and electrolyte movement in these tissues (16,17). Indeed, antisense ClC-2 cDNA reduced native chloride current in the human intestinal cell line Caco-2 and significantly reduced Cl Ϫ -dependent secretion (16).…”

“…The apical location of the ClC-2 channel in rat small intestine, renal, and airway epithelia further suggests that ClC-2 plays a role in regulating fluid and electrolyte movement in these tissues (16,17). Indeed, antisense ClC-2 cDNA reduced native chloride current in the human intestinal cell line Caco-2 and significantly reduced Cl Ϫ -dependent secretion (16). Genetic analysis has provided important and sometimes surprising insights into the function of several chloride channels.…”