To determine the effects of chloride channel 3 (ClC-3) knockdown and overexpression on lysophosphatidic acid (LPA)- and volume-regulated anion channel Cl(-) currents (I(Cl，LPA) and I(Cl，VRAC)， respectively)， cell differentiation， and cell volume regulation， a short hairpin RNA (shRNA) expression system based on a mouse U6 promoter was used to knock down ClC-3 in human corneal keratocytes and human fetal lung fibroblasts. ClC-3 overexpression was achieved by electroporating full-length ClC-3， within a pcDNA3.1 vector， into these two cell lines. RT-PCR and Western blot analysis were used to detect ClC-3 mRNA and protein levels. Whole cell perforated patch-clamp recording was used to measure I(Cl... More
To determine the effects of chloride channel 3 (ClC-3) knockdown and overexpression on lysophosphatidic acid (LPA)- and volume-regulated anion channel Cl(-) currents (I(Cl，LPA) and I(Cl，VRAC)， respectively)， cell differentiation， and cell volume regulation， a short hairpin RNA (shRNA) expression system based on a mouse U6 promoter was used to knock down ClC-3 in human corneal keratocytes and human fetal lung fibroblasts. ClC-3 overexpression was achieved by electroporating full-length ClC-3， within a pcDNA3.1 vector， into these two cell lines. RT-PCR and Western blot analysis were used to detect ClC-3 mRNA and protein levels. Whole cell perforated patch-clamp recording was used to measure I(Cl，LPA) and I(Cl，VRAC) currents， and fluorescence-activated cell sorting analysis was used to measure cell volume regulation. ClC-3 knockdown significantly decreased I(Cl，LPA) and I(Cl，VRAC) activity in the presence of transforming growth factor-beta(1) (TGF-beta(1)) compared with controls， whereas ClC-3 overexpression resulted in increased I(Cl，LPA) activity in the absence of TGF-beta(1). ClC-3 knockdown also resulted in a reduction of alpha-smooth muscle actin (alpha-SMA) protein levels in the presence of TGF-beta(1)， whereas ClC-3 overexpression increased alpha-SMA protein expression in the absence of TGF-beta(1). In addition， keratocytes transfected with ClC-3 shRNA had a significantly blunted regulatory volume decrease response following hyposmotic stimulation compared with controls. These data confirm that ClC-3 is important in VRAC function and cell volume regulation， is associated with the I(Cl，LPA) current activity， and participates in the fibroblast-to-myofibroblast transition.
