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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

ClC-3 is required for LPA-activated Cl^– current activity and fibroblast-to-myofibroblast differentiation

Departments of ¹Physiology and ²Anatomy and Neurobiology, University of Tennessee Health Science Center, Memphis, Tennessee; and ³Department of Pathology, St. Jude Children's Research Hospital, Memphis, Tennessee

Submitted 9 July 2007 ; accepted in final form 7 December 2007

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-β₁ (TGF-β₁) compared with controls, whereas ClC-3 overexpression resulted in increased I_Cl,LPA activity in the absence of TGF-β₁. ClC-3 knockdown also resulted in a reduction of -smooth muscle actin (-SMA) protein levels in the presence of TGF-β₁, whereas ClC-3 overexpression increased -SMA protein expression in the absence of TGF-β₁. 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.

chloride channel-3; lysophosphatidic acid; cornea; lung; keratocyte