The chloride channel ClC-2 contributes to the inwardly rectifying Cl- conductance in cultured porcine choroid plexus epithelial cells

The contribution of ClC-2 protein to the inwardly rectifying Cl- conductanc e in cultured porcine choroid plexus epithelial cells was investigated usin g Western analysis and whole-cell current recordings. Inwardly rectifying currents were elicited by hyperpolarizing voltage at a potential more negative than -50 mV in the presence of intracellular protei n kinase A (PKA). The relative halide selectivity estimated from the shift in the reversal potential (E-rev) was I- > Br- > Cl- > F-. Extracellular vasoactive intestinal peptide (VIP) activated the same curren ts in a dose-dependent manner with a half-maximal concentration of 167.3 nM . H-89 (a PKA inhibitor) interfered with the current activation by VIP. The Cl- channel was inhibited by external Cd2+, B2+ or H+, but only weakly inhibited by known Cl- channel blockers including glibenclamide, NPPB, DIDS and anthracene-9-carboxylic acid (SAC). A specific antibody to ClC-2 detected a 79 kDa protein in porcine choroid p lexus cells, which was reduced in cells treated with antisense oligodeoxynu cleotide for ClC-2. Both PKA and VIP failed to activate the inwardly rectif ying Cl- currents in cells transfected with the antisense oligodeoxynucleot ide, while they activated the currents in cells transfected with GFP alone or the control oligodeoxynucleotide randomized from antisense oligonucleoti de. It is concluded that ClC-2 protein contributes to the inwardly rectifying C l- conductance in porcine choroid plexus epithelial cells.