PKA and arachidonic acid activation of human recombinant ClC-2 chloride channels

Abstract

An HEK-293 cell line stably expressing the human recombinant ClC-2 Cl− channel was used in patch-clamp studies to study its regulation. The relative permeability P x/ P Cl calculated from reversal potentials was I− > Cl− = NO3 − = SCN−≥Br−. The absolute permeability calculated from conductance ratios was Cl− = Br− = NO3 −≥ SCN− > I−. The channel was activated by cAMP-dependent protein kinase (PKA), reduced extracellular pH, oleic acid (C:18 cisΔ9), elaidic acid (C:18 transΔ9), arachidonic acid (AA; C:20 cisΔ5,8,11,14), and by inhibitors of AA metabolism, 5,8,11,14-eicosatetraynoic acid (ETYA; C:20 transΔ5,8,11,14), α-methyl-4-(2-methylpropyl)benzeneacetic acid (ibuprofen), and 2-phenyl-1,2-benzisoselenazol-3-[2H]-one (PZ51, ebselen). ClC-2 Cl− channels were activated by a combination of forskolin plus IBMX and were inhibited by the cell-permeant myristoylated PKA inhibitor (mPKI). Channel activation by reduction of bath pH was increased by PKA and prevented by mPKI. AA activation of the ClC-2 Cl− channel was not inhibited by mPKI or staurosporine and was therefore independent of PKA or protein kinase C activation.