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

An HEK-293 cell line stably expressing the human recombinant ClC-2 Cl- chan nel was used in patch-clamp studies to study its regulation. The relative p ermeability P-x/P-Cl calculated from reversal potentials was I- > Cl- = NO3 - = SCN- greater than or equal to Br-. The absolute permeability calculated from conductance ratios was Cl- = Br- = NO3- greater than or equal to SCN- > I-. The channel was activated by cAMP-dependent protein kinase (PKA), re duced extracellular pH, oleic acid (C:18 cis Delta 9), elaidic acid (C:18 t rans Delta 9), arachidonic acid (AA; C:20 cis Delta 5,8,11,14), and by inhi bitors of AA metabolism, 5,8,11,14-eicosatetraynoic acid (ETYA; C:20 trans Delta 5,8,11,14), alpha-methyl-4-(2-methylpropyl)benzeneacetic acid (ibupro fen), and 2-phenyl-1,2-benzisoselenazol-3-[2H]-one (PZ51, ebselen). ClC-2 C l- 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 m PKI. AA activation of the ClC-2 Cl- channel was not inhibited by mPKI or st aurosporine and was therefore independent of PKA or protein kinase C activa tion.