A Pentasymmetric Open Channel Blocker for Cys-Loop Receptor Channels

γ-Aminobutyric acid type A receptors (GABA(A) receptors) are chloride ion channels composed of five subunits, mediating fast synaptic and tonic inhibition in the mammalian brain. These receptors show near five-fold symmetry that is most pronounced in the second trans-membrane domain M2 lining the Cl(−) ion channel. To take advantage of this inherent symmetry, we screened a variety of aromatic anions with matched symmetry and found an inhibitor, pentacyanocyclopentdienyl anion (PCCP(−)) that exhibited all characteristics of an open channel blocker. Inhibition was strongly dependent on the membrane potential. Through mutagenesis and covalent modification, we identified the region α(1)V256-α(1)T261 in the rat recombinant GABA(A) receptor to be important for PCCP(−) action. Introduction of positive charges into M2 increased the affinity for PCCP(−) while PCCP(−) prevented the access of a positively charged molecule into M2. Interestingly, other anion selective cys-loop receptors were also inhibited by PCCP(−), among them the Drosophila RDL GABA(A) receptor carrying an insecticide resistance mutation, suggesting that PCCP(−) could serve as an insecticide.