A Pentasymmetric Open Channel Blocker for Cys-Loop Receptor Channels

c-Aminobutyric acid type A receptors (GABAA 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 Cl2 ion channel. To take advantage of this inherent symmetry, we screened a variety of aromatic anions with matched symmetry and found an inhibitor, pentacyanocyclo-pentdienyl anion (PCCP2) 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 a1V256-a1T261 in the rat recombinant GABAA receptor to be important for PCCP 2 action. Introduction of positive charges into M2 increased the affinity for PCCP2 while PCCP2 prevented the access of a positively charged molecule into M2. Interestingly, other anion selective cys-loop receptors were also inhibited by PCCP2, among them the Drosophila RDL GABAA receptor carrying a