A pentasymmetric open channel blocker for cys-loop receptor channels

Abstract

γ-Aminobutyric acid type A receptors (GABAAreceptors) 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, pentacyanocyclo-pentdienyl 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 α1V256-α1T261 in the rat recombinant GABAAreceptor 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 GABAAreceptor carrying an insecticide resistance mutation, suggesting that PCCP-could serve as an insecticide.