The ability of members of the nicotinicoid superfamily of ligand-gated ion channels to selectively conduct anions or cations is critical to their function within the central nervous system. Recent work has demonstrated that residues at the intracellular end of the second transmembrane domain, between the -3' and 2' positions, form the ion selectivity filter of these receptors. In this study, the proline residue at the 2' position (Pro-2') at the intracellular end of the second transmembrane domain of the gamma-aminobutyric acid type C rho 1 subunit was mutated to glutamate (rho 1P2'E) and arginine (rho 1P2'R). Dilution potential experiments indicated that the charge selectivity of the rho 1P2'E receptor channels had been inverted, with the channels now becoming predominantly cation selective, indicating the ability of negatively charged residues at this 2' position to control charge selectivity. The mutation was also seen to have significantly decreased agonist potency and intrinsic efficacy. In contrast, the rho 1P2'R receptor channels were anion-selective but were now found to be constitutively open with high holding currents (inhibited by low gamma-aminobutyric acid doses and the competitive antagonist, 1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid alone) and increased agonist activity. Hill coefficients of both mutants were decreased, but competitive antagonist studies indicated that their binding sites were not significantly affected.