Cation permeability and cation-anion interactions in a mutant GABA-gated chloride channel from Drosophila

To investigate the structural basis of anion selectivity of Drosophila GABA -gated Cl- channels, the permeation properties of wild-type and mutant chan nels were studied in Xenopus oocytes. This work focused on asparagine 319, which by homology is one amino acid away from a putative extracellular ring of charge that regulates cation permeation in nicotinic receptors. Mutatio n of this residue to aspartate reduced channel conductance, and mutation to lysine or arginine increased channel conductance. These results are consis tent with an electrostatic interaction between this site and permeating ani ons. The lysine mutant, but not the arginine mutant, formed a channel that is permeable to cations, and this cannot be explained in terms of electrost atics. The lysine mutant had a 25-mV reversal potential in solutions with s ymmetrical Cl- and asymmetrical cations. The permeability ratio of K+ to Cl - was determined as 0.33 from reversal potential measurements in KCI gradie nts. Experiments with large organic cations and anions showed that cation p ermeation can only be seen in the presence of Cl-, but Cl- permeation can b e seen in the absence of permeant cations. Measurements of permeability rat ios of organic anions indicated that the lysine mutant has an increased por e size. The cation permeability of the lysine-containing mutant channel can not be accounted for by a simple electrostatic interaction with permeating ions. It is likely that lysine substitution causes a structural change that extends beyond this one residue to influence the positions of other channe l-forming residues. Thus protein conformation plays an important role in en abling ion channels to distinguish between anions and cations.