Amino acid substitutions in the pore of rat glutamate receptors at sites influencing block by polyamines

1. The effect on polyamine block of mutations at the Q/R site and the conse rved negative charge +4 site in AMPA and kainate receptors was studied usin g the rat glutamate receptor GluR6 expressed in Xenopus oocytes and human e mbryonic kidney (HEK) cells. 2. Introduction of negative charge at the Q/R site increased the equilibriu m dissociation constant at 0 mV (K-d(0)) for spermine from 1.3 to 4.0 mu m (Q590E); the smaller side chains Q590D and Q590N had K-d(0) values of 47 an d 20 mu m. Reductions in spermine affinity were also obtained for the small hydrophobic residues Q590V and Q590A, with K-d(0) values of 3.6 and 8.8 mu m. Positively charged side chains produced outward rectifying responses si milar to those recorded for GluR6(Q) with polyamine-free conditions, sugges ting a complete absence of voltage-dependent block by spermine. 3. Substitution of tryptophan at the Q/R site produced high-affinity block with a K-d(0) of 190 pm. In Xenopus oocytes no outward current was observed at potentials up to +200 mV. A much smaller increase in affinity was obser ved fur Q590F and Q590Y, which had K-d(0) values of 0.28 and 0.83 mu m, res pectively. 4. The Q590H mutant gave weakly birectifying responses strikingly different from those for other mutants. When ionization of the His group was increas ed by raising the external hydrogen ion concentration, responses became out ward rectifying. The ratios of the conductance at 100 mV over that at -100 mV for Q590H were 0.52 at pH 8.3 and 2.5 at pH 5.3. 5. Neutralization of charge or aromatic residues at the +4 site produced a large reduction of spermine affinity, with K-d(0) values for E594N, E594Q a nd E594W of 109, 1020 and 2150 mu m, respectively. In the absence of polyam ines, E594K and E594R produced strongly inward rectifying responses while E 594Q, E594A and E594W were birectifying. 6. A model for permeant block allowed quantitative comparisons between muta nts. Despite large changes in well depth and barrier heights, there was lit tle change in the voltage dependence of block for both Q/R and +4 site muta nts. We propose a model with a distributed binding site for polyamines in w hich the +4 site is located near the: entrance to the channel.