Extracellular vestibule determinants of Ca2+ influx in Ca2+-permeable AMPA receptor channels

At certain synapses in the brain, Ca 2+ -permeable AMPA receptor (AMPAR) channels represent an important pathway for synaptically controlled Ca 2+ entry. However, the molecular determinants of this Ca 2+ influx are poorly defined. In NMDA receptor (NMDAR) channels, where the influx is much greater, the extracellular vestibule, specifically the M3 segment and regions C-terminal to it in the NR1 subunit, contains elements critical to their high Ca 2+ influx under physiological conditions. We therefore investigated the contribution of homologous positions in AMPAR as well as kainate receptor (KAR) subunits to the process of Ca 2+ influx. Substitutions of a conserved asparagine (N) in M3 of AMPAR GluR-B(Q) channels strongly attenuated Ca 2+ permeability measured using reversal potentials under biionic conditions and fractional Ca 2+ currents recorded under physiological conditions. Hence, as in NMDAR channels, the conserved N makes a significant contribution to Ca 2+ influx in AMPAR channels. In addition, C-terminal to M3, substitutions of negatively (glutamate, E) or positively (arginine, R) charged residues also altered Ca 2+ influx. However, in contrast to charged residues occupying homologous positions in NMDAR channels, these effects were about equal and opposite suggesting that this ER in AMPARs does not contribute significantly to the mechanism of Ca 2+ influx. Opposite charge substitutions of two negative residues C-terminal to M3 in KAR GluR-6(Q) subunits had no effect on Ca 2+ permeability. We conclude that the different contribution of residues C-terminal to M3 to Ca 2+ permeation in NMDAR and non-NMDAR channels reflects a different positioning of these residues relative to the tip of the M2 loop.