Extracellular Vestibule Determinants of Ca²⁺ Influx in Ca²⁺‐Permeable AMPA Receptor Channels

Abstract

At certain synapses in the brain, Ca2+‐permeable AMPA receptor (AMPAR) channels represent an important pathway for synaptically controlled Ca2+ entry. However, the molecular determinants of this Ca2+ 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 Ca2+ 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 Ca2+ influx. Substitutions of a conserved asparagine (N) in M3 of AMPAR GluR‐B(Q) channels strongly attenuated Ca2+ permeability measured using reversal potentials under biionic conditions and fractional Ca2+ currents recorded under physiological conditions. Hence, as in NMDAR channels, the conserved N makes a significant contribution to Ca2+ influx in AMPAR channels. In addition, C‐terminal to M3, substitutions of negatively (glutamate, E) or positively (arginine, R) charged residues also altered Ca2+ 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 Ca2+ influx. Opposite charge substitutions of two negative residues C‐terminal to M3 in KAR GluR‐6(Q) subunits had no effect on Ca2+ permeability. We conclude that the different contribution of residues C‐terminal to M3 to Ca2+ permeation in NMDAR and non‐NMDAR channels reflects a different positioning of these residues relative to the tip of the M2 loop.

Atypical Functional Properties of GluK3-Containing Kainate Receptors

“…GluA3 mutated in the Q/R site to an N gave calcium permeable receptors with no rectification (Dingledine et al, 1992). Moreover, GluA2 receptor mutants (N619C and N619K) have much decreased calcium permeability, but their rectification properties are similar to wild-type receptors, suggesting that their spermine sensitivity is the same (Jatzke et al, 2003). In a more physiological situation, addition of stargazin to AMPA receptors decreases their sensitivity to spermine without changing their calcium permeability (Soto et al, 2007).…”

“…(Lomeli et al, 1992). We determined the fractional calcium current by recording the charge going through receptors and the amount of calcium into the cell by fluorescence imaging with fura-2 Jatzke et al, 2003). However, for reliable detection of calcium concentration changes with 1 mM fura-2 large charges going through the receptors are needed.…”

Block of AMPA Receptor Desensitization by a Point Mutation outside the Ligand-Binding Domain

“…The R 3 E substitution introduces an additional negative side chain in the M3-S2 linker and underlies surface charge-like effects on Ca 2ϩ permeability (Jatzke et al, 2003). The nondesensitizing phenotype therefore might arise because of divalent ions such as Ca 2ϩ or Zn 2ϩ more strongly interacting with this highly negative element.…”

“…1). On the other hand, substitutions of these residues in AMPARs (Jatzke et al, 2003) and of homologous positions in the NMDAR NR1 subunit (Watanabe et al, 2002) affect Ca 2ϩ permeation, suggesting that they are associated with the ion conduction pathway. Nevertheless, ER is positioned four amino acid residues external to SYTANLAAF, a motif forming the most extracellular part of the channel (Sobolevsky et al, 2003).…”

“…Notable in this regard is the presence in all subunits of a highly conserved positively charged arginine (R). This arginine and an adjacent negatively charged glutamate (E) in AMPAR subunits influence Ca 2ϩ permeation (Jatzke et al, 2003) and presumably are positioned in the ion conduction pathway. Recent evidence has also suggested that charged residues in linker regions in GABA A (Kash et al, 2003) and voltage-gated channels (Larsson and Elinder, 2000) affect channel gating.…”